7th INTERNATIONAL Conference on HHV-6 & 7

7th INTERNATIONALConference on HHV-6 & 7

February 27th – March 2nd, 2011Hyatt Regency | Reston, Virginia

Hosted By The:

www.HHV-6Foundation.org

PROGRAM BOOKPROGRAM BOOK

SUNDAYFEBRUARY 27

MONDAYFEBRUARY 28

TUESDAYMARCH 1

WEDNESDAYMARCH 2

Registration16:00 - 19:00

Speaker Check-In16:00 - 19:00

Market Street Bar and GrillPrivate Dining Room



Grand Ballroom Foyer







Tea, Coffee, and Muffins08:00 - 08:30






Welcome and Introduction08:30 - 08:50

Presenters: Flamand, YoshikawaPanel Discussion

Epidemiology09:20 - 09:50

Presenters: Bonnafous, GoldfarbHHV-6 and Non-Neurological Diseases

09:50 - 10:30Presenters: Di Luca, Broccolo

Refreshment Break10:45 - 11:00






HHV-6 and Non-Neurological Diseases (cont'd)10:45 - 11:15

Presenters: Bell, Knox

HHV-6 & Multiple Sclerosis10:35 - 11:50

Presenters: Jacobson, Alvarez-Lafuente, Banerjee, Virtanen

HHV-6, DRESS/DIHS & Immunity 11:15 - 12:25

Presenters: Descamps, Eriksson, Moosmann, Calvo-Calle

Diagnostics & Reactivation12:25 - 12:45

Presenter: Yoshikawa

Lunch 12:45 - 14:00

Lunch 12:45 - 14:00

Lunch 12:45 - 14:00

Diagnostics & Reactivation (cont'd)14:00 - 15:15

Presenters: Lautenschlager, Gautheret-Dejean, Govind, Pellett

HHV-6 & 7 in Epilepsy 14:00 - 15:00

Presenters: Niehusmann, Epstein, Zhou

HHV-6 Treatment15:25 - 15:55

Presenters: Agut, Naesens





Welcome Reception 18:00 - 19:00

Market Street Bar and Grill Private Dining Room

Poster Session and Reception18:00 - 19:00

Grand Ballroom

Poster Session and Reception17:30 - 18:30

Grand Ballroom Banquet

19:00 - 22:00McCormick and Schmick's Seafood Restaurant

A walking group will depart from the lobby at 18:50.

HHV-6 Animal Models11:50 - 12:45

Presenters: Wohler, ReynaudPanel Discussion

Panel Discussion: How to Confirm/Refute Suggestions that HHV-6B Plays a Causative

Role in MTLE15:00 - 16:00

Germ-line Chromosomal Integration & Telomeres - Part II

16:10 - 18:00Presenters: Kaufer, Luka, Arbuckle, Pantry,

MedveczkyPanel Discussion

Scientific Advisory Board Meeting16:00 - 18:00

Lake Fairfax B, Second Floor

HHV-6 Treatment (cont'd)16:20 - 17:30

Presenters: Gerdemann, Montoya, Painter, Lassner

Panel Discussion

PROGRAM AT A GLANCE

All general sessions will be held in the Grand Ballroom, Second Floor.

HHV-6 BAC08:30 - 09:20

Presenters: Yu, Mori, Frenkel

HHV-6 Host Cell Interactions8:50 - 10:45

Presenters: Lusso, Coscoy, Mardivirin, Kofod-Olsen, Clark, Prusty, Crawford

HHV-6 Genes and Proteins11:00 - 12:45

Presenters: Gompels, Flamand, Kowalik, Tang, Zeigerman, Kawabata

Germ-line Chromosomal Integration & Telomeres - Part I

14:00 - 15:55Presenters: Luppi, Ward, Hall, Lieberman,

Hendrickson, Riethman

HHV-6 in CNS Disease08:30 - 10:20

Presenters: Komaroff, Zerr, Yolken, Kawamura, Ogata

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TABLE OFCONTENTS

WELCOMEDear Colleagues,

It is with great pride and honour that we have accepted to act as Co-Chairs of the 7th international conference on HHV-6 & HHV-7. One of the main goal of the meeting is to bring together top scientists and clinicians to foster new collaborations, exchange ideas and reagents and discuss the latest developments in HHV-6 & HHV-7 research. The meeting is truly international with over 100 students, scientists and clinicians coming for 15 different countries.

Three years have passed since the Baltimore meeting and many interesting new developments were made during this period. Chromosomally-integrated HHV-6 is certainly one area gaining a lot of attention and an entire session will be devoted to this topic. The International Committee on Taxonomy of Viruses has reached a decision regarding the nomenclature to adopt when referring to HHV-6 variants A and B. The decision will be presented and discussed. Several other important findings such as immune evasion mechanisms utilized by HHV-6 to persist within its host as well as the development of new molecular tools, such as the cloning of the HHV-6 genome in a bacterial artificial chromosome, will greatly facilitate our comprehension of protein functions will be presented. As complement, small animal models permissive to HHV-6 infection are being developed allowing researchers to better understand the pathogenic nature of HHV-6. Lastly, the identification of dominant and potentially protective T cell epitopes is also being reported for the first time, opening the field for anti-HHV-6-based immunotherapies.

Our mandate was greatly facilitated by the continuous and relentless efforts deployed by members of the HHV-6 Foundation, spearheaded by its executive Director Kristin Loomis and its Scientific Director Dharam Ablashi. We also take this opportunity to thank the sessions chairs and co-chairs for helping us put together an exciting scientific program.

We look forward to meet and exchange with you on is promising to be a very informative event.

Louis Flamand and Tetsushi YoshikawaConference Co-Chairs

Welcome . . . . . . . . . . . . . . . .3–4

Chairs, Program Committee, Advisory Board . . . . . . . . . . . .5

HHV-6A and HHV-6B Divergence . . . . . . . . . . . . . . .7

Awards . . . . . . . . . . . . . . . . . 8-9

Workshop Information . . . . 11-14

Workshop Schedule . . . . . .15–22

Monday, February 28 . . . . . .15

Tuesday, March 1 . . . . . . . . .18

Wednesday, March 2 . . . . . .21

Poster Presentations by Topic . . .23

Abstracts . . . . . . . . . . . . . .27–78

Overview . . . . . . . . . . . . . . .29

HHV-6 Host Cell Interactions .30

HHV-6 Genes & Proteins . . . .35

Germ-line Chromosomal Integration . . . . . . . . . . . . . . .40

Telomeres & Integration . . . . .47

HHV-6 BAC . . . . . . . . . . . . .49

Epidemiology . . . . . . . . . . . .51

HHV-6 and Non-Neurological Diseases . . . . . . . . . . . . . . . .53

HHV-6, DRESS/DIHS & Immunity . . . . . . . . . . . . . . .57

Diagnostics & Reactivation . . .60

HHV-6 Treatment . . . . . . . . .64

HHV-6 in CNS Disease . . . . .72

HHV-6 & Multiple Sclerosis . .76

HHV-6 Animal Models . . . . .78

HHV-6 & 7 in Epilepsy . . . . .79

Author Index . . . . . . . . . . . . . .81

Participants Roster . . . . . 86Notes 91

HHV-6 Foundation Repository of Reagents . . . . . . . . . . . . .99

Dharam Ablashi Lifetime Achievement Award Winners . . . . . . . . . .Back cover

Louis Flamand, Ph.D.Université LavalCanada

Tetsushi Yoshikawa, M.D.Fujita Health UniversityJapan

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CONFERENCESPONSORS

WELCOME FROM THE HHV-6 FOUNDATIONDear Colleagues and Invited Guests,

The HHV-6 Foundation was chartered in 2004 as a non-profit Foundation to foster laboratory & clinical research on HHV-6. The foundation offers small grants to groups interested in gathering pilot data and, with the help of many HHV-6 and HHV-7 scientists, we operate a repository of reagents in an effort to facilitate research. If any of you have valuable reagents and would like to share with the scientific community to help promote the study of HHV-6 worldwide, we invite you to deposit these in our Repository.

We would like to thank Dr. Flamand and Dr. Yoshikawa for taking the responsibility of co-chairing this important event. We hope this conference will give you the opportunity to informally meet each other, establish collaborations, and exchange clinical materials so that the science presented here can ultimately be taken to the bedside.

Sincerely,

Dharam, Kristin and the HHV-6 Foundation Team

HHV-6 FOUNDATION TEAM

Dharam V. Ablashi, Scientific DirectorKristin S. Loomis, President and Executive DirectorJudith Anderson, Vice PresidentLouise G. Chatlynne, Senior AdvisorJill Chase, Repository ManagerJoshua Pritchett, Research AssistantMona Elliason, Chair, Board of Directors

Dharam Ablashi, DVMHHV-6 FoundationUSA

Henri Agut, M.D., Ph.DGroupe Hospitalier Pitié-SalpêtrièreFrance

Roberto Alvarez-Lafuente, M.D.Hospital Clinico San CarlosSpain

Ursula Gompels, Ph.DLondon School of Hygiene & Tropical MedicineUnited Kingdom

Anthony Komaroff, M.D.Harvard Medical SchoolUSA

Peter Medveczky, M.D.University of South FloridaUSA

Yasuko Mori, M.D.Kobe University Graduate School of MedicineJapan

Lieve Naesens, Ph.DRega Institute, Katholieke Universiteit LeuvenBelgium

Philip Pellett, Ph.DWayne State University, School of MedicineUSA

CHAIR

Dharam Ablashi, DVMHHV-6 FoundationUSA

COMMITTEE

Henri Agut, M.D., Ph.D.Groupe Hospitalier Pitié-SalpêtrièreFrance

Roberto Alvarez-LaFuente, M.D.Hospital Clinico San CarlosSpain

Leon Epstein, M.D.Northwestern Medical SchoolUSA

Louis Flamand, Ph.D.Université LavalCanada

Ursula Gompels, Ph.D.London School of Hygiene & Tropical MedicineUnited Kingdom

Steven Jacobson, Ph.D.National Institute of Neurological Disorders and Stroke, NIHUSA

Anthony Komaroff, M.D.Harvard Medical School,USA

Irmeli Lautenschlager, M.D., Ph.D.University of HelsinkiFinland

Paolo Lusso, M.D.San Raffaele Scientific InstituteItaly

Peter Medveczky, M.D.University of South FloridaUSA

Jose Montoya, M.D., Ph.D.Stanford UniversityUSA

Yasuko Mori, M.D., Ph.D.Kobe University Graduate School of MedicineJapan

Lieve Naesens, Ph.D.Rega Institute, Katholieke Universiteit LeuvenBelgium

Masao Ogata, M.D., Ph.D.Oita University School of MedicineJapan

Philip Pellett, Ph.D.Wayne State University, School of MedicineUSA

Koichi Yamanishi, M.D., Ph.D.National Institute of Biomedical InnovationJapan

Tetsushi Yoshikawa, M.D.Fujita Health UniversityJapan

Kristin Loomis, Executive DirectorHHV-6 FoundationUSA

Conference Co-Chairs

Program Committee

Conference Secretariat

2011 - 2012 HHV-6 Foundation Scientific Advisory Board

CHAIRS, PROGRAM COMMITTEE, ADVISORY BOARD

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Louis Flamand, Ph.D.Université Laval

Canada

Tetsushi Yoshikawa, M.D.Fujita Health University

Japan

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HHV-6A and HHV-6B DIVERGENCE The HHV-6 Foundation would like to thank Phil Pellett, Chair of the Herpesvirales Working Group of the International Committee on Taxonomy of Viruses for considering our petition to formally recognize HHV-6A and HHV-6B divergence. We would also like to thank Dario Diluca and the Ad Hoc Committee on HHV-6A & HHV-6B Genomic Divergence that worked very hard on the petition submitted in January 2010. Phil Pellett will announce the Subcommittee’s recommendation Monday morning.

Ad Hoc Committee:Dharam Ablashi (Co-Chair)Dario Di Luca (Co-Chair)

Henri AgutYoshizo AsanoRoberto Alvarez-LaFuenteDuncan Clark Steve DewhurstLouis Flamand Niza FrenkelRobert Gallo Ursula Gompels

Caroline HallSteve JacobsonKazuhiro KondoMario LuppiPaolo Lusso Yasauki MoriKoichi YamanishiTetsushi Yoshikawa

Philip Pellett, Chair, Herpesvirales Working Group

Dario Di Luca, Co-Chair, Ad Hoc Committee

Dharam Ablashi, Co-Chair, Ad Hoc Committee

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AWARDS

Jesse ArbuckleUniversity of South Florida College of Medicine

USA

Pinaki BanerjeeThe Children’s Hospital of Philadelphia

USA

Adam BellUniversity of Glasgow

United Kingdom

David ClarkLondon School of Hygiene & Tropical Medicine

United Kingdom

Ulrike GerdemannBaylor College of Medicine

USA

Emil Kofod-OlsenAarhus University

Denmark

Bhupesh PrustyUniversity of Würzburg

Germany

Joséphine ReynaudINSERMFrance

Huamin TangNational Institute of Biomedical Innovation

Japan

Haim ZeigermanTel Aviv University

Israel

2011 Outstanding Trainee Abstract Awards

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2011 – Kinetics of cytokine and chemokine responses in patients with primary human herpesvirus 6 infection.

2010 – Direct detection of human herpesvirus 6 DNA in serum by variant specific loop-mediated isothermal amplification in hematopoietic stem cell transplant recipients.

2009 – Exanthem subitum-associated encephalitis: nationwide survey in Japan.

– Relationship between U83 gene variation in human herpesvirus 6 and secretion of the U83 gene product.

2008 – Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B.

– Elevated serum cytokine levels are associated with human herpesvirus 6 reactivation in hematopoietic stem cell transplantation recipients.

– Human herpesvirus 6 infection in adult living related liver transplant recipients.

2007 – Direct detection of human herpesvirus 6 DNA in serum by the loop mediated isothermal amplification method.

2006 – Human herpesvirus 6 reactivation and inflammatory cytokine production in patients with drug-induced hypersensitivity syndrome.

– Time course characteristics of human herpesvirus 6 specific cellular immune response and natural killer cell activity in patients with exanthema subitum.

– Drug-induced hypersensitivity syndrome due to mexiletine hydrochloride associated with reactivation of human herpesvirus 7.

– Latent infection of human herpesvirus 7 in CD4(+) T lymphocytes.

2005 – Postinfectious myeloradiculoneuropathy with cranial nerve involvements associated with human herpesvirus 7 infection.

– Analysis of shedding of 3 beta-herpesviruses in saliva from patients with connective tissue diseases.

– In vitro and in vivo analysis of human herpesvirus-6 U90 protein expression.

2004 – Human herpesvirus 6 fulminant hepatic failure treated by living donor liver transplantation.

– Atypical clinical features of a human herpesvirus-6 infection in a neonate.

– Detection of human herpesvirus 7 DNA by loop mediated isothermal amplification.

– Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification.

2003 – Variation of human herpesvirus 7 shedding in saliva.

– Human herpesvirus 6 infection of human epidermal cell line: pathogenesis of skin manifestations.

– Human herpesvirus 7-associated meningitis and optic neuritis in a patient after allogeneic stem cell transplantation.

– Evaluation of active human herpesvirus 6 infection by reverse transcription-PCR.

2002 – Serological examination of human herpesvirus 6 and 7 in patients with coronary artery disease.

– Reactivation of human herpesvirus 6 and 7 in pregnant women.

– Guillain-Barre syndrome after exanthem subitum. – Human herpesvirus 6 viremia in bone marrow

transplant recipients: clinical features and risk factors.

– Latent infection of human herpesvirus 6 in astrocytoma cell line and alteration of cytokine synthesis.

– Influenza encephalopathy associated with infection with human herpesvirus 6 and/or human herpesvirus 7.

– Fatal adult case of severe lymphocytopenia associated with reactivation of human herpesvirus 6.

– Monitoring of active HHV-6 infection in bone marrow transplant recipients by real time PCR; comparison to detection of viral DNA in plasma by qualitative PCR.

2001 – Fatal acute myocarditis in an infant with human herpesvirus 6 infection.

– Quantitation of human herpesvirus 6 DNA in infant with exanthem subitum by microplate PCR-hybridization assay.

– Correlation between HHV-6 infection and skin rash after allogeneic bone marrow transplantation.

– Primary human herpesvirus 6 infection in liver transplant recipients.

– Transfer of human herpesvirus 6 and 7 antibodies from mothers to their offspring.

– Rapid loss of insulin secretion in a patient with fulminant type 1 diabetes mellitus and carbamazepine hypersensitivity syndrome.

– Comparison of specific serological assays for diagnosing human herpesvirus 6 infection after liver transplantation.

– Correlation between human herpesvirus 6 and 7 infections after living related liver transplantation.

2000 – Monitoring four herpesviruses in unrelated cord blood transplantation.

– Human herpesvirus 6 infection after living related liver transplantation.

– Invasion by human herpesvirus 6 and human herpesvirus 7 of the central nervous system in patients with neurological signs and symptoms.

– Central nervous system complications in human herpesvirus-6 infection.

– Clinical characteristics of febrile convulsions during primary HHV-6 infection.

1999 – Human herpesvirus 6 latently infects mononuclear cells but not liver tissue.

1998 – Prediction of human herpesvirus 6 infection after allogeneic bone marrow transplantation.

– Prospective study of persistence and excretion of human herpesvirus-6 in patients with exanthem subitum and their parents.

– Five cases of thrombocytopenia induced by primary human herpesvirus 6 infection.

– Four cases of human herpesvirus 6 variant B infection after pediatric liver transplantation.

– Clinical features of primary HHV-6 and HHV-7 infections.

1997 – Human herpesvirus 6 and 7 infections. – Clinical features and virological findings in

children with primary human herpesvirus 7 infection.

1996 – Distribution of human herpesvirus 6 and varicella-zoster virus in organs of a fatal case with exanthem subitum and varicella.

– Development and application of HHV-6 antigen capture assay for the detection of HHV-6 infections.

– Human herpesviruses 6 and 7.

1995 – Human herpesvirus-7 (HHV-7): current status. – Detection of human herpesvirus 6 DNAs in

samples from several body sites of patients with exanthem subitum and their mothers by polymerase chain reaction assay.

– Detection of human herpesvirus 6 in plasma of children with primary infection and immunosuppressed patients by polymerase chain reaction.

– Clinical features and viral excretion in an infant with primary human herpesvirus 7 infection.

1994 – Bone marrow transplant recipients harbor the B variant of human herpesvirus 6.

– Human herpesvirus-7 (HHV-7). – Clinical features of infants with primary human

herpesvirus 6 infection (exanthem subitum, roseola infantum).

1993 – Seroepidemiology of human herpesvirus 7 in healthy children and adults in Japan.

– Clinical features of primary human herpesvirus-6 infection in an infant with acute lymphoblastic leukemia.

– Clinical and virological analyses of 21 infants with exanthem subitum (roseola infantum) and central nervous system complications.

– Epidemiology of acute childhood encephalitis. – Exacerbation of idiopathic thrombocytopenic

purpura by primary human herpesvirus 6 infection.

– Human herpesvirus-6 and parvovirus B19 infections in children.

1992 – Fatal encephalitis/encephalopathy in primary human herpesvirus-6 infection.

– Activation of human herpesvirus-6 in children with acute measles.

– A prospective study of human herpesvirus-6 infection in renal transplantation.

– Primary human herpesvirus-6 infection (exanthem subitum) in the newborn.

– Human herpesvirus-6 infection and bone marrow transplantation.

– Endonuclease analyses of DNA of human herpesvirus-6 isolated from blood before and after bone marrow transplantation.

– Human herpesvirus-6 DNA in cerebrospinal fluid of a child with exanthem subitum and meningoencephalitis.

– IgM neutralizing antibody responses to human herpesvirus-6 in patients with exanthem subitum or organ transplantation.

Plus 14 additional publications from 1988-1991

Dharam Ablashi Lifetime Achievment Award

YOSHIZO ASANO’S 90 HHV-6 AND HHV-7 PUBLICATIONS BY YEAR:

2011 Recipient of the HHV-6 Foundation Board’s

Yoshizo Asano, M.D., Ph.DProfessor for the Zambia Project

Research Center for Zoonosis ControlHokkaido University

Formally Chair, Pediatrics Department, Fujita Health University, Japan

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CONFIDENTIALITY NOTICEPlease note that this is a conference that presents information ahead of publication. Respect all information communicated by all presenters by acknowledging information that is presented as a “personal communication”. Please seek the approval of the presenter before quoting unpublished research results or using data as a basis for further investigations. Photography of presentations and posters is not allowed.

WORKSHOP INFORMATION

CONFERENCE VENUEHyatt Regency Reston1800 Presidents StreetReston, Virginia 20190Phone: +1.703.709.1234 Fax: +1.703.925.8295

WORKSHOP REGISTRATION AND INFORMATION DESKMarket Street Bar and Grill, Private Dining Room FoyerSunday, February 27 . . . . . . . . . . 16:00 – 19:00

Grand Ballroom Foyer, Second FloorMonday, February 28 . . . . . . . . . 07:30 – 18:30Tuesday, March 1. . . . . . . . . . . . . 08:00 – 18:30Wednesday, March 2 . . . . . . . . . . 08:00 – 16:00

GENERAL SESSIONSAll general sessions will be held in the Grand Ballroom on the second floor.

SPEAKER CHECK-IN Grand Ballroom Foyer, Second Floor

All speakers must use computers provided by the conference; personal laptops may not be used. Computers provided by the conference will be equipped with the latest version operating system. PowerPoint is the only acceptable presentation format.

Bring your presentation on a CD or USB stick to the speaker check-in desk according to the schedule below.

Presentation Date Presentation SubmissionMonday, February 28 . . . . . . . . . Sunday, February 27 . . . . . . .16:00 – 19:00Tuesday, March 1. . . . . . . . . . . . . Monday, February 28 . . . . . .07:30 – 15:45Wednesday, March 2 . . . . . . . . . . Tuesday, March 1 . . . . . . . . .08:00 – 15:30

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POSTER SETUP, DISMANTLE AND SESSIONSGrand Ballroom, Second Floor

Setup Sunday, February 27 . . . . . . . . . . 15:00 – 18:00

DismantleTuesday, March 1. . . . . . . . . . . . . 18:30 – 19:00

*All posters must be removed prior to the final plenary session on Wednesday, March 2. The remaining posters will be discarded during the removal of the poster boards.

Poster Schedule General Viewing Presentations Monday, February 28 . . . . . . . . . 08:00 - 21:00. . . . . . . . . . . . . . .18:00 – 19:00 Tuesday, March 1. . . . . . . . . . . . . 08:00 - 21:00. . . . . . . . . . . . . . .17:30 – 18:30

All posters, in all topics, will be attended by the presenting author in the evening poster sessions.

CONFERENCE MEALS AND RECEPTIONSThe following meals are included in your conference registration:

Welcome Reception Sunday, February 27 . . . . . . . . . . . . . . . . . . . . . . . 18:00 – 19:00

The Sunday Welcome Reception will be held in the private dining room of Market Street Bar and Grill at the Hyatt Regency Reston. Light fare and beverages will be served.

Poster ReceptionsGrand Ballroom Foyer, Second FloorMonday, February 28 . . . . . . . . . . . . . . . . . . . . . . 18:00 – 19:00Tuesday, March 1. . . . . . . . . . . . . . . . . . . . . . . . . . 17:30 – 18:30

Tea, Coffee and MuffinsGrand Ballroom Foyer, Second FloorMonday, Tuesday, Wednesday . . . . . . . . . . . . . . 08:00 – 08:30

BanquetTuesday, March 1. . . . . . . . . . . . . . . . . . . . . . . . . . 19:00 – 22:00

McCormick & Schmick’s Seafood Restaurant11920 Democracy DriveReston, Virginia 20190Phone: +1.703.481.6600

The restaurant is less than a quarter mile from the hotel. A group walk will depart from the hotel lobby at 18:50. Walking directions are available at the registration desk.

INDEPENDENT MEALS Lunch and dinner, with the exception of the Tuesday banquet, are not included in the conference registration fee. The dining guide distributed at registration has a map of near-by dining options. A partial list of restaurants appears at the end of this section.

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TRANSPORTATION TO WASHINGTON DULLES INTERNATIONAL AIRPORT (IAD) ShuttleA complimentary shuttle to Dulles Airport is available for all guests of the Hyatt Regency Reston. The shuttle van departs from the lower level of the hotel lobby for the airport at forty-five minutes after the hour, every hour, between 05:45 and 21:45.

TaxiA taxi is approximately $25 USD from the hotel to Washington Dulles International Airport.

BANKING/ATMAn ATM is located in the lobby of the Hyatt Regency

INSURANCE/LIABILITYThe conference organizers cannot accept liability for injuries or losses arising from accidents or other situations during or as a result of the conference.

INTERNET ACCESSWiFi is not included in the room rate but can be purchased for $9.95 per 24 hours. The purchased wireless connection will provide you with internet access in your guest room, conference meeting rooms and common space, such as the lobby.

Panera Bread Bakery Cafe, located in the lobby of the hotel, has complimentary wireless access for patrons.

LANGUAGEEnglish is the official language of the conference.

MEDICAL ASSISTANCEIn case of emergency, please dial 911 from any house or mobile phone. The Reston Hospital Center is located directly across from the hotel.

Reston Hospital Center1850 Town Center ParkwayReston, Virginia 20190Phone: +1.703. 689.9000Fax: +1.703. 689.0840

MOBILE PHONE POLICYPlease ensure your mobile phone is turned off during all session so as not to disrupt the presenters or other conference attendees.

SMOKING POLICYThe Hyatt Regency Reston is a non-smoking hotel.

TIME ZONEReston, Virginia is on Eastern Standard Time.

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61 | American Tap Room (classic American grill menu) 703-834-0400 www .americantaproom .com73 | Big Bowl (Mongolian grill/Asian) 703-787-8852 www .bigbowl .com 8 | Busara Thai Restaurant & Lounge (all natural Thai) 703-435-4188 www .busara .com32 | Chipotle Mexican Grill (burritos, tacos) 703-435-5795 www .chipotle .com53 | Clyde’s of Reston (American food) 703-787-6601 www .clydes .com80 | Community Canteen (sandwiches, soups, salads) 703-707-9442 www .communitycanteen .com70 | Cosi (sandwiches, soups, salads, pizza) 703-435-7600 www .getcosi .com83 | The Counter (custom built burgers) 703-796-1008 www .thecounterburger .com74 | Jackson’s Mighty Fine Food (casual all-American food) 703-437-0800 www .greatamericanrestaurants .com67 | M & S Grill (American grill, upscale dining) 703-787-7766 www .mandsgrill .com41 | Market Street Bar & Grill (American grill, upscale dining) 703-925-8250 www .msbg .net65 | McCormick & Schmick’s Seafood Restaurant (seafood, upscale dining) 703-481-6600 mccormickandschmicks .com79 | Mon Ami Gabi (French steakhouse & bistro) 703-707-0233 www .monamigabi .com11 | Morton’s, The Steakhouse (upscale steakhouse) 703-796-0128 www .mortons .com14 | Obi Sushi (sushi and Japanese cuisine) 703-766-7874 www .obisushi .com48 | Panera Bread Bakery-Café (sandwiches, soups, salads) 703-437-6022 www .panerabread .com31 | Paolo’s Ristorante (Italian cuisine, casual dining) 703-318-8920 paolosristorante .com78 | Passion Fish (world class seafood) 703-230-3474 www .passionfishreston .com27 | Potbelly Sandwich Works (sandwiches, soups, salads) 703-481-5080 www .potbelly .com62 | Uncle Julio’s Rio Grande Café (Mexican food) 703-904-0703 www .unclejulios .com15 | Uno Chicago Grill (Chicago style pizza) 703-742-8667 www .unos .com51 | Yogen Fruz (frozen yogurt, smoothies) 571-926-9567 www .yogenfruz .com

Restaurants within a short walk of the Hyatt Regency

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08:30 – 08:50 Opening Session

Session Chairs: Dharam Ablashi, Louis Flamand, and Tetsushi Yoshikawa

Welcome and Introduction by the Conference Co-Chairs Louis Flamand and Tetsushi Yoshikawa

Status of the Effort to Have HHV-6A and HHV-6B Recognized as Distinct 1-01 Herpesvirus Species P.E. Pellett

08:50 – 10:45 HHV-6 Host Cell Interactions Session Chairs: Paolo Lusso and Yasuko Mori

Immunomodulation and Immunosuppression by HHV-6 2-01 P. Lusso

Downregulation of the T-cell Receptor by the HHV6-Encoded U24 Protein 2-02 L. Coscoy

Reed-Sternberg-like Deregulations in BJAB Cells Expressing DR7 Protein 2-03 From HHV-6B L. Mardivirin, A. Lacroix, M. Marie, A. Pinon, C. Lepage, S. Collot-Teixeira, A. Jaccard, S. Rogez

Regulation of Apoptosis by HHV-6B Infection 2-04 P. Hollsberg, E. Kofod-Olsen

Molecular Determinants of Strain Variants A and B Specificity Identified by 2-05 Analyses of the HHV-6 Chemokine U83 D.J. Clark, U. Gompels

In Vitro Co-Infection of HHV-6 Alters Infectivity of Chlamydia Trachomatis 2-06 B.K. Prusty, L. Böhme, T. Rudel

Differential Gene Expression Profiling of Acute and Chronic HHV-6A-Infected 2-09 Human Malignant Astrocytes J. Crawford, K.Yao, Y. Cho, M. McCormick, S. Jacobson

10:45 – 11:00 Coffee Break Grand Ballroom Foyer

Monday, February 28

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11:00 – 12:45 HHV-6 Genes & Proteins Session Chairs: Louis Flamand and Koichi Yamanashi

HHV-6 Cellular Biology - An Overview 3-01 U. Gompels

Innate Immune Modulation by Human Herpesvirus 6 Immediate-Early 1 Protein 3-02 J. Jaworska, A. Gravel, L. Flamand

Deep Sequencing of HHV-6A and HHV-6B Strains Reveals Mixed 3-03 Populations Structures T. Kowalik, B. Bhattacharjee, N. Renzette

The Functional Analysis Of Glycoprotein O During HHV-6 Infection: HHV-6 gO is 3-04 Non- Essential for the Virus Growth in CBMCs H. Tang, H. Oyaizu, T. Maeki, K. Yamanishi, Y. Mori

Analyses of the Terminal Direct Repeats (DRs) of Human Herpevirus 6A DNA 3-05 H. Zeigerman, R. Borenstein, N. Frenkel

Identification of the Epitopes on Neutralizing MAb for Human Herpesvirus 6 3-06 Glucoprotein Q1 A. Kawabata, E. Hayashi, M. Hayashi, H. Oyaizu, H. Tang, K. Yamanishi, Y. Mori

12:45 – 14:00 Lunch Break

14:00 – 16:00 Germ-line Chromosomal Integration & Telomeres - Part 1

Session Chairs: Mario Luppi and Peter Medveczky

Historical Overview on CIHHV-6 4-01 M. Luppi

The Importance of HHV-6 Chromosomal Integration in Clinical Practice 4-02 K. Ward

Neurodevelopmental Outcome of Children with Congenital HHV-6 Infection 4-03 C.B. Hall, M.T. Caserta, R.L. Canfield, H. Wang, P.W. Davidson

Viruses and Telomeres 5-01 P. Lieberman

The Role of Ku86 in Human Telomere Maintenance 5-02 E. Hendrickson

Human Telomeric and Subtelomeric DNA Sequence Features and Function 5-03 H. Riethman


Monday, February 28

16:10 – 18:00 Germ-line Chromosomal Integration & Telomeres - Part 2

Session Chairs: Mario Luppi and Peter Medveczky

Herpesvirus Telomeric Repeats Facilitate Genomic Integration into 4-04 Host Telomeres and Mobilization of Viral DNA During Reactivation B.B. Kaufer, A. Egerer, K.W. Jarosinski, N. Osterrieder

Integration, Reactivation and Mutation of Human Herpesvirus-6 Genom 4-05 In Vitro and In Vivo J. Luka, J. Gubin-Jurgens

The Characterization and Mapping of the Integrated Human Herpesvirus-6A 4-06 and 6B genome J.H. Arbuckle, M.M. Medveczky, S. Pantry, D.V. Ablashi, P.G. Medveczky

Characterization of the Nucleotide Sequence of an HHV-6A Strain Isolated from 4-07 a Patient with Germline Integrated HHV-6 S.N. Pantry, J. Luka, J.H. Arbuckle, M.M. Medveczky, R. Renne, D. Ablashi

Is Integration of the Human Herpesvirus 6 Genome in Telomeres Irreversible or 4-08 it Represents Latency? M.M. Medveczky, J.H. Arbuckle, S.N. Pantry, J. Luka, D. Ablashi, P.G. Medveczky

Panel Discussion L. Flamand, E. Hendrickson, P. Lieberman, M. Luppi, P. Medveczky, P. Pellett, H. Riethman

18:00 – 19:00 Poster Session and Reception Grand Ballroom

Monday, February 28

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18

08:30 – 09:20 HHV-6 BAC Session Chairs: Niza Frenkel and Philip Pellett

Using Infectious Bacterial Artificial Chromosomes to Dissect the Molecular Biology 6-01 of Beta Herpesviruses A. Paredes, D. Yu

HHV-6 Glycoprotein Complex Formation is Required for the Folding and Trafficking 6-02 of the Complex, gH/gL/gQ1/ gQ2 and its Cellular Receptor Binding; gQ1 Y. Mori

Cloning of the HHV-6A Genome Into BACs, Viral-Host Interactions and the 6-03 Derivation of Amplicon-6 Vectors for Potential Gene Therapy N. Frenkel, R. Borenstein, H. Zeigerman, E. Sharon

09:20 – 09:50 Epidemiology Session Chairs: Ursula Gompels and Caroline Hall

Quantitation of Human Herpesvirus -6 and -7 DNAs in Different Blood Fractions 7-01 from Blood Donors P. Bonnafous, B. Géraudie, M. Charrier, D. Heurté, M. Desmonet, M. Bartoletti, C. Penasse, H. Agut, A. Gautheret-Dejean

Human Herpesvirus-6 in Children Undergoing Cancer Chemotherapy 7-02 J. Goldfarb, N. Borges, B. Yen-Lieberman, K. Gowans, C. Woodard, D. Kohn, L. Danziger-Isakov, P. Pellett

09:50 – 11:15 HHV-6 and Non-Neurological Diseases Session Chairs: Yoshizo Asano and Dario Di Luca

HHV-6 and Autoimmune Thyroiditis 8-01 D. Di Luca, E. Caselli

Serum Viremia Of Human Herpesvirus 6 (HHV-6) In Patients With Active Autoimmune 8-02 Connective Tissue Diseases: Epiphenomenon Or Causative Role in Disease Development? F. Broccolo, G. Cassina, A. Fava, L. Fusetti, S. Paolino, B. Matteoli, E. Zaccaria, S. Franchini, A. Parodi, P. Lusso, L. Ceccherini-Nelli, M. Grazia Sabbadini, F. Drago, M. Malnati


Identification of HHV-6B Sequences in EBV-negative Hodgkin Lymphoma Using 8-03 Next Generation Sequencing A.J. Bell, A. Gallagher, L. Shield, D. Gatherer, R.F. Jarrett

Systemic Leukotropic Herpesvirus Infections and Autoantibodies in Patients with 8-04 Myalgic Encephalomyelitis – Chronic Fatigue Syndrome K. Knox, D. Peterson, A. Knox, J. Weismann, D. Carrigan

Tuesday, March 1

11:15 – 12:25 HHV-6, DRESS/DIHS & Immunity Session Chairs: Paolo Lusso and Vincent Descamps

Drug Reaction with Eosinophilia and Systemic Symptoms: a Multiorgan 9-01 Antiviral T Cell Response V. Descamps

Can HHV-6 Protect Children from Allergic Disease? 9-02 K. Eriksson

Identification of HHV6B-specific CD8+ T cells 9-03 A. Schub, L. Martin, S. Dillinger, A. Moosmann

Identification of HHV-6 Epitopes Targeted by CD4+ T Cell Responses in 9-04 Healthy Donors J.M. Calvo-Calle, M.D. Nastke, A.R. Becerra-Artiles, L. Yin, O.A. Dominguez-Amorocho, L. Gibson, L.J. Stern

12:25 – 12:45 Diagnostics & Reactivation 14:00 – 15:15

Session Chairs: Henri Agut and Irmeli Lautenschlager

Diagnosis of HHV-6 Reactivation in Transplant Recipients; Critical Point to 10-01 Determine Precise Role of the Virus in Causing Disease T. Yoshikawa


Breakthrough HHV-6B Infections During Antiviral Prophylaxis After 10-02 Liver Transplantation I. Lautenschlager

Which Tools to Diagnose and Monitor HHV-6 Infections: From Theory to Practice 10-03 A. Gautheret-Dejean

Do Latent HHV-6 and HHV-7 Infection Impact on Successful Aging? 10-04 S. Govind, W. Mitchell, R. Aspinall

Chromosomally Integrated HHV-6: Q & A for physicians 10-05 P. Pellett, D. Ablashi, H. Agut, M. Caserta, V. Descamps, L. Flamand, A. Gautheret-Dejean, C. Hall, U. Kuhl, D. Lassner, I. Lautenschlager, K. Loomis, M. Luppi, P. Medveczky, J. Montoya, Y. Mori, M. Ogata, R. Razonable, E. Seto, T. Yoshikawa

Development and Validation of a Q-RT-PCR based TCID50 Assay for HHV-6A 10-07 R.K. Gustafsson, E.E. Engdahl, A. Fogdell-Hahn

Tuesday, March 1

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15:25 – 17:30 HHV-6 Treatment Session Chairs: Jose Montoya and Lieve Naesens

The Challenge of Measuring the Clinical Impact of Acute HHV-6 Infections 11-01 H. Agut

Identification of Bicyclic Sulfone Inhibitors of HHV-6 Targeting the 11-02 HHV-6 U77 Helicase L. Naesens, G. Andrei, R. Snoeck, D. Gerry, J.E. Banning, P.D. Wilkerson, P.M. Johnson, H. Shah, Z.M. Renew, C.E. Stephens


Adoptive Immunotherapy to Prevent and Treat HHV-6 Reactivation 11-03 Post Allogenic Stem Cell Transplant U. Gerdemann, L. Keukens, U.L. Katari, J.M. Keirnan, A.P. de Pagter, H.E. Heslop, C.M. Rooney, A.M. Leen

Chromosomally Integrated Human Herpesvirus 6 in Patients with 11-04 Acquired Cardiomyopathies and Heart Failure Symptoms D. Lassner, M. Rhode, U. Kühl, U.M. Gross, G.F. Krüger, B. Seeberg, F. Escher, H.P. Schultheiss

CMX001, A Novel Antiviral for the Treatment of dsDNA Viral Diseases 11-05 W.P. Painter, J. Kurtzberg, H. Mommeja-Marin, V. Prasad

The Challenges of Treating HHV-6 CNS Infections 11-06 J.G. Montoya

17:30 – 18:30 Poster Session and Reception Grand Ballroom

Tuesday, March 1

08:30 – 10:20 HHV-6 in CNS Disease Session Chairs: Anthony Komaroff and Tetsushi Yoshikawa

Overview of HHV-6 in CNS disease A. Komaroff

HHV-6 Reactivation and its Effect on Delirium and Cognitive Functioning in 12-01 Hematopoietic Cell Transplant Recipients D. Zerr

Elevated Levels of HHV-6 Antibodies in Individuals with Psychiatric Disorders 12-02 R. Yolken, F. Dickerson

Different Characteristics of Human Herpesvirus 6 Encephalitis Between 12-03 Primary Infection and Viral Reactivation Y. Kawamura

Plasma HHV-6 DNA Loads and IL-6 Concentration as Factors in the Development 12-04 of HHV-6 Encephalitis After Allogeneic Stem Cell Transplantation M. Ogata

Panel Discussion: S. Jacobson B. Lavenstein , T., Komaroff, M. Ogata, R. Yolken T. Yoshikawa, D. Zerr


10:35 – 11:45 HHV-6 & Multiple Sclerosis Session Chairs: Roberto Alvarez-Lafuente and Steven Jacobson

Multiple Sclerosis Studies S. Jacobson

Human Herpesvirus 6a Genes Interaction in Multiple Sclerosis Patients 13-01 R. Alvarez-Lafuente

IFN-γ-Dependent Demyelinating Activity by Natural Killer Cells is Regulated 13-02 by HLA-G Upregulation on Oligodendrocytes During HHV-6 Infection P.P. Banerjee, S.S. Soldan, S. Miah, A. Esinberg, S. Maru, Y. Rosenberg-Hasson, K.S. Campbell, D. Ablashi, J.S. Orange

Oligoclonal Antibody Response in Multiple Sclerosis Includes 13-03 Antibodies to Herpesviruses J.O. Virtanen, K. Fenton, I. Cortese, B. Bielekova, S. Jacobson

Panel Discussion: R. Alvarez-Lafuente, S. Jacobson, P. Banerjee, JO Virtanen

Wednesday, March 2

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11:50 – 12:45 HHV-6 Animal Models Session Chairs: Dharam Ablashi and Andrew Goodman

Effects of HHV-6A Infection in the Common Marmoset 14-01 J.E. Wohler, M.I. Gaitan, E. Harberts, A.C. Silva, D.S. Reich, S. Jacobson

Development of a Transgenic Murine Model for Human Herpesvirus-6 Infection 14-02 J. Reynaud, J. Jégou, J. Welsch, B. Horvat

Panel Discussion D. Ablashi, A. Goodman, S. Jacobsen, J. Reynaud, W. Theodore, J. Wohler


14:00 – 16:00 HHV-6 & 7 in Epilepsy and Brainstorming Session on HHV-6B & MTLE

Session Chairs: W. Theodore, S. Jacobson

Analysis of HHV-6 Presence in Brain Tissue from Well Defined Subgroups of 15-01 Patients with Temporal Lobe Epilepsy P. Niehusmann, T. Mittelstaedt, C.G. Bien, J.F. Drexler, A. Grote, S. Schoch, A.J. Becker

The Role of Human Herpesvirus 6 and 7 in Febrile Status Epilepticus: 15-02 The FEBSTAT study L. Epstein

Detection of human herpes virus 6B in patients with mesial temporal lobe epilepsy 15-03 in China and the possible association with elevated NF-KappaB expression D. Zhou

Panel Discussion: How to Confirm/Refute Suggestions that HHV-6B Plays a Causative Role in MTLE

M. Caserta, L. Epstein, S. Jacobsen, J. Kapur, S. Shinnar, W. Theodore

Wednesday, March 2

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HHV-6 Host Cell Interactions

2-07 HHV-6 Prevents Fusion Between Autophagic Vacuoles and Lysosomes in Human T Cells O. Debbeche, A. Iannello, S. Samarani, A. Ahmad

2-08 Characterization of the T Cell Response to HHV-6 Immunodominant Proteins in Healthy Donors A.R. Becerra Artiles, O.A. Dominguez Amorocho, L.J. Stern, J.M. Calvo Calle

HHV-6 Genes & Proteins

3-08 HHV-6B Protein U19 Stabilizes and Inactivates p53 in the Cytoplasm and Rescues Cells from p53-dependent Cell Death E. Kofod-Olsen, J.M. Møller, B. Bundgaard, R. Bak, J.G. Mikkelsen, P. Höllsberg

3-09 The Human Herpesvirus 6 U27 Gene Product Negatively Regulates the Transactivation Activity of Immediate-Early 2 Protein A. Gravel, L. Flamand

Germ-line Chromosomal Integration

4-09 Human Herpesvirus-6 Chromosomal Integration and Bing-Neel Syndrome Revealing a Waldenstrom’s Disease: Incidental Association? M. Giroux, A. Gautheret-Dejean, A. Dewilde, O. Outteryck, C. Maurage, B. Onraed, L. Terriou, P. Vermersch

4-10 Frequency of Chromosomally-Integrated Human Herpesvirus 6 in Children with Acute Lymphoblastic Leukemia A. Gravel, D. Sinnett, L. Flamand

4-11 Detection of HHV-6 Antigen and Herpesvirus Particles in Individuals with Chromosomally Integrated HHV-6 (ciHHV-6) V. Strenger, I. Lautenschlager, S. Richter, W. Schwinger, E. Engdahl, J. Ahlqvist, A. Fogdell-Hahn, S.W. Aberle, T. Popow-Kraupp, E.P. Nacheva, G. Krueger, C. Urban

4-12 A Simple and Fast Method to Diagnose Chromosomal Integration of Human Herpesvirus 6 in Circulating Mononuclear Cells M. Malnati, F. Broccolo, E. Di Marco, M. Grazia Marazzi, P. Lusso

Poster Presentations by Topic

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Epidemiology

7-03 Human Herpesvirus 6 (HHV-6) in the Genesis of Acute Leukemia F. Nefsi, A. Gautheret-Dejean, N. Ben Fredj, A. Khlif, H. Agut, S. Feki, M. Aouni

HHV-6 and Non-Neurological Diseases

8-05 Treatment of Uveitis and blindness associated with HHV-6 infection by Valganciclovir (Valcyte) and Immunoprop. D. Enlander

8-06 Presence and Activity of Human Herpesvirus-6 and 7 Infections in Patients with Autoimmune Thyroid Diseases Z. Nora-Krukle, A. Sultanova, S. Gravelsina, S. Chapenko, M. Chistyakov, E. Cunskis, M. Murovska

8-07 HHV-6 Etiopathogenetic Role in the Development of Cervical Cancer by Combining Immunohistochemistry, Laser Microdissection and Single Cell-Qpcr F. Broccolo

HHV-6, DRESS/DIHS & Immunity

9-05 A Study of Chromosomally Integrated HHV-6 in Severe Cutaneous Adverse Drug Reactions W. Chung

Diagnostics & Reactivation

10-06 Analysis of Viral Load and Transcripts in Blood Leukocyte Subpopulations from Patients with Different Types of HHV-6 Infection A. Milovanovitch, S. Nguyen, C. Bigaillon, P. Bonnafous, N. Dhédin, M. Stern, V. Descamps, H. Agut, A. Gautheret-Dejean

10-07 HHV-6 And HHV-7 Infection in the Patients with Autologous Peripheral Blood Stem Cell Transplantation S. Chapenko, I. Trocjukas, S. Gravelsina, M. Chistyakov, A. Sultanova, S. Donina, Z. Nora-Krukle, S. Lejniece, M. Murovska

10-08 HHV-6-DNAemia After Liver Transplantation Monitored By Two Quantitative Real-Time PCR Methods T. Karlsson, R. Loginov, L. Mannonen, K. Höckerstedt, I. Lautenschlager

10-09 High Intrahepatic HHV-6 DNA Load is Associated with Decreased Graft Survival in Liver Transplant Recipients with Graft Hepatitis S. Pischke, J. Gösling, I. Engelmann, J. Schlue, B. Wölk, C. Schmitt, C. Strassburg, H. Barg-Hock, T. Becker, M. Manns, T. Schulz, H. Wedemeyer, A. Heim


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HHV-6 Treatment

11-07 Treatment of HHV6 infection in ME/CFS with Valganciclovir (Valcyte) and Immunoprop/ Immunoplus D. Enlander

11-08 Diversity of T cell Responses to Human Herpesvirus 6B L. Martin, A. Schub, A. Moosmann

HHV-6 in CNS Disease

12-06 HHV-6 and EBV Infections in Multiple Sclerosis: an Analysis by Calibrated Ultrasensitive QPCR Assays F. Broccolo, S. Matà, T. Biagioli, G. Cassina, L. Fusetti, B. Matteoli, L. Ceccherini-Nelli, P. Lusso, M. Malnati

12-07 Recognition of Human Herpesvirus 6 Rhombencephalitis in Immunocompetent Children: Clinical and Neuroimaging Features B. Lavenstein, J. Crawford


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Abstracts

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Overview

Oral 1-01Status of the Effort to Have HHV-6A and HHV-6B Recognized as Distinct Herpesvirus Species

P.E. Pellett1([email protected])1Wayne State University, Detroit, MI, USA

Dr. Pellett will summarize recent changes in the definition of herpesvirus species and species nomenclature, as well as the status of a proposal to the International Committee for Taxonomy of Viruses for HHV-6A and HHV-6B to be formally recognized as distinct herpesvirus species.

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2-01 OralImmunomodulation and Immunosuppression by HHV-6

P. Lusso1([email protected])1LIR, NIAID, NIIH, Bethesda, MD, USA

Clinical and experimental evidence suggests that HHV-6 exploits a variety of strategies to interfere with the host immunologic function, thereby favoring its own spread and persistence in vivo. Besides its ability to directly infect and destroy critical cells of the immune system, most notably CD4+ T lymphocytes, HHV-6 can also act via indirect mechanisms including manipulation of the cytokine network. Recent studies in our laboratory suggest that interaction of HHV-6 with the CD46 receptor, which constitutes an important trait d’union between the innate and the adaptive immune systems, triggers a cascade of immunosuppressive events including blockade of IL-12 production by mononuclear phagocytic cells and induction of a T-regulatory-like phenotype in naive CD4+ T cells. A deeper understanding of the complex interactions between HHV-6 and the immune system may lead to the development of novel approaches for the prevention and treatment of HHV-6-associated diseases.

2-02 OralDownregulation of the T-cell Receptor by the HHV6-Encoded U24 Protein

L. Coscoy1([email protected])1University of California, Berkeley, Berkeley, CA, USA

The biological activity of T cells is tightly regulated by the T-cell receptor (TCR) complex expressed at the cell surface. Engagement of the TCR complex results in CD4+ T-cell activation through a series of biochemical events mediated by the CD3 molecules. Since CD4+ T cells play a central role in regulating the antiviral response, it is not surprising that many viruses have evolved mechanisms to modulate T-cell function. For example, Nef, a protein encoded by the human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV), downmodulates TCR/CD3 from infected T cells, blocking their responsiveness to activation. This property is thought to have evolved to maintain viral persistence in the context of an intact host immune system. Similar to HIV-infected cells, HHV-6-infected CD4+ T cells express low levels of TCR-CD3 molecules suggesting that HHV-6 might encode a protein that modulates CD3 expression. To test this hypothesis, we generated a library of HHV-6 candidate genes and tested whether one of these genes modulates the expression of the TCR/CD3 complex. We found that transfection of the U24 open reading frame in T-cell lines mediates a rapid relocalization of the CD3 and TCR molecules from the plasma membrane to early endosomes, a phenotype due to an active inhibition of CD3 recycling to the cell surface. As a consequence of CD3 downregulation, U24-expressing cells are impaired in their ability to become activated. We show that U24 is a tail-anchored protein that depends on TRC40/Asna-1 for its function and that a PPXY motif near the amino terminus of U24 is essential to its function. Finally, we found that U24 is specific to roseoloviruses, with U24 sequence and function being conserved in the two subtypes of HHV-6 and in HHV-7.

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Oral 2-03Reed-Sternberg-like Deregulations in BJAB Cells Expressing DR7 Protein from HHV-6B

L. Mardivirin1([email protected]), A. Lacroix1, M. Marie1, A. Pinon2, C. Lepage3, S. Collot-Teixeira1, A. Jaccard4, S. Rogez1

1Microbiologie, Faculté de Pharmacie de Limoges, Limoges, France; 2Chimie Minérale, Faculté de Pharmacie de Limoges, France; 3Biochimie, Faculté de Pharmacie de Limoges, France; 4Hématologie Clinique, CHU Dupuytren, Limoges, FranceBackground: If the role of HHV-6 in haematological malignancies is now demonstrated, the mechanisms involved are still unknown. Previous works in our group demonstrated specifically an involvement of HHV-6 variant B in classical Hodgkin’s lymphoma and the presence of DR7 viral oncoprotein in Reed-Sternberg cells. Objectives: The aim of this study was to determine the effects of viral DR7B protein expression in mature B cells. Proliferation, resistance to apoptosis and Reed-Sternberg cell markers expression were studied after stable transfection.Methods: BJAB cell line, a mature B lymphocyte cell line, free from EBV and HHV-6, was stably transfected with HHV-6 variant B dr7 gene by electroporation and neomycin selection. Proliferation was studied using MTT assay, and apoptosis, induced after exposition to UV, was measured by flow cytometry after or without incubation with BAY11-7082, an inhibitor of cytokine-induced IkB-alpha phosphorylation. Protein expression levels of Bcl2, Bax, CD99, CD83, CD30 and Gcet1 were determined by western-blot analysis. Each assay was realised in triplicate. Mock-transfected cell line and cell line transfected with empty vector were used as controls.Results: Compared to controls, BJAB cells expressing DR7B showed increased proliferation and higher resistance to UV-induced apoptosis in a NFkB-dependent manner. Western-blot analysis revealed that DR7B expression induced up-regulation of Bcl2, CD83, CD30 and Gcet1, and down-regulation of CD99 and Bax expression levels.Conclusion: These data show that in mature B cells, HHV-6 DR7B protein expression is able to induce cellular deregulations similar to those observed in Reed-Sternberg cells, and argue in favour of a role of HHV-6 in the pathogenesis of Hodgkin’s lymphoma.

Oral 2-04Regulation of Apoptosis by HHV-6B Infection

P. Hollsberg1, E. Kofod-Olsen1([email protected])1Aarhus University, Aarhus, Denmark

The immune system targets virus-infected cells by different means. One of the essential antiviral mechanisms is apoptosis induced by a family of closely related receptors, termed Tumour Necrosis Factor Receptor Super Family (TNFRSF). These receptors can be activated by membrane bound ligands or secreted cytokines. Among these receptors is a conserved group of death receptors (DRs). Members of this group include a C-terminal death domain (DD), which is responsible for binding of death domain-associated factors. These factors are responsible for induction of programmed death and may together constitute two known death inducing signaling complexes (DISCs), termed complex I and II. A third receptor-independent DISC platform that signals through programmed necrosis has also been described. We found that HHV-6B was able to inhibit signaling from three well-described DRs: TNFR1, FAS/CD95 and TRAIL and located the inhibition to be at the level of the DISC formation. HHV-6B infection was able to induce signaling through the poorly described receptor-independent DISC platform and caused rapid cell death through programmed necrosis when co-stimulated with a known inducer of receptor-independent DISC signaling. Our data disclose a novel mechanism by which HHV-6B modulate apoptosis induction.

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2-05 Oral Molecular Determinants of Strain Variants A and B Specificity Identified by Analyses of the HHV-6 Chemokine U83

D.J. Clark1([email protected]), U. Gompels1

1London School of Hygiene and Tropical Medicine, London, United Kingdom (Great Britain)HHV-6 infects T-lymphocytes lytically and monocytic/macrophage cells latently. For an infection to persist in these

immune response cells, and other cells, the virus has evolved with genes encoding immunomodulatory proteins; a key member of these genes, U83, encodes a viral chemokine, which mediates inflammatory responses. U83, unique to HHV-6, is a major divergent molecule between strain variants HHV-6A and HHV6-B. We have previously shown U83A is a highly potent chemokine, specific for receptors CCR1, CCR4, CCR5, CCR6 and CCR8(1,2) while U83B is weaker and specific for only CCR2. Moreover, we showed U83A is uncontrolled by endogenous regulatory receptors(3). These distinct viral chemokines recruit different leukocyte subsets with these receptors, and are thus key in tropism and pathological differences between variants. HHV-6A is an emerging infection linked with neuro-inflammatory disease, multiple sclerosis and encephalitis. Mature full length U83A binds and signals, inducing chemotaxis, while a spliced mRNA transcript is translated into a truncated molecule, U83A-N, that binds yet does not signal. In this study, based on analyses of naturally occurring strain variants, N-terminal peptides were designed and utilised to examine specificity differences between U83A and U83B compared to full length (mature) and spliced forms. The results show the N-terminal region has the main residue differences which could confer the specificity differences between U83A and U83B. Ex vivo human leukocytes were used in chemotaxis assays, with migrated cells further analysed by flow cytometry for other cellular markers. Key amino acids were identified which dictated strain variant specificity. These findings indicate the N terminal sequence and its variation, can affect leukocyte infection, linked inflammatory pathology and identifies targets for therapy.(1)Dewin DR, Catusse J, Gompels UA. (2006) Journal of Immunology. 176:544.(2)Catusse J, Parry CM, Dewin DR, Gompels UA. (2007) Blood. 109:3633. (3)Catusse J, Clark DJ, Gompels UA. (2009) Journal of Inflammation. 6:22

2-06 Oral In Vitro Co-Infection of HHV-6 Alters Infectivity of Chlamydia Trachomatis

B.K. Prusty1([email protected]), L. Böhme1, T. Rudel11Department of Microbiology, Biozentrum, University of Würzburg, Würzburg, Germany

Both human herpes virus 6 (HHV-6) and Chlamydia (C. trachomatis or C. pneumoniae) are detected together in different human disorders including chronic fatigues syndrome (CFS) and autoimmune disorders like multiple sclerosis (MS). The possible interaction between these two types of pathogens and its influence on their fate inside host cell is still unknown. Here we demonstrate the mutual interference of HHV-6 (both A & B) and C. trachomatis infection. Under in vitro growth conditions, acute HHV-6 infection reduced the chlamydial infectivity. On the contrary, bacterial replication and infectivity was strongly up-regulated in latent HHV6-containing HeLa229 cells. Interestingly, C. trachomatis favored enhanced entry and survival of HHV-6. In a co-infection model, both pathogens affected each other’s entry, survival as well as infectivity and influenced the replication and transcription pattern of each other. Our findings strongly argue for an increased biological significance of co-infection of these pathogens.

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2-07HHV-6 Prevents Fusion Between Autophagic Vacuoles and Lysosomes in Human T Cells

O. Debbeche1([email protected]), A. Iannello1, S. Samarani1, A. Ahmad1

1Centre de Recherche de L’hopital St-Justine, Montreal, CanadaThe Human herpesvirus (HHV)-6 is a ubiquitously occurring pathogen that infects humans in their early childhood.

The virus causes exanthum subitum, commonly known as roseola, in children. It has also been associated with different lymphoproliferative disorders, clinical manifestations of graft-versus-host disease and several chronic diseases in humans. The host cells usually respond to viral infections by inducing autophagic response (virophagy), and viruses tend to modify this host response and may also exploit it to increase viral replication. HHV-6 is no exception to this paradigm. We show that the HHV-6 infection of HSB-2, an established human T cell line, induces autophagic response in the infected cells. Autophagic vacuoles appear in the virus-infected cells. However, the virus modifies this response and prevents fusion between autophagic vacuoles and lysosomes. Furthermore, we provide evidence to demonstrate that the virus uses this process to enhance its replication. Our results provide important novel insights on the HHV-6-induced modification of the host’s autophagic response.

2-08Characterization of the T Cell Response to HHV-6 Immunodominant Proteins in Healthy Donors

A.R. Becerra Artiles1([email protected]), O.A. Dominguez Amorocho1, L.J. Stern1, J.M. Calvo Calle1

1University of Massachusetts Medical School, Worcester, MA, USAAlthough T cells responses to HHV-6 can be observed in healthy donors using basic immunological techniques, major

targets of the T cell response to this virus have not been defined. Moreover, there are not HHV-6-specific T cell epitopes reported in the literature. The large number of proteins encoded by this virus is a major roadblock. In order to indentify major targets of the T cell response, proteins in a HHV-6 virus pellet obtained from supernatant of infected cell cultures were fractionated by SDS-PAGE. Subsequent to the separation each lane was sliced in 10 fractions. Each fraction was used as a source of antigens to assess the IFN-gamma response of PBMCs from healthy donors. Fractions which induced significant cytokine response were also analyzed by mass spectrometry to identify viral proteins. In this manner 8 viral proteins were identified as potential targets of the T cell response to HHV-6 in healthy donors. To map T cell epitopes, we used a set of 369 predicted peptides from these 8 proteins. After the screening of the IFN-gamma response in healthy donors using ELISpot, a total 35 T cell epitope candidates have been identified so far. ELIspot responses to these peptides indicated that the response to HHV-6 virus is complex and variable between individuals. The response was dominated by peptides from two proteins, namely the major capsid protein and the glycoprotein H, but responses to other proteins also were observed. Phenotypic characterization and cytokine profiling of the cells involved in the response to the candidate T cell epitopes are in progress.


2-09 Oral Differential Gene Expression Profiling of Acute and Chronic HHV-6A-Infected Human Malignant Astrocytes

J. Crawford1([email protected]), K.Yao2, Y. Cho3, M. McCormick2, S. Jacobson2

1University of California, San Diego, San Diego, CA, USA; 2Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; 3Department of Neurology, Harvard Medical School, USAObjective: To determine whether HHV-6A-infected malignant astrocytes have distinct genetic expression profiles during acute and chronic infection.Background: Human herpesvirus-6 (HHV-6) variants A and B (HHV-6A, HHV-6B) have been detected in a number of CNS diseases including encephalitis, epilepsy, multiple sclerosis, and brain tumors. Relatively little data exists to explain how HHV-6 variants (A and B) can promote or alter CNS disease. We sought to determine whether HHV-6A (U1102) infection of human malignant astrocytes during acute and chronic infection is associated with distinct gene expression profiles that may provide a mechanistic hypothesis of HHV-6 pathogenesis in central nervous system (CNS) disease.Design/Methods: U251 malignant astrocyte cell line was infected with HHV-6A U1102 variant over a 3 week time course along with uninfected astrocytes. Cells were collected in triplicate at days 1-4, 7, 14, and 21 and analyzed by RT-PCR, primary/nested DNA PCR, and in situ hybridization to confirm and quantify HHV-6A infection. RNA samples extracted in triplicate from uninfected and HHV-6A-infected U251 cells were assessed for gene expression using Affymetrix U133 microarrays.Results: HHV-6A U12/U16 RNA peaked at day 3 of infection (1X104 viral copies/million cells) and was undetectable by day 7. HHV6-A U57 DNA was present in HHV-6A infected U251 cells by nested PCR throughout the 21 day entirety, signifying low level chronic infection. The majority of gene expression changes by heat map analysis occurred during the first 3 days of infection. Time course canonical pathway analysis revealed significant changes in integrin and P53 gene expression pathways among several others. Conclusions: HHV-6A appears to have distinct genetic expression profiles during both acute and chronic infection of human astrocytes involving a multitude of cellular and metabolic pathways. The distinct expression signatures may provide clues of HHV-6A infection and pathogenetic disease mechanisms worthy of further study.

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Oral 3-01HHV-6 Cellular Biology - An Overview

U. Gompels1([email protected])1London School of Hygiene & Tropical Medicine, London, United Kingdom (Great Britain)

Roseolovirus are formed of HHV-6, variants A and B, plus HHV-7. Together with human cytomegalovirus they represent the human Betaherpesvirus subgroup. Roseolovirus differ from Cytomegalovirus in prevalence of neuro-inflammatory complications, such as encephalitis or encephalopathy, with HHV-6A having increased neurotropism. Highlights from many laboratories show that virus cellular-biology reflects pathological differences. HHV-6, unlike other herpesvirus, can integrate their genomes in about 1% of people, and may affect lytic/latent infections. Comparisons between herpesvirus genomes identify conserved or variable genes encoding proteins mediating cellular interactions specific for Betaherpesvirus, Roseolovirus, HHV-6 or the variants. Betaherpesviruses have conserved glycoprotein gO genes encoding proteins which complex with Herpesvirus conserved gH/gL and direct cellular entry and host transmission, by cell fusion. The HHV-6 complex binds CD46 with varying affinity between the variants. A further complex is Roseolovirus specific, gH/gL/gQ. After cellular entry, the IE genes, Betaherpesvirus homologues, control transcription, but there are differential spliced IE in the HHV-6 variants. IE1A remains sensitive to type I interferon, whereas IE1B can evade signalling to stimulate interferon responsive genes. Later in the replicative cycle, the virus encodes cellular homologues of immune inflammatory mediators, chemokines and their receptors, which can divert the inflammatory response. The Betaherpesvirus conserved, U51 and U12 chemokine receptors, are also conserved in the variants. However, their specificities are unique, with combinations of inducible and constitutive signalling affecting immune regulators. The chemokine U83 is specific for HHV-6. Different specificity of the variants U83 for the HIV co-receptor CCR5 and CCR2 chemokine receptors can affect virus spread in vivo and correlate with variant specific differences encoded at hypervariable sites on the molecule. Thus, divergent activities of these chemokines and their strain variation can affect both infection and related inflammatory pathology. This is consistent with some epidemiological studies in infected children, particularly HIV affected, or immunosuppressed adults.

Oral 3-02

Innate Immune Modulation by Human Herpesvirus 6 Immediate-Early 1 Protein J. Jaworska1, A. Gravel1, L. Flamand1([email protected])

1CHUQ Research center and Laval University, Quebec, CanadaHuman herpesvirus 6 (HHV-6) is a very-well adapted pathogen infecting and persisting in nearly 100% of the human

population. This not only suggests that HHV-6 is transmitted easily and very efficiently but also that this virus has developed measures to successfully counteract the initial phase of the immune response allowing it to infect and persist within its host. During viral infections, the initial immune response involves the synthesis and secretion of interferon (IFNs), proteins that have potent antiviral activities and capable of restricting the growth of many viruses. Over the last few years, we have spent considerable time and efforts at determining whether HHV-6 can modulate the synthesis and the effects of IFNs. Our results indicate that cells infected with laboratory strains and primary isolates of HHV-6B are resistant to IFN-α/β antiviral actions as a result of improper IFN-stimulated genes (ISGs) expression. In contrast, HHV-6A-infected cells were responsive to IFN-α/β with pronounced antiviral effects observed. Type II IFN (γ)-signaling was unaltered in cells infected by either variant. Resistance to IFN-α/β could be mapped to HHV-6B immediate-early 1 (IE1), one of the least conserved protein between HHV-6 A and B variants. By physically interacting with STAT2, IE1B prevents the assembly and the binding of ISGF3 to IFN-responsive gene promoters, causing ISG silencing and improper establishment of the antiviral state. IE1 from variants A and B also behaved differently with regard to their abilities to interfere with IFN-α genes expression. In contrast to IE1A, IE1B expression prevents IFN-α genes expression by precluding the expression of IRF7, a transcription factor that is essential for IFN-α genes expression. The possible mechanisms by which IE1B impairs IRF7 expression will be discussed. In summary, our work highlights major biological differences between HHV-6 variants with IE1 playing a central role in type I IFN modulation discrepancies.

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3-03 Oral Deep Sequencing of HHV-6A and HHV-6B Strains Reveals Mixed Populations Structures

T. Kowalik1([email protected]), B. Bhattacharjee1, N. Renzette1

1UMass Medical School, Worcester, MA, USAEvidence suggests that beta-herpesviruses are genetically variable, which raises the possibility that diversity may

contribute to disease. To better understand variability on genome-wide scales, we have coupled deep sequencing technology with a custom data analysis pipeline to sequence HHV-6 genomes from virions and infected cells. Using a set of 24 common and 8 unique primer sets, we enriched for, processed, and sequenced HHV-6A (GS strain) and HHV-6B (Z29 strain) genomes from infected cells and virions. This approach resulted in an average coverage of 97% for cell free and 92% for cell associated, viral genomes. Average read depth was 578 for the 4 samples. Our resequencing of HHV-6B Z29 strain revealed 90 polymorphisms relative to the published sequence. The polymorphisms were spread across 34 ORFs with 72% being C-T/G-A transitions. Such a large fraction of C-T/G-A transitions can be indicative of methylation events. The HHV-6A GS strain had not been sequenced before, so the HHV-6A U1102 strain was used as a reference. There were 953 polymorphisms spread across 74 ORFs in GS relative to U1102. Seventy one percent of the polymorphisms were transitions of which 48% were C-T/G-A. Surprisingly, the HHV-6A and HHV-6B populations generated in infected cell cultures were not homogenous. Analysis of the HHV-6B Z29 and HHV-6A GS sequence datasets revealed nucleotide diversity scores of 0.1% and >0.5%, respectively. These scores are unexpectedly high for dsDNA genomes, but are similar to genome-wide sequencing results for HCMV (0.22%) collected from urines of congenitally infected neonates. Thus, evidence for a population of HHV-6 genome types in cell culture may be indicative of extensive population variability within humans. Deep sequencing of clinical specimens will provide insight into the diversity of HHV-6 populations between and within individuals and offer a greater detailed picture of the relationship(s) between HHV-6 genetics and disease.

3-04 OralThe Functional Analysis of Glycoprotein O During HHV-6 Infection: HHV-6 gO is Non- Essential for the Virus Growth in CBMCs.

H. Tang1([email protected]), H. Oyaizu1, T. Maeki1, K. Yamanishi1, Y. Mori21National Institute of Biomedical Innovation, Ibaraki, Japan; 2 Kobe University Graduate School of Medicine, JapanBackground: A glycoprotein O (gO) is conserved among betaherpesvirus subfamily and forms a complex with gH and gL in these herpesviruses. Comparing with many elucidated functions of gO in HCMV and MCMV infections, its function in HHV-6’s infection is still unknown. In addition, its maturation process remains to be investigated because only one of low molecular weight is incorporated into HHV-6 virions, although two forms of gO are expressed in HHV-6 infected cells.Objective: The aim of this study is to elucidate the maturation process of HHV-6 gO and to define its function during HHV-6 infection.Methods: To investigate gO’s maturation process, we used HHV-6 gO transient expression system to determine whether the other viral protein(s) is required for this process. As well, we tried to determine the role of the C-terminal of gO during gO’s maturation. To elucidate gO’s function during HHV-6 infection, we constructed gQ-deleted HHV-6BAC (bacterial artificial chromosome) in E.coli and examined whether the infectious virus was reconstituted in CBMCs. Result: The maturation of the gO required the interaction with gH and gL, and the transmembrane domain of gH was essential for gO’s maturation. Moreover, we succeeded to reconstitute the virus using gO-deleted HHV-6 BAC, indicating that gO was not essential for virus growth in CBMCs. Other phenotypes of the mutant virus are under investigating.

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Oral 3-05 Analyses of the Terminal Direct Repeats (DRs) of Human Herpevirus 6A DNA

H. Zeigerman1([email protected]), R. Borenstein1, N. Frenkel11Tel Aviv University, Tel Aviv, Israel

In a separate abstract for this conference we describe the construction of HHV-6A-BAC clones derived from large cocatemeric replicative intermediates of viral genomes. We have shown that the HHV-6A BACs as well as the replicating viral genomes contained a single direct repeat (DR) rather than the DR-DR junction. We now show that in the HHV-6A BAC clones, as well as in the entire HHV-6A current virus stock the DR was of size ~2.7 kb rather then the known 8-10 kb DR. We have made an elaborate comparison in the DRs of our present virus and the DRs of additional virus isolations, obtained from others, all over the world, as well as the DR of HHV-6A virus propagated in our lab in 1992. We found that whereas the other viruses had an intact DR our present virus had a deletion, including the imperfect telomeric repeats, the DR1 orf up to the first exon of DR6 orf. Remarkably, the pac-2/pac-1 packaging signals, the DR7 orf and DR6 second exon were conserved. The mature DNA of BACs parental HHV-6A strain contained two identical short terminal DRs. Furthermore, we tested whether the DR deletion occurred upon repeated serial passaging of the early viral strains in cord blood and then in SupT1 T cells for 26 times. Furthermore and remarkably, we show that both the DRs were not shortened. We conclude that (i) the deletion occurred once and virus with short DRs “conquered” the population. (ii) duplication of the DR left and right most likely occurred during the cleavage and packaging of viral DNA by mechanism ensuring precise coping of retained DR sequence. (iii) The deleted DR sequences, including the imperfect telomeric repeats, the DR1 orf and the first exon of DR6 orf are not essential for HHV-6A propagation in tissue culture.

Oral 3-06 Identification of the Epitopes on Neutralizing MAb for Human Herpesvirus 6 Glucoprotein Q1

A. Kawabata1([email protected]), E. Hayashi1, M. Hayashi1, H. Oyaizu1, H. Tang1, K. Yamanishi1, Y. Mori11Kobe University Graduate School of Medicine, Kobe, Japan

Human herpesvirus 6 (HHV-6) is a T lymphotropic herpes virus that is categorized into two variants, HHV-6A and HHV-6B, on the basis of distinct genetic, immunological and biological characteristics. HHV-6B is the causative agent of exanthem subitum, but the role of HHV-6A in human disease is less clear. HHV-6A uses human CD46 as a cellular receptor. A multiple glycoprotein composed of glycoprotein H (gH) – gL-gQ1-gQ2 is a viral ligand for the human CD46 receptor in HHV-6A, while that of HHV-6B is not. The cellular receptor for HHV-6B has not been identified yet. Glycoproteins on the viral envelope play an essential role in viral infection, especially in the process of virion entry. In addition, the glycoproteins elicit neutralizing antibodies and thus become major targets of the host immune response. In this study, we isolated a hybridoma clone producing MAb named as KH-1, which had a neutralizing activity for HHV-6B infection and was capable of reacting specifically with HHV-6B gQ1. We have mapped the HHV-6B-specific neutralizing epitope on gQ1 using a series of C-terminal deletion and point mutants of HHV-6B gQ1. These results suggested that the recognition site of MAb KH-1 was located on the sequence between amino acid residues 484 and 496 of HHV-6B gQ1. Therefore, we demonstrate that as well as HHV-6A, HHV-6B gQ1 plays an important role for viral entry.

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3-08HHV-6B Protein U19 Stabilizes and Inactivates p53 in the Cytoplasm and Rescues Cells from p53-dependent Cell Death

E. Kofod-Olsen1([email protected]), J.M. Møller1, B. Bundgaard1, R. Bak1, J.G. Mikkelsen1, P. Höllsberg1

1Aarhus University, Aarhus, DenmarkUpon a viral infection, the host cell may respond by immediate cell-cycle arrest and subsequent apoptosis. This

response is controlled by the regulatory protein p53, which rapidly accumulates when its negative suppressor MDM2 is inhibited. Many viruses have attained mechanisms for inhibiting the activities of p53. We here study the evolutionarily well-adapted human herpesvirus (HHV)-6B and its ability to prevent the activities of p53. It has previously been shown that HHV-6B leads to a stabilization and accumulation of p53 after infection with HHV-6B. We now show that HHV-6B completely inactivates the p53 response to DNA damage, but does not protect the cells against DNA damage-induced, p53-independent apoptosis. We identify the HHV-6B protein U19 as an important regulator for p53 stability and activity, possibly through direct interaction with MDM2. Overexpression of U19 inhibited p53-induced effector functions in a manner similar to infection with HHV-6B. Our data suggest that U19 may be an important protein for establishing HHV-6B infection.

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3-09The Human Herpesvirus 6 U27 Gene Product Negatively Regulates the Transactivation Activity of Immediate-Early 2 Protein

A. Gravel1([email protected]), L. Flamand1

1CHUQ Research Center and Laval University, Quebec, CanadaThe immediate-early 2 (IE2) protein of human herpesvirus 6A (HHV-6A) is a potent transactivator of cellular and

viral promoters. However, no information is available on the possible role of other HHV-6 proteins on its transactivation abilities. In this study, the activity of HHV-6A U27 gene product on promoter transactivation mediated by IE2 was investigated. HHV-6 U27 encodes a early phosphonuclear protein of 41 kDa, that possesses a viral DNA polymerase-associated stimulatory factor activity. Transactivation of HHV-6 immediate-early and DNA polymerase promoters by IE2, in the presence or absence of HHV-6 p41 protein was determined in 293T cells through transient transfection experiments. Some experiments were also performed in 293T-p41-inducible cell line. The basal transactivation activity of all tested promoters was not influenced by the presence of p41. On the contrary, the transactivation observed in the presence of IE2 was strongly downregulated by p41, in a dose-dependent manner. Immunofluorescence studies in transiently and stably transfected cells showed a perfect colocalization between IE2 and p41. Same results were also observed in HHV-6-infected cells. Taken together, these results suggest that during infection, p41 is located near IE2 and presumably interacts with it, inhibiting its transactivation activity, in order to facilitate the replication of viral DNA and infection progression.

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4-01 Oral Historical Overview on CIHHV-6

M. Luppi1([email protected])1University of Modena and Reggio Emilia, Modena, Italy

Human herpesvirus 6 (HHV-6) exhibits a form of latency consisting of the integration of the whole viral genome into the host’s chromosomes. Individuals carrying HHV-6 chromosomal integration (CIHHV-6) bear such high levels of HHV-6 DNA either in plasma (>3.5 log10 copies/ml), peripheral whole blood (>6 log10 copies/ml) or in cerebrospinal fluid (4.0 log10 copies/ml), that they may be misinterpreted as patients with active HHV-6 infection. Human herpesvirus 6 chromosomal integration occurs at a similar prevalence, ranging from 1 to 3.5%, with only small differences related to different geographical areas, in both adults and children, suggesting that the main route of acquisition of CIHHV-6 is vertical transmission. Both A and B viral variants have been found to be integrated, both as whole and deleted/rearranged genomes. Moreover, early studies by Southern blot analysis and fluorescent in situ hybridization, performed on peripheral blood and bone marrow cells from patients with lymphoproliferative diseases and multiple sclerosis, showed the integration of the HHV-6 genome (or a portion thereof) in only a proportion of host cells, although the inheritance of CIHHV-6 was not formally ruled out by analysis in patients’ hair follicles. Although CIHHV-6 may be located on several chromosomes and not always linked to the short- or long-arms sites, the insertion of the viral genome has invariably been found in the proximity of telomeres. In infants with CIHHV-6, the detection of humoral immune responses to HHV-6 at mean titers similar to those present in children without congenital infection, and their persistence, even after the expected decline of passive antibody levels, suggested that the integrated genome may produce viral particles to which infants with CIHHV-6 are able to mount a humoral immune response. The improvement in neurologic status and the decrease in HHV-6 viral loads after the administration of ganciclovir and foscarnet in one patient with encephalomyelitis and CIHHV-6, in the absence of any other etiology of the neurologic impairment, has provided a further indirect hint that the integrated virus is able to replicate. More recently, Arbuckle and colleagues (P.N.A.S., 2010) performed in vitro experiments demonstrating that variant A CIHHV-6 may be inducible following chemical stimulation, such as with trichostatin A and 12-O-tetradecanoyl-13 acetate, which may produce fully competent virus and infect permissive cells. Furthermore, they have supposed that the chromosomal integration may represent the sole way through which HHV-6A achieves latency, by showing that: HHV-6A genome may be found covalently linked to chromosomes, soon after infection; that no free circular or linear viral genomes may be detected in permissive latently infected cells; and that HHV-6A has been found integrated in three out of three cell clones deriving from infected permissive cells (Morissette G, Flamand L. J Virol 2010). An historical overview of the studies addressing the issues related to CIHHV-6 will be provided.

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Oral 4-02 The Importance of HHV-6 Chromosomal Integration in Clinical Practice

K. Ward1([email protected])1Royal Free University College Medical School, London, United Kingdom (Great Britain)

Human herpesvirus-6 (HHV-6) exists as two closely related variants (A & B). Whereas there is no disease yet firmly associated with HHV-6A, variant B causes febrile illness in young children and is pathogenic in the immunosuppressed transplant recipient. HHV-6 differs from all other human herpesviruses because it has the unique ability for its genome to integrate under natural conditions into the chromosomes of some individuals. Chromosomally-integrated HHV-6, either variant A or B, occurs in about 1% of persons. Since chromosomally integrated HHV-6 DNA sequences are inherited through the germline, viral DNA is in every nucleated cell in the body and hence can be found in a range of body fluids such as whole blood, serum, plasma and cerebrospinal fluid. This phenomenon has the potential to confound the diagnosis of active HHV-6 infection because there are characteristically very high viral loads in whole blood (6 log10 HHV-6 genomes/ml) and serum (5 log10 HHV-6 genomes/ml) which are frequently misinterpreted in the clinical setting.The implications of this situation in the diagnostic virology laboratory will be discussed with particular reference to the findings after haematopoietic stem cell transplant.

Oral 4-03Neurodevelopmental Outcome of Children with Congenital HHV-6 Infection

C.B. Hall1([email protected]), M.T. Caserta1, R.L. Canfield2, H. Wang1, P.W. Davidson1

1University of Rochester Medical Center, Rochester, NY, USA; 2Cornell University, USAWhat is the clinical outcome of children with congenital HHV-6 infection? This is the major question, especially for

those with chromosomally integrated HHV-6 (ciHHV-6). Our studies indicate that most (86%) of all congenital HHV-6 infections are caused by ciHHV-6. Fourteen percent are transplacentally acquired HHV-6 congenital infection and even most, possibly all, transplacental congenital infections may be due to maternal ciHHV-6.We therefore followed children with congenital HHV-6 infection, determined by detection of HHV-6 in cord blood mononuclear cells, and matched controls to determine their neurodevelopmental outcome. Deficits, if similar to CMV, may develop postnatally and be progressive.Infants of 36 weeks gestational age or greater were followed up to 30 months of age. Neurodevelopmental testing included audiometric testing, the Bayley Scales of Infant Development, the Fagan Test of Infant Intelligence (FTII), and Visual Expectation Paradigm (VEXP) which measures saccade reaction time, anticipatory behavior, and off-task behavior. Factors potentially affecting developmental outcomes, including socioeconomic status, maternal intelligence and stress, were also measured.The results showed 1) No significant differences existed in the demographic and baseline characteristics of the controls (n = 193) and congenitally infected children (n = 42). 2) All infants appeared clinically normal at birth. 3) Development of hearing loss was not detected in either group. 4) Analyses for primary and secondary outcomes of the VEXP and FTII showed no significant differences between the two groups. 5) The Bayley scores, however, of the congenitally infected children were significantly lower at 12 months of age.Further follow-up is needed to confirm these findings. Studies are currently underway to assess developmental outcomes at later ages.

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4-04 Oral Herpesvirus Telomeric Repeats Facilitate Genomic Integration into Host Telomeres and Mobilization of Viral DNA during Reactivation

B.B. Kaufer1([email protected]), A. Egerer1, K.W. Jarosinski2, N. Osterrieder1

1Freie Universität Berlin - Institut for Virology, Berlin, Germany; 2Cornell University, Department of Microbiology and Immunology, NY, USA

Several lymphotropic herpesviruses, including Epstein Barr virus (EBV), human herpesvirus 6 (HHV-6) and Marek’s disease virus (MDV), are capable of integrating their genomes into host chromosomes. For the latter two viruses, integration of viral DNA was found at the distal end of host chromosomes, where telomeres consisting of telomeric repeat sequences and telomere associated proteins form structures that prevent chromosomal instability and degeneration. Intriguingly, HHV-6, MDV and other herpesviruses harbor telomeric repeats identical to host telomere sequences (TTAGGG)n at either end of the linear viral genomes; however, the role of these genetic elements has remained enigmatic. In order to investigate the role of the herpesvirus telomeric repeats, we mutated or deleted the hexameric repeats in MDV, a virus that infects chickens and serves as a natural virus-host model for herpesvirus induced lymphomagenesis. We could demonstrate that absence of the telomeric repeats leads to a significant decrease in mortality and tumor incidence in infected chickens. Tumor cells derived from animals infected with telomere mutant viruses were found to contain only a single integration site that was not located in host telomeres. Furthermore, we determined that mutant viruses integrate as concatemers and that reactivation from tumor cells occur only at low levels upon induction. Our study provides the first evidence that the MDV telomeric repeats, and possibly telomeric repeats of other herpesviruses (e.g. HHV-6 and HHV-7), mediate integration into the host genome and crucially contribute to transformation, tumor formation and reactivation.

4-05 Oral Integration, Reactivation and Mutation of Human Herpesvirus-6 Genome In Vitro and In Vivo

J. Luka1([email protected]), J. Gubin-Jurgens2

1Bioworld Consulting Laboratories, LLC, New Windsor, MD, USA; 2University of Nebraska Medical Center, USAGenomic polymorphisms have been identified among wild type HHV-6 isolates. Despite numerous studies of

genomic and protein variation between strains, little is known about the ability of a single HHV-6 strain to mutate and change with replication. Similarly, nothing is known about the role of the integration of the HHV-6 genome into the host chromosome neither in vitro nor in vivo.Preliminary studies will be presented indicating that certain regions of the HHV-6 genome, near the immediately early genes are able to mutate by deletions or additions of specific sequences to this area of the viral genome. These mutations can be detected both in vitro and in vivo in patients with chronic HHV-6 replication. Mutations associated with this part of the viral genome are effecting both replication and latency of the virus in vitro, and are associated with changes in the host cell specificity.Data will also be presented, demonstrating that in vitro integration of HHV-6 genome into the host chromosome is a precondition for HHV-6 replication and integrated viral genome can also be reactivated by various treatments. Whether integration is a prerequisite for in vivo replication, is still under investigation.The mutation of regulatory elements and acquisition of genes by HHV-6 are relevant to several fields of HHV-6 research and biology. The ability of HHV-6 to delete genes and tolerate the presence of additional DNA sequences may have implication in genetic manipulation of HHV-6. Mutations in HHV-6 could also change the virus ability to replicate or infect new cell types; or increase the virus capability to be more pathogenic.

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Oral 4-06The Characterization and Mapping of the Integrated Human Herpesvirus-6A and 6B Genome

J.H. Arbuckle1([email protected]), M.M. Medveczky1, S. Pantry1, D.V. Ablashi2, P.G. Medveczky1

1University of South Florida College of Medicine, Tampa, FL, USA; 2The HHV-6 Foundation, Santa Barbara, CA, USAPrevious research by our laboratory and others has definitively illustrated the integration of human herpesvirus-6

(HHV-6) into host cell chromosomes in vitro and in vivo, while the viral genome can be vertically transmitted through the germ line (gli-HHV-6). Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DRR) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DRL), we established a working hypothesis that HHV-6 integrates into telomeres via homologous recombination. Amplification and sequencing of the HHV-6A and more recently HHV-6B viral-chromosome junction identified DRR integrated into the telomere directly adjacent to the subtelomere of the chromosome. After mapping the DRR of gli-HHV-6, we subsequently focused on determining if the DRL was present in the integrated genome and whether the remaining telomere sequence of the chromosome was extended beyond the DRL. Southern hybridization of PCR amplified CI-HHV-6 cell lines and gli-HHV-6 patients PBMCs indicate the presence of DRL within the integrated viral genome. Moreover, the tandem array of telomere repeats [(TTAGGG)n] at the end of chromosome was shown to extend beyond the DRL of gli-HHV-6. Therefore, the structure of the gli-HHV-6 is as follows: chromosome-subtelomere-(TTAGGG)5-41-DRR-UL-DRL-(TTAGGG)n. Furthermore, we have previously shown trichostatin-A treatment of PBMCs and in vitro integrated HEK-293 cells induced the reactivation of HHV-6 from its latent integrated state. Here we show the induction of integrated HHV-6 with trichostatin-A lead to the excision of the integrated genome and generation of the DRR-DRL junction which signifies circularization and concatemer formation of the viral genome through rolling-circle replication. Taken together, the data suggests that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency, while at the same time the chromosome’s telomere remains stably intact during viral integration.

Oral 4-07 Characterization of the Nucleotide Sequence of an HHV-6A Strain Isolated from a Patient with Germline Integrated HHV-6

S.N. Pantry1([email protected]), J. Luka2, J.H. Arbuckle1, M.M. Medveczky1, R. Renne3, D. Ablashi41University of South Florida, Tampa, FL, USA; 2Bioworld Consulting Laboratories, Mt. Airy, MD, USA; 3University of Florida, Gainesville, FL, USA; 4The HHV-6 Foundation, Santa Barbara, CA, USA

Human herpesvirus-6, a member of the Betaherpesvirus subfamily, is the causative agent of roseola infantum. The genome of HHV-6 includes a central unique region flanked on both sides by direct repeats and an array of telomeric repeats, which allows integration of the viral genome into the telomeres of host cells. This is in contrast to other herpesviruses, in which the viral genome typically persists as a circular episome during latency. In the case of HHV-6, integration readily occurs in vitro as well as in vivo; recent studies by Arbuckle et al. reported germline integrated HHV-6 (gliHHV-6) in chromosome 18 of three members of family. Here we have characterized and analyzed the nucleotide sequence of reactivated virus from these patients. To this end, peripheral blood mononuclear cells from these patients were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydrocortisone. Reactivation resulted in the production of an infectious, replication competent virus as well as linear replicating HHV-6 DNA. Viral DNA isolated from the supernatant of cells infected with the reactivated virus purified and sequenced by ultra high-throughput 454 sequencing. Sequencing results indicate that the reactivated virus is colinear to, but distinctly different from that of the prototype U1102 strain. Additionally, there is a high level of sequence conservation between the reactivated virus and U1102 in the central unique region of the HHV-6 genome, while the greatest amount of variability is within the direct repeats. These data provide further evidence that gliHHV-6 can reactivated from its integrated form.

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4-08 Oral Is Integration of the Human Herpesvirus 6 Genome in Telomeres Irreversible or it Represents Latency?

M.M. Medveczky3, J.H. Arbuckle3, S.N. Pantry3, J. Luka1, D. Ablashi2, P.G.Medveczky3([email protected])1Bioworld Consulting Laboratories, Mt. Airy, MD, USA; 2The HHV-6 Foundation, Santa Barbara, CA., USA; 3University of South Florida College of Medicine, Tampa, FL, USA

Several reports documented the integration of Human Herpesvirus-6 (HHV-6) genome in malignant cells. Integration of the viral genome was also demonstrated in all somatic cells in individuals either suffering from chronic fatigue syndrome, other diseases of the central nervous system, and from individuals that are apparently healthy. This form of integration is passed through the germ line and is commonly referred to as “chromosomally integrated” or ciHHV-6. Most reports have suggested that integration of HHV-6 is an irreversible fate of the viral genome. In contrast, our group has published and now presents additional data that supports HHV-6 integration following infection is a common event detectable in all cell types tested. Moreover, several complementary assays demonstrate that the latent virus can be reactivated from its telomeric integration site after induction with chemicals. We now present a working model that could explain the contradictory findings published in the literature.

4-09Human Herpesvirus-6 Chromosomal Integration and Bing-Neel Syndrome Revealing a Waldenstrom’s Disease: Incidental Association?

M. Giroux1, A. Gautheret-Dejean2([email protected]), A. Dewilde3, O. Outteryck1, C. Maurage4, B. Onraed5, L. Terriou6, P. Vermersch1

1Université Lille Nord de France, Department of Neurology, CHRU Lille, France; 2Service de Virologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 3Laboratory of Virology, CHRU Lille, France; 4Université Lille Nord de France, Laboratory of Anatomopath CHRU Lille, France; 5Université Lille Nord de France, Laboratory of Biochemistry CHRU Lille, France; 6Université Lille Nord de France, Department of Hematology, CHRU Lille, France

Detection of Human Herpesvirus-6 (HHV-6) viremia can be related to viral reactivation or chromosomal integration (CI) of HHV-6 DNA. CI HHV-6 is rare and concerns about 0.2 to 2.8% of general population. A 35-year-old patient has developed headache, photophobia and blurred vision over ten months. Fundus oculi showed papillary oedema. Brain magnetic resonance imaging and cerebrospinal fluid (CSF) analysis showed right parieto-occipital pachymeningitis and lymphocytic pleiocytosis. Other investigations were negative except presence of monoclonal Immunoglobulin M Kappa dysglobulinemia with a level of 3.5 g/l. Anatomopathological analysis of cerebromeningeal biopsy showed lymphocytic infiltration with normal B-lymphocytes. HHV-6 viral load was quantified by real-time PCR was high on this biopsy (3,343,923 copies per million cells (cop/Mc)) and also in blood and CSF. Intravenous treatment by ganciclovir was performed during 15 days. After treatment, HHV-6 viral load remained stable in blood (2,716,233 cop/Mc) and CSF (3,947,090 cop/Mc). PCR was also positive in hair follicles (8,425,287 cop/Mc). Complementary molecular analysis of the biopsy and the bone marrow found a malignant B-lymphocytic clonality. CSF flow cytometry disclosed a population of kappa-positive B cells CD5-, CD10- and CD23-. Diagnosis of Waldenstrom’s macroglobulinemia with primary involvement of central nervous system (Bing-Neel syndrome) was made.Persistence of high HHV-6 viral load in blood, CSF, brain and hair follicles proved in this patient CI HHV-6. High HHV-6 viral load due to CI can lead to a misdiagnosis with potentially unsuitable toxic treatments. Unlike Epstein-Barr virus, HHV-6 seems not to be an accepted risk factor of hematological proliferation. However in our case is the CI HHV-6 really fortuitous?

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4-10Frequency of Chromosomally-Integrated Human Herpesvirus 6 in Children with Acute Lymphoblastic Leukemia

A. Gravel1([email protected]), D. Sinnett2, L. Flamand1

1CHUQ Research Center and Laval University, Quebec, Canada; 2CHU Sainte-Justine, Montreal, Quebec, CanadaIntroduction: Human herpesvirus 6 (HHV-6) is a ubiquitous pathogen infecting nearly 100% of the human population. Of these individuals, between 0.2% and 1% of them carry chromosomally-integrated HHV-6 (ciHHV-6). The consequences of chromosomal integration by HHV-6 remain unknown.Objective: To determine and compare the frequency of ciHHV-6 in Canadian children with lymphoblastic leukemia to healthy blood donors.Methodology: A total of 293 DNA samples from children with pre-B (n=255), pre-pre-B (n=4), pre-T (n=26) and undetermined (n=8) leukemia were analyzed for ciHHV-6 by quantitative TaqMan PCR (QPCR) using HHV-6 specific primers and probe. As control, DNA samples from 288 healthy individuals were used. Primers and probe specific to the cellular GAPDH gene were used to estimate integrity and DNA content. Results: Out of 293 DNA samples from the leukemic cohort, 287 contained amplifiable DNA. Of these, 1 (0.35%) contained ciHHV-6. Variant typing indicates that the ciHHV-6 corresponds to variant A. None of the 288 DNA samples from healthy individuals contained ciHHV-6.Conclusion: The frequency of ciHHV-6 in children with acute lymphoblastic leukemia is similar (p=0.47) to that of healthy individuals. These results suggest that lymphoblastic leukemia does not originate as a consequence to integration of HHV-6 within the chromosomes.

4-11Detection of HHV-6 Antigen and Herpesvirus Particles in Individuals with Chromosomally Integrated HHV-6 (ciHHV-6)

V. Strenger1([email protected]), I. Lautenschlager2, S. Richter3, W. Schwinger1, E. Engdahl4, J. Ahlqvist1, A. Fogdell-Hahn1, S.W. Aberle1, T. Popow-Kraupp1, E.P. Nacheva1, G. Krueger1, C. Urban1

1Medical University of Graz, Graz, Austria; 2Central Hospital and University of Helsinki, Finland; 3Austrian Agency for Health and Food Safety (AGES), Austria, 4Karolinska Institutet, Stockholm, SwedenBackground: Up to now, the ability to replicate chromosomally integrated HHV-6 genome (ciHHV-6) and its clinical consequences are unclear. Methods: We performed HHV-6 antigen detection by means of an indirect immunoperoxidase staining (MAB 8535, Chemicon, Inc., Temecula, CA, and Ref-11-242, Argene Biosoft, Varilhes, France) in PBMCs and examined serum and PBMCs by electron microscopy (EM) in 10 individuals (out of 5 families) with FISH proven ciHHV-6 (3 persons with ciHHV-6A, 1 acquired ciHHV-6A by stem cell transplantation). Blood from 13 controls without ciHHV-6 served as negative controls for EM. EM investigator was blinded. To prove that the observed particles are HHV-6 virions we performed EM with immunogold labeled antibodies (iEM, gp116/54/64 and gp60).Results: In 7 of 10 individuals with ciHHV-6 HHV-6 antigen was detectable at least once. Four persons were repeatedly tested. Antigen detection varied over time. In addition, herpestype particles of intracytoplasmatic virions of different degrees of maturation were found in 9 of these 10 individuals, mature herpesvirus particles, yet were found in serum of only 2 of these individuals. Structural characteristics and measurements of observed particles matched criteria for HHV-6. In specimens of negative controls no herpesvirus particles were found. Currently, results of iEM are pending and will be presented at the conference.Discussion: Our results indicate synthesis of viral proteins and mostly incomplete (abortive) replication of HHV-6 in humans with ciHHV-6. Pathophysiological role of this phenomenon has to be clarified.

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4-12A Simple and Fast Method to Diagnose Chromosomal Integration of Human Herpesvirus 6 in Circulating Mononuclear Cells

M. Malnati1([email protected]), F. Broccolo2, E. Di Marco3, M. Grazia Marazzi3, P. Lusso4

1San Raffaele Scientific Institute, Milan, Italy; 2Università Milano-Bicocca, Italy; 3University of Genoa, Italy; 4Laboratory of Immunoregulation NIAID, NIH Bethesda, USA

Chromosomal integration of HHV-6 is a common event occurring with a frequency estimated between 0.5 and 1% of the human population. Therefore, it is pivotal to distinguish contamination by cell-derived chromosomally integrated HHV-6 (CIHHV-6) DNA from the presence of virion-associated DNA (a marker of active infection) when HHV-6 DNAemia is detected in biological fluids. To distinguish between CIHHV-6 (nuclear) and episomal (cytoplasmic) HHV-6 DNA, we developed a new method for the separation of nuclei and cytosolic fractions by testing different types of lysis buffer, incubation time and centrifugation speed, while monitoring the levels of cytosolic genomic DNA contamination and the nuclear recovery by a real-time QPCR system developed on the human CCR5 gene. The optimized system showed ≤ 0.0003% residual DNA contamination of the cytosolic fraction, allowing for a recovery of the nuclear fraction (≥99% pure) in less than 2.5 hours. The method was validated by testing productively and non-productively HHV-6-infected cells (PBMC, HSB-2, Molt-3, Hela), as well as PBMC derived from 10 individuals with high HHV-6 DNA load (>106 GeQ/106 cells). The system has been successfully used to diagnose CIHHV-6 integration and vertical transmission in a child suffering of encephalytis, migraine and cognitive disorders.

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Telomeres & Integration

Oral 5-01Viruses and Telomeres

P. Lieberman1([email protected])1The Wistar Institute, Philadelphia, PA, USA

HHV6, Marek’s Disease Virus (MDV), and Epstein-Barr Virus (EBV) have captured components of cellular telomeres and telomere regulatory factors. HHV6 and MDV have telomere repeats within the viral termini, while EBV has telomere repeats flanking the viral origin of plasmid replication (OriP). Cellular telomere repeat binding factors (TRF1 and TRF2) can bind to these repeats and modulate viral and cellular genome behavior. In HHV6, telomere repeats appear to function in telomere integration, while in EBV the TRF2 binding sites may prevent telomere integration. One major difference between the repeats in HHV6 and EBV is that the repeats in HHV6 may be transcribed into RNA, which can facilitate homologous recombination. We have investigated the function of the cellular telomere repeat containing RNA, termed TERRA, and have observed that it is highly over-expressed in cells where homologous recombination is the primary mechanism for maintaining telomere length through the alternative lenghthening of telomeres (ALT) pathway. We have also found that EBV infection can suppress TERRA RNA expression from cellular telomeres, and promote telomerase (hTERT) gene expression, consistent with the idea that the hTERT and TERRA have antagonistic activities at telomeres. While the precise function of TERRA at telomeres is not yet known, we have evidence to suggest that TERRA may promote homologous recombination and inhibit DNA damage repair pathways. The potential for HHV6 to express a viral TERRA like molecule and how this may regulate HHV6 reactivation and integration at telomeres will be discussed. The mechanisms that regulate cellular TERRA RNA expression will also be discussed, as well as how other viruses, like EBV, may deregulate normal telomere functions.

Oral 5-02The Role of Ku86 in Human Telomere Maintenance

E. Hendrickson1([email protected])1University of Minnesota, Minneapolis, MN, USA

Telomeres are the nucleoprotein structures located at the ends of human chromosomes and they are important for proper chromosomal stability and replication. The DNA component of human telomeres consists of a hexameric sequence, T2AG3, that is repeated for many kilobases. Interestingly, the chromosomal integration of human herpes virus 6 (ciHHV-6) appears to occur preferentially into telomeric regions. It has generally been assumed that this is the result of homologous recombination (HR) between the telomeric-like repeats located in the termini of HHV-6 and telomeres, although this has not been rigorously established.

Our laboratory has utilized a different virus, recombinant adeno-associated virus (rAAV), which is capable of facilitating gene targeting in human cells at high frequency, to construct a large series of isogenic human somatic cell lines that are defective for genes involved in either HR, or in the competing pathways of classic or alternative non-homologous end joining (C-NHEJ or A-NHEJ, respectively). In particular, we have focused our recent investigations on a critical C-NHEJ factor, Ku86. Since Ku86 is an essential gene, we constructed a human cell line that contains a single functional conditionally-null allele of Ku86 that is dependent upon the expression of the Cre recombinase. In the presence of Cre, the residual Ku86 allele is deleted from the chromosome, which ultimately results in the demise of the cells. Molecular analyses of these cells demonstrated that death ensued due to severe telomere dysfunction. Specifically, our work demonstrated that Ku86 is a potent suppressor of HR at telomeres and that in the absence of Ku86, aberrantly hyperactive HR results in lethal telomeric recombination.

These studies have identified a gene, ku86, and pathways, HR and C-NHEJ, that are critical for regulating HR events at human telomeres and as such they are likely to be relevant to HHV-6 chromosomal integration.

Telomeres & Integration

5-03 Oral Human Telomeric and Subtelomeric DNA Sequence Features and Function

H. Riethman1([email protected])1The Wistar Institute, Philadelphia, PA, USA

Our lab has focused primarily upon fundamental questions surrounding human telomeric and subtelomeric DNA structure, sequence organization, and variation. As part of the human genome project, we collaborated with the genome mapping and sequencing community to finish and annotate subtelomere regions, incorporating bioinformatic and computational approaches to classify and annotate subtelomeric segmental duplications and subterminal sequence features. We have continued to investigate subterminal structural variation as well as more conventional allelic variation in telomere-adjacent regions, using a set of fosmid libraries created as part of the Structural Variation Project as a major resource. Most recently, we have begun developing novel methods for detecting telomeric structural changes in the human genome using next-generation paired-end sequencing approaches, and applying the methods to somatic telomere length changes and mutations associated with aging and cancer. Integration of the HHV-6 genome at the subtelomere-telomere boundary raises a host of fascinating questions for telomere biology; among these are the potential impact upon telomere integrity and length regulation, and how the introduction of foreign sequence features affects transcription of subterminal RNAs such as TERRA and the WASH gene family. I will present a summary of our subterminal sequence annotation and variation studies, and speculate on the potential consequences of HHV-6 integration for telomere function.

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HHV-6 BAC

Oral 6-01Using Infectious Bacterial Artificial Chromosomes to Dissect the Molecular Biology of Beta Herpesviruses

A. Paredes1, D. Yu1([email protected])1Washington University School of Medicine, Saint Louis, MO, USA

Our understanding of the molecular biology of beta herpesviruses, including cytomegalovirus (CMV), human herpesvirus 6A and 6B (HHV-6A and HHV-6B), and human herpesvirus 7, has long been hindered by the lack of an effective viral genetic system. Cloning a viral genome as an infectious bacterial artificial chromosome (BAC), an approach first developed for CMV, has allowed us to overcome this barrier. We have generated infectious BAC clones of several CMV strains and, together with other labs, have developed the methods for efficient manipulation of BAC-cloned viral genomes and creation of recombinant viruses. Using the BAC technology, we have investigated the role of several previously uncharacterized viral genes in CMV biology. The lessons learned from BAC-based CMV genetics should also help investigators apply this genetic approach to other herpesviruses.

While BAC genetics have pushed CMV research forward, an infectious BAC clone of HHV-6 has not been available until recently. This made it difficult for a comparative analysis between CMV and HHV-6 in order to determine general principles conserved among the beta herpesvirus family as well as features unique to individual members. We have isolated the genome of the HHV-6B Z29 strain as BAC clones from viral replication intermediates in infected cells. Initial characterization indicated that two clones had the intact viral genome but contained only a single direct repeat (DR). Indeed, while the mature virion DNA was flanked by two terminal DRs as previously reported, the vast majority of HHV-6B replication intermediates contained junctions composed of only a single DR, similar to that reported for HHV-6A. Efforts to reconstitute HHV-6B virus from BAC clones are ongoing. Recently, several other labs have successfully cloned the HHV-6A genome as BAC clones. It is anticipated that these collective efforts will significantly advance the understanding of HHV-6 molecular biology and pathogenesis in the foreseeable future.

Oral 6-02HHV-6 Glycoprotein Complex Formation is Required for the Folding and Trafficking of the Complex, gH/gL/gQ1/ gQ2 and its Cellular Receptor Binding; gQ1

Y. Mori1([email protected])1Kobe University Graduate School of Medicine, Kobe, Japan

HHV-6 unique glycoprotein complex, gH/gL/gQ1/gQ2 has been shown to be important for its cellular receptor, human CD46 binding. However, whether the entire complex or one containing subset of the components binds to CD46 has been unclear, and the mechanism for appropriate folding of the complex itself is also unknown. The gQ1 protein which is a component of the complex, is glycosylated and that at least two products of gQ1 with different molecular weights, 80 kDa (gQ1-80K) and 74 kDa (gQ1-74K) are expressed in HHV-6A-infected cells. Only gQ1-80K is incorporated into mature virions, and gQ1-80K is part of the gH/gL/gQ1/gQ2 complex, which act as a viral ligand for the cellular receptor, CD46. So far, gQ1-74K, but not gQ1-80K, has been detected in cells transfected with only the plasmid for gQ1. However, it is unknown whether gQ1-80K and gQ1-74K are expressed from the same transcript, and the detailed functions of the gQ1s are also unknown. To examine the maturation of gQ1, we transfected 293T cells with several combinations of the individual molecules, gH, gL, gQ1, and gQ2, and examined the modifications of gQ1. Surprisingly, only when all four molecules were expressed, a substantial amount of gQ1 was clearly detected as an 80k-Da band, the same molecular weight as in virions. These results suggest that the presence of all the molecules, gQ2, gH, and gL, was necessary and sufficient for gQ1 maturation. Furthermore, we found that the tetrameric complex formation was required for its receptor binding and the gQ1 was essential for the virus growth.

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HHV-6 BAC

6-03 Oral Cloning of the HHV-6A Genome into BACs, Viral-host Interactions and the Derivation of Amplicon-6 Vectors for Potential Gene Therapy

N. Frenkel1([email protected]), R. Borebstein1, H. Zeigerman1, E. Sharon1

1The Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, IsraelThe studies described can be briefly summarized as follows: (i) Cloning of the 160 kb HHV-6A genome into a modified

BAC vector, by a single step, employing concatemeric replicative intermeiates, arising most likely by the rolling circle DNA replication mechanism. We have cleaved the head-to-tail replication intermediates with SfiI that cleaves once in the viral genome. The resultant unit length genome was cloned into a modified BAC made to carry the SfiI site. The BAC cloning enabled potential molecular analyses. We found that the cloned HHV-6A BAC, as well as equivalent replicating viral genomes in the infected cells contained a single direct repeat (DR), rather than the DR-DR junction, predicted to arise by circularization of parental viral genomes with a DR at each terminus. (ii)We found alterations in cellular pathways: E2F1 presence was increased, Rb phosphorylation was changed and there was cell cycle arrest. Moreover, we found activation of molecular stress functions leading to induced cell death. (iii) We have previously derived HHV-6 amplicon vectors containing mixtures of mutant helper virus and defective genomes of size 160 kb, containing multiple reiterations of the DNA replication origin, the packaging signals along with the selected new transgene(s). The large gene capacity and the gene reiterations allow efficient gene expression. We show that HHV-6 amplicon vectors can be potentially employed for lymphotropic cancer gene therapy, by inserting into the amplicon the tk gene and showing cell death in response to ganciclovir treatment. Additionally, amplico-6 can be potentially employed for vaccination. We have shown that amplicon vectors carrying the gD of HSV express the protein very efficiently at the T cell membrane. Vectors carrying the gD mutant with deleted transmembrane region produced most efficiently a secreted gD. Current studies are designed to test the vaccination efficiency.

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Epidemiology

Oral 7-01Quantitation of Human Herpesvirus -6 and -7 DNAs in Different Blood Fractions from Blood Donors

P. Bonnafous1([email protected]), B. Géraudie1, M. Charrier1, D.Heurté1, M. Desmonet1, M. Bartoletti1, C. Penasse2, H. Agut1, A. Gautheret-Dejean1

1Service de Virologie, ER1 DETIV, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 2Etablissement Français du Sang, Site Pitié-Salpêtrière, Paris, FranceObjective: The diagnosis of human herpesvirus-6 (HHV-6) or -7 (HHV-7) infections is based on the quantitation of viral genomic DNA in blood. The aim of our study was to define usual values of HHV-6 and HHV-7 loads in different blood fractions (whole blood [WB], mononuclear cells [PBMCs], polymorphonuclear cells [PMNLs]) of blood donors.Method: For 200 blood donors, 10 mL of blood were sampled on EDTA. After putting aside aliquots of WB, PBMCs and PMNLs were separated using Ficoll and dextran gradients, respectively. Nucleic acids were extracted using QIAamp® DNA blood mini kit. Quantitation of HHV-6 and HHV-7 DNAs was carried out using real-time PCR assays and viral loads were expressed as the number of viral genomic equivalent copies per million of cells (EqCop/M).Results: HHV-6 DNA was detected in 8% of WB, 10.5% of PMNLs and 16.5% of PBMCs samples, whereas HHV-7 DNA was detected 51.5% of WB and PMNLs and 62% of PBMCs samples. Median loads were 81 EqCop/M in WB, 34.5 EqCop/M in PMNLs and 62 EqCop/M in PBMCs for HHV-6, and 129 EqCop/M in WB, 62 EqCop/M in PMNLs and 225 EqCop/M in PBMCs for HHV-7. One subject had chromosomally integrated HHV-6 with high viral loads: 2.23 x 10^6 EqCop/M in WB, 2.55 x 10^6 EqCop/M in PMNLs, 2.84 x 10^6 EqCop/M in PBMCs, and 3.21 x 10^6 in plasma.Conclusion: These results confirm that PBMCs are the main compartment for HHV-6 and -7 in blood, the presence of viral genome in PMNL probably corresponding to phagocytosis phenomenon. These results allow us to define usual median viral load values in WB, 100 EqCop/M for HHV-6, 150 EqCop/M for HHV-7, commonly encountered in immunocompetent patients who control the viral infection. Finally, the prevalence of chromosomally integrated HHV-6 in France is 0.5%.

Oral 7-02Human Herpesvirus-6 in Children Undergoing Cancer Chemotherapy

J. Goldfarb1([email protected]), N. Borges2, B. Yen-Lieberman3, K. Gowans1, C. Woodard4, D. Kohn3, L. Danziger-Isakov1, P. Pellett41Children’s Hospital Cleveland Clinic, Cleveland, OH, USA; 2Cleveland Clinic Lerner College of Medicine, USA; 3Cleveland Clinic Clinical Virology Laboratory, USA; 4Wayne State School of Medicine Department of Immunology and Microbiology, USA

HHV-6 is ubiquitous, with most of children infected by 2 years of age. The virus has not been well studied in children with cancer to determine whether the immune suppression accompanying treatment of malignancies is associated with disease from re-activation of the virus (as appears to happen with solid organ transplantation). Our objective was to determine if HHV-6 reactivation is associated with disease in these children. We longitudinally evaluated for the presence of HHV-6 from the onset of chemotherapy throughout treatment in children with newly diagnosed malignancies. Associations between viral replication and clinical symptoms over the course of the study were assessed to determine if HHV-6 is a true pathogen in these children. We enrolled 77 children with a new diagnosis of cancer who were to be treated with chemotherapy and followed them prospectively for 2 years. The data presented represent 74 children with data for the first year of the study. Blood and clinical data were collected every 2 weeks from the start of therapy for cancer for 6 months and then every month for the next 6 months as well as during acute episodes of illness. DNA was extracted and evaluated by PCR for HHV-6, HHV-7, Epstein Barr Virus, and Cytomegalovirus; serology to HHV-6 was measured in serum. Data were analyzed by logistic regression with generalized estimating equations using SAS 9.2 software. Results: HHV-6 viremia was associated with younger age but was not associated with acute symptoms or illness. Serology also did not suggest a host response to the viremia. While HHV-6 positivity occurred in 47% of subjects at some time, it occurred with equal frequency during acute and routine visits. Conclusion: If HHV-6 PCR is not measured longitudinally, clinical events may be misattributed to this virus.

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Epidemiology

7-03 Human Herpesvirus 6 (HHV-6) in the Genesis of Acute Leukemia

F. Nefsi1, A. Gautheret-Dejean2([email protected]), N. Ben Fredj1, A. Khlif3, H. Agut2, S. Feki4, M. Aouni11Service of Infectious Diseases and Biological Agents, Monastir, Tunisia; 2Service of Virology, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 3Service of Clinical Hematology, Sousse, Tunisia; 4Service of Clinical Biology, Faculty of Pharmacy, Monastir, Tunisia

Infectious etiology in lymphoproliferative diseases is suspected. The pathogenic roles of HHV-6 in acute leukemia have been of continued interest. Discordant results to establish link between HHV-6 activation and genesis of acute leukemia were observed. HHV-6 viral load was quantified by quantitative real-time PCR in bone marrow or blood samples obtained from 17 children and 30 adults with acute leukemia: 13 B acute lymphoblastic leukemia (B-ALL), 5 T acute lymphoblastic leukemia (T-ALL), 27 acute myeloid leukemia (AML) and 2 Acute biphenotypic leukemia at diagnosis, aplasia, remission and relapse. HHV-6 was detected in 12% of samples at diagnosis. At aplasia, 8% of patients were positive for HHV-6. At remission, 25% of samples were positive and at relapse HHV-6 was evidenced in 24% of patients. Viral loads were 137 copies per million cells (Cop/M) at diagnosis, 68 Cop/M at aplasia, 57 Cop/M at remission and 14 Cop/M at relapse. HHV-6 was present in 29 patients, 41% of children with viral load of 137 Cop/M and in 7% of adults with viral load of 50 Cop/M. Twenty-six patients, 6% with B-ALL were positive for HHV-6 with viral load of 205 Cop/M compared to 8% with AML with viral load of 50 Cop/M. No HHV-6 was found for patients with T-ALL. All HHV-6 detected were variant B. No link was shown between neither the clinical symptoms specific of HHV-6 nor the abnormal karyotype and HHV-6 activation. A case of HHV-6 chromosomal integration (CI-HHV-6) was shown in a patient with AML. Our results suggest also that CD34+ hematopoietic progenitors carry latent HHV-6, but the virus can’t infect leukemic cells. In conclusion, this study supports the absence of HHV-6 role in the genesis of acute leukemia and shows that HHV-6 can reactivated in immunocompromised patients after chemotherapy treatment.

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Oral 8-01 HHV-6 and Autoimmune thyroiditis

D. Di Luca1([email protected]), E. Caselli11University of Ferrara, Ferrara, Italy

We are studying a potential association of HHV-6 with chronic lymphocytic thyroiditis, or Hashimoto’s thyroiditis (HT). HT is the most common cause of hypothyroidism in the Western world. It is an autoimmune disease, where thyroid follicles are gradually destroyed by a variety of cell and antibody mediated immune processes. HT is characterized by invasion of the thyroid tissue by leukocytes, mainly T-lymphocytes. We analysed fine needle biopsies from HT patients for the presence and the transcriptional state of HHV-6. Controls, represented by benign follicular epithelial lesions and thyroid neoplasias, showed a low prevalence of HHV-6, with low viral load and the transcriptional pattern showed a latent infection. Instead, samples from HT had a high prevalence of infection (81 vs 14%, p 0.002), significantly higher viral load, and low-grade acute infection in the majority of samples. HHV-6 was latent in the blood of the HT same patients with acute thyroid infection. Preliminary results suggest that HHV-6 is present in thyroid cells and not in the more abundant lymphocitic infiltration. The tropism of HHV-6 for thyroid cells was verified by infection of Nthy-ori3-1, a thyroid follicular epithelial cell line. The results show that Nthy-ori3-1 cells are permissive to HHV-6 replication. Both HHV-6 variants support productive infection for the first 7 days p.i., and subsequently persist establishing latency. The observations that Nthy-ori3-1 cells infected with HHV-6 become target for innate NK cell killing and that HT patients have altered T-cell responses to HHV-6 strongly support a potential role for HHV-6 in the development or the triggering of HT.

Oral 8-02 Serum Viremia of Human Herpesvirus 6 (HHV-6) in Patients with Active Autoimmune Connective Tissue Diseases: Epiphenomenon or Causative Role in Disease Development?

F. Broccolo1([email protected]), G. Cassina2, A. Fava2, L. Fusetti3, S. Paolino4, B. Matteoli3, E. Zaccaria4, S. Franchini2, A. Parodi4, P. Lusso5, L. Ceccherini-Nelli3, M. Grazia Sabbadini2, F. Drago4, M. Malnati21Department of Clinical Medicine, Prevention and Biotechnology, University of Milano-Bicocca, Milan, Italy; 2San Raffaele Scientific Institute, Italy; 3University of Pisa, Italy; 4University of Genoa, Italy; 5Laboratory of Immunoregulation NIAID, NIH Bethesda, USA

Autoimune connective tissue diseases (ACTDs) encompass a heterogeneous group of diseases including systemic sclerosis (SSc), systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE) and dermatomyositis (DM)..To evaluate whether active infections of the lymphotropic human herpesviruses Epstein-Barr virus (EBV), HHV-6 and HHV-7 occur in patients with ACTDs, viral DNA loads were assessed in serum and peripheral blood mononuclear cells (PBMCs) obtained from 83 ACTD patients, 35 patients affected by other Autoimmune Chronic inflammatory (ACIDs) including psoriasis (PS) and rheumatoid arthritis (RA) and 38 healthy subjects (HS). Anti-HHV-6 and anti-EBV antibody titers were measured.Active HHV-6 infection was observed in a significantly higher proportion of ACTD patients (30/83, [36%]) compared to HS (2/61 [3.3%]) and subjects with other ACIDs (4/35 [11.4%]), with the highest reactivation frequency (42.5%) observed in patients with SLE or DLE; EBV was detected in serum of 5 patients with ACTD and in 2 patients with other ACIDs. The frequency of EBV and HHV-6 detection in PBMC did not significantly differed amongst the 3 cohorts of individuals. HHV-6 or EBV reactivation was not the consequence of an immune suppressive drug treatment since HHV-6 and/or EBV serum viremia was detected in 13/39 ACTD patients under therapy and in 22/45 ACTD patients without therapy. HHV-6 reactivation was associated with disease activity: HHV-6 serum viremia was detected in 23/40 patients with active disease and only in 7/44 patients with an inactive disease state (p < 0.05; χ2 test). Although, significantly higher HHV-6 IgG titers and anti-EBV EA IgG titers were present in ACTD patients as compared to HS viral Dnaemia was not associated with the titers of these antibodies.Our data suggest that HHV-6 may act as a pathogenic factor predisposing patients to the development of ACTD, especially LE or, conversely, that ACTD may predispose patients to HHV-6 reactivation.

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8-03 Oral Identification of HHV-6B Sequences in EBV-negative Hodgkin Lymphoma Using Next Generation Sequencing

A.J. Bell1([email protected]), A. Gallagher1, L. Shield1, D. Gatherer1, R.F. Jarrett11MRC University of Glasgow Centre for Virology Research, Glasgow, United Kingdom (Great Britain)

A proportion of cases of classical Hodgkin lymphoma (cHL) are causally associated with the Epstein-Barr virus (EBV) but the aetiology of EBV-negative cHL cases is poorly understood. Epidemiological studies suggest that delayed exposure to a common infectious agent(s) may be involved in these cases. The aim of this study was to search for a novel virus in Hodgkin and Reed-Sternberg (HRS) cells, the tumour cells in cHL. HRS cells were enriched from three cases of cHL, including two EBV-negative cHL cases, and cDNA was amplified and subjected to sequencing using the Roche 454 platform. Comparisons with viral sequence databases revealed human herpesvirus 6B (HHV-6B) sequences in one case of EBV-negative cHL. We therefore initiated a further analysis of the association between HHV-6 and cHL to determine: the frequency of HHV-6 detection in cHL; the cellular localisation of the HHV-6 sequences; and whether integrated and/or deleted HHV-6 genomes are present in cHL tumours. HHV-6B, but not A, pol gene sequences were detected in 39/67 (59%) of cHL biopsies but in only 1/21 reactive nodes. EBV-ve cHL cases were more likely to be HHV-6B-positive than EBV-positive cases but differences were not statistically significant (p=0.11). Four cases with HHV-6B genomes above a pre-defined copy number level were all EBV-negative. Two cases were negative for HHV-6B pol sequences but contained DR6 sequences suggesting that deleted genomes may be present in some cases. Immunohistochemistry using the HHV-6 p41 antibody revealed staining of HRS cells in one case with a high level of HHV-6B genomes. Further analysis of this intriguing association between HHV-6 and cHL is ongoing.

8-04 Oral Systemic Leukotropic Herpesvirus Infections and Autoantibodies in Patients with Myalgic Encephalomyelitis – Chronic Fatigue Syndrome

K. Knox1([email protected]), D. Peterson2, A. Knox1, J. Weismann2, D. Carrigan1

1Wisconsin Viral Research Group, Milwaukee, WI, USA; 2Sierra Internal Medicine, Incline Village, Nevada, USAThe investigations described here studied individuals suffering prolonged fatigue with systemic signs and symptoms

consistent with an active ongoing infection. This infective process is believed to involve active disseminated infection with one or more leukotropic herpesviruses. The goal of these studies is to prevent the misdiagnosis of their illness as idiopathic chronic fatigue syndrome (CFS) and to justify new strategies for effective treatments such as antiviral drug intervention.Our virological data has demonstrated three distinct, but overlapping, patient populations. Methods used for the detection of active human herpesvirus six (HHV-6) and human cytomegalovirus (HCMV) infections were viral isolation from blood leukocytes (PBL) using human fibroblasts as the viral target and detection of viral DNA in serum or plasma by nested PCR. Active Epstein Barr virus (EBV) infections were detected by means of an approved serologic assay targeting the viral EBNA1 protein. Active HHV-6 infections were detected in 28% (54/195) of the patients, active HCMV infections were found in 29% (71/249), and active EBV infections were present in 52% (80/153) of the patients. Interestingly, dual positivity for active HHV-6 and active HCMV infections was significantly (p<0.025) observed. Additional analyses revealed that active EBV infections significantly (p<0.045) correlated with the presence of autoantibodies in the patients’ plasmas with antibodies directed at thyroid peroxidase (TPO) and parietal cells being the most common. Also, active HCMV infections independently correlated (p<0.013) with anti-TPO autoantibodies, and EBNA1 antibody titers were significantly (p<0.05) higher in patients with either active HHV-6 or HCMV infections. These studies identified a subpopulation of individuals with chronic fatiguing illness whose disease is associated with active infection with one or more leukotropic herpesviruses and raised the possibility that these viruses may predispose to the development of various autoimmune diseases. Possible pathogenic mechanisms involved in the disease development will be discussed.

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8-05Treatment of Uveitis and blindness associated with HHV-6 infection by Valganciclovir (Valcyte) and Immunoprop.

D. Enlander1([email protected]), S. Chang2

1ME /CFS Clinic of New York, New York, NY, USA; 2Opthalmology Department, Columbia University Medical Center, New York, NY USAIntroduction: Uveitis can be caused by several infections and HHV-6 has been reported to be associated with uveitis on rare occasions. Methods: A fifty nine year old female who with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS ) was diagnosed with viral uveitis and clinical blindness. She was found to have elevated antibody titers against serology was 1:1280 (Focus Diagnostics). After topical ophthalmic medicines failed to resolve the uveitis, the patient received a course of valganciclovir (Valcyte) an oral antiviral and ImmunoPlus, a non-prescription supplement containing trace minerals and vitamins reported to act on the meythylation cycle and support immune response.Antiviral therapy consisted of 1800 mg per day valganciclovir (Valcyte) for 3 weeks followed by 900 mg per day for six months. The immune stimulants were Immunoprop and ImmunoPlus (Immunoprop Pharma Inc) containing trace minerals and vitamins reported to act on the meythylation cycle and support immune response, each given the the patient once daily.Results: The patient’s serum HHV6 IgG decreased fourfold from 1:1280 to 1:80 . The ME/CFS condition also improved as seen by the Karnofsky score increasing 20%. The patient recovered from her blindness and was determined to have 20/20 vision. The leukocytes in the optic fluid resolved with treatment.Conclusion: Antiviral therapy could be helpful for a subset of patients with active HHV-6 infections.

Disclosured: Immunoprop Inc supported the study.

8-06Presence and Activity of Human Herpesvirus-6 and 7 Infections in Patients with Autoimmune Thyroid Diseases

Z. Nora-Krukle1([email protected]), A. Sultanova1, S. Gravelsina1, S. Chapenko1, M. Chistyakov1, E. Cunskis2, M. Murovska1

1RSU A.Kirchenstein Institute of Microbiology and Virology, Riga, Latvia; 2Riga Eastern Hospital, Clinic “Linezers”, LatviaIntroduction: HHV-6 and HHV-7 are widespread lymphotropic, immunosuppressive and immunomodulating beta-herpesviruses. Reactivation of these viruses may lead to the development of different pathologies. Viral infections are often considered major environmental factors related to etiopathogenesis of sub-acute thyroiditis and autoimmune thyroid diseases. Aim: To evaluate latent/persistent and active HHV-6 and HHV-7 infection in patients with autoimmune thyroid diseases.Methods: The study included 18 patients with autoimmune thyroiditis after surgery. Presence of HHV-6 and HHV-7 genomic sequences were detected by nPCR using DNA isolated from peripheral blood leukocytes (PBL), thyroid gland tissues, and cell free blood plasma as a template (markers of latent/persistent and active infection, respectively). HHV-6 viral load was detected using Real time PCR, HHV-6 variants – nPCR with variant-specific primers.Results: Single latent/persistent HHV-7 infection was found in 6 out of 18 and simultaneous (HHV-6, HHV-7) infection - in 9 out of 18 patients. Analyzing thyroid tissue samples 7 out of 9 patients with previously detected viral sequences in PBL were positive for HHV-6 and 8 out of 15 – for HHV-7, but in 7 cases HHV-6 and in 2 cases HHV-7 sequences were found in tissue DNA samples only. In 6 cases double infection was detected in tissue samples. No simultaneous activation of viruses was found. Single HHV-6 activation was detected in 1out of 9 and single HHV-7 activation in 9 out of 15 patients. In all samples HHV-6 viral load was higher in DNA isolated from thyroid gland tissues than from PBL. In all cases HHV-6 B variant was detected. Conclusion: The presence of HHV-6 and HHV-7 genomic sequences in DNA isolated from thyroid tissues of patients with autoimmune thyroiditis is common. To clarify the role of these viruses in the etiopathogenesis of the disease the number of examined patients should be increased and compared with non autoimmune thyroid gland diseases.

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8-07HHV-6 Etiopathogenetic Role in the Development of Cervical Cancer by Combining Immunohistochemistry, Laser Microdissection and Single Cell-Qpcr

F. Broccolo1([email protected])1Università Milano-Bicocca, Monza, Italy

The long latent period (20 years) between initial HPV infection and emergence of Cervical Carcinoma (CC) suggest that HPV alone is not sufficient for malignant transformation and additional factors might be required for the progression of HPV infected cells to a neoplastic phenotype.

To evaluate whether HHV-6 genital infections, besides those caused by oncogenic HPV genotypes, could be implicated in the development of cancerous lesions and/or CC, archival paraffin-embedded tissue sections (precancerous lesions) and biopsies negative for Cervical Intraephitelial Neoplasia (CIN) were analyzed for HHV-6. First, a morphological evaluation of specimens using ematossilin-eosin staining was carried out; successively, the cynlindrical cores were taken from the macrobiospsy for the Tissue Microarrays technology (TMA) utilized to analyze simultaneously more tissue samples taken from specific areas of interest from paraffin-embedded tissue blocks on one slide. IHC for HHV-6 early, intermiedate early and late antigenes were performed by TMA. Subsequently, single or small groups of HHV-6 positive cells in the cylindrical core were isolated by laser microdissection; the presence of HHV-6 A /B variants was confirmed by an ultrasensitive HHV-6A/B specific Real-time PCR assay. In particular, we observed the expression of HHV-6 early, intermediate early and late antigens in muciparous glands cells of biopsies positive for CIN; the presence of HHV-6 in the muciparous glands cells was confirmed by experiments of laser microdissection combinated to single cell-real-time PCR analysis for HHV-6 DNA.

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Oral 9-01 Drug Reaction with Eosinophilia and Systemic Symptoms: a Multiorgan Antiviral T cell Response

V. Descamps1([email protected])1Bichat Claude Bernard Hospital, Paris, France

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, drug-induced reaction that involves both the skin and the viscera. Evidence for reactivation of herpes family viruses has been seen in some DRESS patients. To understand the immunological components of DRESS and their relationship to viral reactivation, we prospectively assessed 40 patients exhibiting DRESS in response to carbamazepine, allopurinol, or sulfamethoxazole. Peripheral blood T lymphocytes from the patients were evaluated for phenotype, cytokine secretion, and repertoire of CD4 and CD8 and for viral reactivation. We found Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), or HHV-7 reactivation in 76% of the patients. In all patients, circulating CD8 T lymphocytes were activated, exhibited increased cutaneous homing markers, and secreted large amounts of tumor necrosis factor-alpha and interferon-gamma. The production of these cytokines was particularly high in patients with the most severe visceral involvement. In addition, expanded populations of CD8 T lymphocytes sharing the same T cell receptor repertoire were detected in the blood, skin, liver, and lungs of patients. Nearly half of these expanded blood CD8 T lymphocytes specifically recognized one of several EBV epitopes. Finally, we found that the culprit drugs triggered the production of EBV in patients’ EBV-transformed B lymphocytes. Thus, cutaneous and visceral symptoms of DRESS are mediated by activated CD8 T lymphocytes, which are largely directed against herpes viruses such as EBV.

Oral 9-02Can HHV-6 Protect Children from Allergic Disease?

K. Eriksson1([email protected])1University of Gothenburg, Göteborg, Sweden

Epidemiological studies points to an inverse relationship between microbial exposure and the prevalence of allergic diseases. The underlying mechanism for this observation remains largely unknown, as does the nature of the microbes involved. The objective of this study was to investigate if HHV-6 could affect Th2 immune responses and the development of allergic disease. This was done ex situ in blood from young children, in vivo in a mouse model of allergic asthma and in vitro in cord blood mononuclear cells. In 18-month-old children we assessed the correlation between the presence of IgE to common allergens and seropositivity to HHV-6. In mice we investigated the impact of HHV-6 exposure on the development of experimentally induced allergic asthma. In cord blood from new-born children we tried to identify the underlying mechanism for HHV-6s ability to interfere with Th2 development.We found that children seropositive at 18 months of age to HHV-6 were significantly less often IgE sensitized than seronegative children (OR: 0.08, 95% CI: 0.009-0.68), which implies that HHV-6 could protect against allergic sensitization. This was confirmed in mice where animals exposed to HHV-6 at the time of allergen sensitization had significantly lower frequencies of allergen-specific IgE, significantly decreased levels of Th2 cytokines and significantly reduced numbers of inflammatory cells (including eosinophils) in their bronchoalveolar lavage fluid following airway allergen challenge. In vitro, we found that HHV-6 induced strong TLR9-mediated IFN-alpha and IFN-lambda responses in plasmacytoid dendritic cells, and simultaneously decreased the production of Th2-associated cytokines in responding T-cells. However, IFN-alpha and IFN-lambda were not mediating the HHV-6-induced interference with Th2 responses. The data indicates that an early childhood infection with HHV-6 could down-regulate Th2 responses and reduce allergen-specific IgE formation in the young child and, thus, perhaps prevent the establishment of allergic disease.

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9-03 Oral Identification of HHV6B-specific CD8+ T cells

A. Schub1, L. Martin1, S. Dillinger1, A. Moosmann1([email protected])1University of Munich / Helmholtz Center Munich, Munich, Germany

For several members of the human herpesvirus family, it is known that they are under control of antigen-specific T cells, with CD8+ cytotoxic T cells playing a prominent role. For HHV-6, however, little is known on antigen-specific T cell responses. In order to identify HHV-6B-specific CD8+ T cells, we screened the amino acid sequences of three HHV-6B proteins, U11, U54, and U90, for subsequences that might represent potential CD8+ T cell targets. We chose 30 peptides with conserved anchor residues predicting their binding to frequent HLA class I allotypes, including 12 peptides potentially presented by HLA-A*0201. Peptide-specific T cells could not directly be detected in peripheral blood of healthy HHV-6 carriers, but they could be efficiently enriched by in vitro stimulation with peptide-loaded autologous B cell lines, resulting in CD8+ T-cell lines and clones specific for three peptides from U11 and two peptides from U54. CD8+ T cells specific for U11 peptides did not recognize cells expressing U11 antigen. In contrast, CD8+ T cells specific for each of the two U54 peptides recognized epithelial cells expressing the U54 protein and CD4+ T-cell cultures infected with HHV-6B. Recognition resulted in HLA-A*0201-restricted secretion of IFN-g and specific killing of infected target cells. Thus, we identified U54 as an HHV-6 antigen that is presented by infected cells and recognized by antigen-specific T cells that are present in the blood of healthy carriers. U54-specific CD8+ T cells may contribute to control of HHV-6 infection in vivo and may be useful for immunotherapy of HHV-6-associated disease.

9-04 Oral Identification of HHV-6 Epitopes Targeted by CD4+ T Cell Responses in Healthy Donors

J.M. Calvo-Calle1([email protected]), M.D. Nastke1, A.R. Becerra-Artiles1, L. Yin1, O.A. Dominguez-Amorocho1, L. Gibson2, L.J. Stern1

1University of Massachusetts, Worcester, MA, USA; 2University of Massachusetts, Department of Pediatrics, USAVery little is known about the nature and the fine specificity of the T cell response to HHV-6. Cross-reactive T cell

responses with other herpesviruses preclude the use of the whole virus to study HHV-6-specifc T cell responses. A comprehensive analysis of T cell responses would require evaluation of over 40 thousand potential HHV-6 epitopes We approached the characterization of HHV-6 T-cell responses using an algorithm that predicts binding to MHC class II DRB1*0101 molecules. Two sets of peptides were synthesized: The first set included 112 peptides from the entire translated HHV-6 genome and a second set included 220 peptides from proteins found in a proteomics analysis of the HHV-6 virions. Screening of synthetic peptides was performed using as a readout IFN-gamma since HHV-6 induces a robust response by this cytokine in PBMCs and T cell lines, although analysis of T cell lines suggests that IL-10 and IL-4 also could be used. Eleven class II MHC-restricted T-cell epitopes were identified, mainly from abundant virion components found by proteomics analysis. The overall frequency of peptide-specific CD4+ T cells is rather low, but it could be easily expanded by single in vitro expansion with synthetic peptides. A significant fraction of the IFN-gamma producing cells also express the cytotoxic marker CD107a/b. IL-10 producing cells were also observed in PBMCs stimulated with some peptides. To follow HHV-6 antigen-specific T cell responses we constructed MHC tetramers for the eleven epitopes. Staining of PBMCs from healthy donors was observed after enrichment of tetramer positive cells. It is expected that these reagents will be of great use in patients with active virus replication.

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9-05A Study of Chromosomally Integrated HHV-6 in Severe Cutaneous Adverse Drug Reactions

W. Chung1([email protected])1Department of Dermatology, Chang Gung Memorial Hospital, Taiwan

Adverse drug reactions are frequent clinical problems and most often affect the skin which may range from mild rash to severe fatal hypersensitivity reactions. Among them, Stevens-Johnson syndrome/toxic epidermal necrosis (SJS/TEN) and drug induced hypersensitivity syndrome (DIHS) or drug rash with eosinophilia and systemic symptoms (DRESS) are of the most life-threatening severe cutaneous adverse reactions (SCARs), which carry 10-40% mortality rate. The MHC-restricted presentation of a drug or its metabolites for T-cell activation is now supported by our recent findings of strong genetic association between HLA-B alleles (e.g. HLA-B*1502 to carbamazepne-SJS/TEN and HLA-B*5801 to allopurinol, DIHS/SJS/TEN). In addition to genetic factors, there are still many other environmental factors are suspected to be involved in the pathogenesis of SCARs; especially HHV6-reactivation in DIHS/DRESS.

To understand whether persons with chromosomally integrated HHV-6 (CIHHV-6) are at greater risk to develop SCARs after taking certain drugs that may activate HHV-6, we conducted a CiHHV-6 study by using the quantitative PCR to detect HHV-6 DNA load on DNA isolated from PBMC of patients with SCARS at disease active or recovery stage. Different drugs (e.g. carbamazepine, phenytoin, allopurinol) induced SCARS are included in this study and are compared to healthy or drugs tolerant controls. Further study of the predisposing factors for SCARs is recommended

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Oral 10-01Diagnosis of HHV-6 Reactivation in Transplant Recipients; Critical Point to Determine Precise Role of the Virus in Causing Disease

T. Yoshikawa1([email protected])1Fujita Health University, Toyoake, Japan

Monitoring of active human herpesvirus 6 (HHV-6) infection is important for distinguishing between reactivation and latency of virus. We have reported that HHV-6 could be involved in fever and skin rash or acute graft versus host disease in hematopoietic stem cell transplant (HSCT) recipients on the basis of virological analysis such as real-time PCR and virus isolation. Additionally, an association between HHV-6 and encephalitis is evident in transplant recipients. Measurement of HHV-6 DNA copy numbers using real-time PCR is a popular method for monitoring HHV-6 infection after transplant. However, according to the result of DNA real-time PCR in our institute, relatively high level of HHV-6 DNA was continuously detected in peripheral blood mononuclear cells (PBMCs) after viral isolation in several recipients. Furthermore, it has been suggested that chromosomally integrated (CI) HHV-6 may cause miss diagnosis of HHV-6 infection in transplant recipients. Therefore, we tried to develop real-time reverse transcription polymerase chain reaction (real-time RT PCR) for diagnosis of active viral infection. The primers and probes were designed for detecting three different classes of HHV-6 mRNAs (U90, U12, U100). Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1 ng/reaction to 100 ng/reaction) extracted from HHV-6 infected cord blood mononuclear cells and copy numbers of each gene transcript. According to the experiment using clinical samples obtained from HSCT recipients, U90 real-time RT-PCR demonstrated the highest assay sensitivity and specificity. Moreover, although high copies of HHV-6 DNA was continuously detected in PBMCs serially collected from the recipient received graft from CI HHV-6 donor, no viral transcript was detected in those samples. In addition to the basic real-time PCR, monitoring of HHV-6 transcripts using real-time RT-PCR could be useful tool for determining precise clinical features caused by HHV-6 reactivation in immunocompromised patients.

10-02 Oral Breakthrough HHV-6B Infections During Antiviral Prophylaxis after Liver Transplantation

I. Lautenschlager1([email protected])1Helsinki University Hospital, Helsinki, Finland

Human herpesvirus-6 (HHV-6) activation, mostly of HHV-6B, is common after liver transplantation. Most HHV-6 reactivations are asymptomatic, but encephalitis, hepatitis or graft dysfunction have been described. Clinical experience on antiviral therapy is very limited. Based on in vitro studies, the drugs effective against cytomegalovirus (CMV), have also activity against HHV-6. We investigated the efficiency of valganciclovir prophylaxis, given to the CMV-seronegative risk patients receiving a liver graft from the seropositive donor, in preventing HHV-6 reactivation.Of 196 consecutive adult liver transplant patients 32 belonging to the CMV high risk group received antiviral prophylaxis (valganciclovir 900 mg daily and/or i.v. ganciclovir 5mg/kg/d for induction) up to 3 months after transplantation. The patients were frequently monitored for CMV by real-time quantitative PCR and HHV-6 reactivations were demonstrated by the antigenemia test in peripheral blood mononuclear cells (PBMC) using immunoperoxidase staining and monoclonal antibodies against against HHV-6A and HHV-6B (Argene) and HHV-6B (variant B late antigen, Chemicon). Intragraft HHV-6 infection was demonstrated in liver biopsy frozen sections by the same antibodies and immunostaining.During antiviral prophylaxis, no break-through CMV infections were recorded. On the contrary, HHV-6 antigenemia was detected in 12/32 (37%) patients appearing mean 12 days (range 7-22 days) after transplantation. All reactivations were caused by HHV-6B. In three cases HHV-6 antigens were also detected in the liver biopsy obtained due to graft dysfunction. In two cases intragraft HHV-6 was associated with allograft rejection. Biopsies of other 6 patients, obtained during antigenemia, were negative for the viral antigens. No other clinical signs were recorded. HHV-6 antigenemia usually lasted 2-4 weeks and subsided without any additional therapy. In conclusion, HHV-6B reactivations were common during valganciclovir prophylaxis after liver transplantation. At least in three cases also the transplant was infected. Valganciclovir (ganciclovir) prophylaxis did not prevent HHV-6B infections in adult liver recipients.

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10-03 Oral Which Tools to Diagnose and Monitor HHV-6 Infections: From Theory to Practice

A. Gautheret-Dejean1([email protected])1Laboratoire de Virologie, Hôpital Pitié-Salpêtrière, Paris, France

Until recently, pathophysiology of HHV-6 infections was considered as classic: primary infection occuring during the first year of life asymptomatically or as exanthema subitum, asymptomatic latency, and reactivations sometimes associated to severe pathologies in immunocompromised patients. By homology to cytomegalovirus, we consider HHV-6 associated with a disease when it multiplies actively. Since some of these disorders can lead to death of patients, diagnosis of active infection is essential to start antiviral treatment with ganciclovir, cidofovir or foscarnet, and monitor its effectiveness.Different markers are available and involve direct or indirect viral diagnosis. Due to a prevalence above 90% in adults, serology is not contributory. Detection of viral antigens in tissues is of major interest but these methods are cumbersome and difficult to integrate into the laboratory routine. The measure of viral load (VL) in different biological compartments has supplanted the other methods. So far, diagnosis of active infection is based on a high viral load in blood and / or a biological fluid. However, the existence of chromosomal integration of HHV-6 genome in human chromosomes without any pathology interferes with interpretation.With the help of different clinical cases, we propose a classification of HHV-6 infections according to VL level and its kinetics during follow-up. Expression as genomic equivalent copies per million cells (CopEq/M) overcomes variations of differential leucokyte count. In blood:1- 25VL10,000 CopEq/M : usual infection (asymptomatic subject, non CIHHV-6)2- 10,000VL1,000,000 CopEq/M : active infection3- VL1,000,000 CopEq/M : 1- reduction under anti-HHV-6 drug, variation on consecutive samples: active infection. 2- Stability under anti-HHV-6 drug and on consecutive samples, identical VL in hair follicles: CI HHV-6In cerebrospinal fluid: detection is not enough. 1- VL CSF(CopEq/M) VL in whole blood (CopEq/M) with symptoms: probable encephalitis. 2- VL CSF(CopEq/M) = VL in whole blood (CopEq/M), values 1,000,000 CopEq/M CI HHV-6

Oral 10-04Do Latent HHV-6 and HHV-7 Infection Impact on Successful Aging?

S. Govind1([email protected]), W. Mitchell1, R. Aspinall11Cranfield University, Milton Keynes, United Kingdom (Great Britain)

Chronic Human herpes 6 infection in immune compromised individuals has been well documented, and has also been linked with a number of disorders including mononucleosis, multiple sclerosis and encephalitis. However the effects of latent asymptomatic HHV-6A, HHV-6B and HHV-7 viral infections in the ageing population are poorly understood. As we age immune efficacy is compromised, not only due to thymic involution, but is also exacerbated by changes in immune response efficacy, culminating in what is commonly known as immunosenescence.Our study is part of FP7 MARK-AGE project aimed at identifying age related biomarkers. CMV/HHV-5 infection has been implicated in immunosenescence and poor vaccine responsiveness in the elderly. However the impact of persistent HHV-6 and HHV-7 infection on the immunological profile remains to be defined. In the current study, we have designed a non-invasive quantitative PCR strategy to measure HHV-6A, HHV-6B and HHV-7 viral load from urine samples, thus potentially providing a rapid diagnostic tool to determine an individual’s viral status. Furthermore, combining results obtained from single joint T-cell rearrangement excision circles (sjTRECs) quantification, a by-product of TCR gene rearrangement, with our HHV-6 and HHV-7 viral load data, we describe a novel quantifiable biomarker of successful aging. Collectively these data aim to address the effects of latent HHV-6 and HHV-7 infection on biological aging. We envisage that this large-scale population study spanning across Europe encompassing the general population, including individuals with a familial longevity bias, will provide a unique insight into the immunological impact of these emerging members of the herpes family, which may be of particular interest in the care of an ever-growing elderly population.

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10-05 Oral Chromosomally Integrated HHV-6: Q & A for Physicians

P. Pellett1([email protected]), D. Ablashi2, H. Agut3, M. Caserta4, V. Descamps5, L. Flamand6, A. Gautheret-Dejean7, C. Hall4, U. Kuhl8, D. Lassner9, I. Lautenschlager10, K. Loomis2, M. Luppi11, P. Medveczky12, J. Montoya13, Y. Mori14, M. Ogata15, R. Razonable16, E. Seto17, T. Yoshikawa18

1Wayne State University, MI, USA; 2HHV-6 Foundation, USA; 3Groupe Hospitalier Pitié-Salpêtrière, France; 4University of Rochester School of Medicine, USA; 5Bichat Claude Bernard Hospital, France; 6Université Laval, Canada; 7Groupe Hospitalier Pitie-Salpetriere, France; 8Charite University Berlin, Germany; 9Institute for Cardiac Diagnosis & Treatment, Germany; 10Helsinki University Hospital, Finland; 11University of Modena and Reggio Emilia, Italy; 12University of South Florida, USA; 13Stanford University, USA; 14Kobe University Graduate School of Medicine; 15Hematology, Blood Transfusion Center, Oita University, Japan; 16Infectious Disease Department, Mayo Clinic, USA; 17Moffitt Cancer Center & Research, USA; 18Fujita Health University, Japan

As part of an effort to disseminate information about chromosomally integrated HHV-6 (ciHHV-6), an international collaborative has assembled a set of Questions and Answers relating to the definition of ciHHV-6, methods for its detection, interpretation of diagnostic assays in the context of ciHHV-6, and management of patients with ciHHV-6. In addition, the document provides background information related to the possibility that drugs used to treat diseases unrelated to HHV-6 might activate the virus from latency. Given the many gaps in our understanding of the biology of ciHHV-6, and the limited information available related to management of patients with ciHHV-6, the document also includes a list of areas where research is needed. The document will be summarized and will be open for discussion.

10-06 Analysis of Viral Load and Transcripts in Blood Leukocyte Subpopulations from Patients with Different Types of HHV-6 Infection

A. Milovanovitch6, S. Nguyen1, C. Bigaillon2, P. Bonnafous6, N. Dhédin3, M. Stern4, V. Descamps5, H. Agut6, A. Gautheret-Dejean6([email protected])1Service d’Hématologie Clinique, Groupe Hospitalier Pitié-Salpêtrière, France; 2Laboratoire de Biologie Médicale, HIA Bégin, Saint Mandé, France, France; 3Service d’Hématologie Clinique, GH Pitié-Salpêtrière, Paris, France, France; 4Service de Pneumologie, Hôpital Foch, Suresnes, France; 5Service de Dermatologie, Hôpital Bichat-Claude Bernard, Paris, France; 6Service de Virologie, ER1 DETIV, Groupe Hospitalier Pitié-Salpêtrière, Paris, FranceObjectives: Human herpesvirus-6 (HHV-6) is the cause of active, latent or chromosomally integrated infections. Viral load in whole blood constitutes a usefull tool to diagnose and follow HHV-6 infections. The aim of this study was to analyze viral load and transcripts in blood leukocyte subpopulations from patients with different types of HHV-6 infection.Methods: Six patients with HHV-6 infection were seleted. Four patients were stem cell transplant recipients, one patient was lung recipient and the last had a DRESS syndrome. From whole blood, 5 subpopulations of leukocytes were sorted using Miltenyi columns ® with antibodies (polymorphonuclear cells [CD15], monocytes [CD14], T lymphocytes [CD3], B lymphocytes [CD19] and NK cells [CD56]). Viral load (VL), expressed as the number of viral genomic copies per million of cells (cop/M), was determined all fractions and whole blood. Presence of immediate early (U42) and late (U100) transcripts was analyzed in leukocytes fractions HHV-6 positive. Infection was highly active for one patient (VL 217332 cop/M), at low levels for four patients (1899<VL<9018 cop/M) and chromosomally integrated (CIHHV-6) for one (2,2 x 10^6 cop /M).Results: For all patients, CD3 + fraction has the highest VL. Fractions CD14, CD15, CD19 and CD56 are rarely positive, except for highly active infection and CIHHV-6. Transcripts U42 and U100 were detected during highly active infection in CD3+ and CD56+ fractions. For low levels of infection, only U100 transcript was detected in the CD3+ fraction for one patient. Conclusion: Whatever the level of viral infection, CD3+ fraction constitutes the main target of HHV-6. The virus is present in CD15 fraction when the level of infection is important. U100 and U42 transcripts are detectable only in fractions CD3 + and CD56 + in case of highly active infection, and exceptionally in CD3 + fraction at low level of infection.

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10-07HHV-6 and HHV-7 Infection in the Patients with Autologous Peripheral Blood Stem Cell Transplantation

S. Chapenko2, I. Trocjukas1, S. Gravelsina2, M. Chistyakov2, A. Sultanova2, S. Donina2, Z. Nora-Krukle2, S. Lejniece1, M. Murovska2([email protected])1Riga Eastern Hospital, the National Centre of Haematology, Latvia; 2RSU A.Kirchenstein Institute of Microbiology and Virology, Riga, LatviaIntroduction: HHV-6 and HHV-7 are “lymphotropic” immunomodulating beta-herpesviruses that may reactivate from latency under immunosuppression. Relationship between these viruses reactivation and complication development after autologous peripheral blood stem cell transplantation (auto-PBSCT) is unequally evaluated. Objective:. Evaluate the frequency of HHV-6 and HHV-7 reactivation and development of post-transplant complications as well as ascertain potential interactions between these viruses.Methods: 44 patients before and after auto-PBSCT were studied on HHV-6 and HHV-7 infection. Anti-HHV-6 specific antibodies were detected by ELISA, latent/persistent and active viral infection - by qualitative (nPCR) and quantitative (qPCR). Endonuclease restriction analysis was used to detect HHV-6 variants. ELISA and solid-phase competitive chemiluminiscent enzyme immunoassay were used to detect TNF-alpha, IL-1beta, IL-6, sIL-2R expression level in serum/plasma.Results: Specific anti-HHV-6 antibodies were revealed in 81.8% serum/plasma samples. The frequency of plasma viremia (PV) was significantly higher in patients after transplantation than before transplantation (18/38, 47.4%; 5/38, 13.2%). Simultaneous HHV-6 and HHV-7 PV was prevalent after transplantation (10/18, 55.6%; 1/5, 20.0%) and HHV-7 PV preceded HHV-6 PV. A significant difference was found between HHV-6 loads in PBL DNS in cases of latent/persistent infection and PV (6780.8±3910 vs. 54675.5±9141.2 copies/µg DNA). In all cases HHV-6B variant was identified in PBL and plasma samples. Significantly higher serum levels of TNF-alpha, IL-6, IL-1β and sIL-2R were found in cases of active viral infection. The complications after transplantation developed in 12/44 (27.3%) patients and the viruses reactivation was detected in all of them. Conclusion: High frequency of simultaneous HHV-6 and HHV-7 plasma viremia and simultaneous increase of pro-inflammatory cytokines’ serum levels suggest that both viruses may be involved in the complication development after auto-PBSCT via their immunomodulatory ability. The kinetics of the viruses’ reactivation may reflect the potential role of HHV-7 as co-factor of HHV-6 reactivation.

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10-08HHV-6-DNAemia after Liver Transplantation Monitored by Two Quantitative Real-Time PCR Methods

T. Karlsson1([email protected]), R. Loginov1, L. Mannonen1, K. Höckerstedt2, I. Lautenschlager1

1Department of Virology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland; 2Department of Surgery, Helsinki University Hospital, University of Helsinki , Finland

HHV-6 is a ubiquitous virus with a high seroprevalence (>95%). Like other herpesviruses HHV-6 can reactivate under immunosuppression. In liver transplantation, most HHV-6 reactivations are asymptomatic, but symptoms such as encephalitis, hepatitis or graft dysfunction have also been described. The aim of the study was to compare two quantitative HHV-6 PCR methods and correlate with HHV-6 antigenemia in liver transplant patients. Thirty-six adult liver allograft recipients were frequently monitored for HHV-6 during the first 6 months after transplantation. HHV-6 reactivations were diagnosed by the antigenemia test in PBMC using indirect immunoperoxidase staining and monoclonal antibodies against HHV-6. Altogether, 223 whole blood specimens (mean 6 samples/patient) were analyzed. HHV-6 DNA was tested by using automated sample preparation system MagNa Pure LC and quantitative Taqman-based “in-house” real-time PCR and commercial quantitative Argene CMV, HHV6, 7, 8 R-geneTM kit. Argene´s test amplifies a sequence of viral U57 gene and the “in-house” assay amplifies a sequence of viral U67 gene, detecting both HHV-6A and HHV-6B variants. HHV-6-antigenemia was recorded in 21/36 (58%) patients and HHV-6 DNAemia in 18/36 (50%). HHV-6-DNAemia usually occurred during the first weeks after transplantation. The viral loads by the “in-house” Taqman test varied between 280-19700 copies/ml (median 1200) and by Argene’s test 120-24070 copies/ml (median 458). The correlation of viral loads between two quantitative tests was good (R= 0.94, p<0.01). HHV-6 reactivations were common after liver transplantation. The results of the both PCR methods correlated well with HHV-6 antigenemia, and the correlation between the two quantitative PCR assays was good.

10-09High Intrahepatic HHV-6 DNA Load is Associated with Decreased Graft Survival in Liver Transplant Recipients with Graft Hepatitis

S. Pischke1, J. Gösling3, I. Engelmann3, J. Schlue2, B. Wölk3, C. Schmitt3([email protected]), C. Strassburg4, H. Barg-Hock5, T. Becker6, M. Manns4, T. Schulz3, H. Wedemeyer4, A. Heim3

1Hannover Medical School, Department of Gastroenterology, Germany; 2Hannover Medical School, Institute of Pathology, Germany; 3Hannover Medical School, Institute of Virology, Hannover, Germany; 4Hannover Medical School, Department of Gastroenterology, USA; 5Hannover Medical School, Department of Abdominal Surgery, Germany; 6Hannover Medical School, Department of Abdominal Surgery, USA

While the impact of CMV and EBV infections on the outcome of liver transplant recipients is well established, the clinical relevance of HHV-6 in liver transplant recipients is not well defined. In this study, 170 liver transplant recipients with graft hepatitis were included. Viral loads of CMV, EBV and HHV-6 were determined in blood and liver biopsies by quantitative PCR. The median time of follow-up after liver biopsy was 23.8 months (range 0-56 months). HHV-6 DNA, CMV DNA and EBV DNA were detected in 58%, 14% and 44% of liver samples, respectively, with double and triple infections in 34%. High intrahepatic HHV-6 DNA levels (> 75th percentile of 11.27 copies/ 1000 cells) were associated with a significant lower graft survival (p=0.024). Multivariate analysis confirmed high intrahepatic HHV-6 load as an independent factor associated with graft loss (p=0.015, adjusted Odds ratio 4.37, 95% confidence interval 1.33-14.36). In contrast, detection of CMV and EBV in liver biopsies was not associated with graft loss. In HCV infected patients, negative effects of HHV-6 on graft survival seemed to be enhanced.In conclusion, our data indicated that intrahepatic HHV-6 reactivation contributed to graft hepatitis and graft survival because only high intrahepatic HHV-6-DNA levels were associated with graft loss.

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Oral 10-10Development and Validation of a Q-RT-PCR Based TCID50 Assay for HHV-6A

R.K. Gustafsson1([email protected]), E.E. Engdahl1, A. Fogdell-Hahn1

1Karolinska Institutet, Stockholm, SwedenThe standard 50% tissue culture infectivity dose (TCID50) assay for human herpesvirus 6A (HHV-6A), where

infection is assessed by ocular inspection for syncytia, is widely used at date. However, syncytia might be difficult to identify, especially at the border line of infection, and therefore it is hard to obtain unanimous results. We developed an alternative method based on quantitative real-time PCR (Q-RT-PCR) analysis. Ten thousand HSB-2 cells per well were inoculated with HHV-6A (GS strain) in five-fold dilution series and sextuplicate for each dilution step and incubated for seven days. Samples were taken from every well at 0 and at 7 days post infection (dpi) and subjected to DNA extraction before quantitative real-time PCR (Q-RT-PCR) analysis. Wells were considered infected if the viral DNA load increased with at least one log10 between day 0 and day 7. For comparisons the plates were read in parallel by ocular inspection for syncytia by two assessors. Cells were also mounted on glass slides and monitored by immunofluorescence assay (IFA) targeting the viral protein gp116. The titers where calculated with the formula of Reed and Muench. The procedure was performed three times for one virus batch. On average the TCID50-titers were 2.2 times higher when using Q-RT-PCR readout than when using ocular inspection. In addition, nearly all wells that were infected, as seen by Q-RT-PCR, contained infected cells as evident by IFA. The intra assay reproducibility revealed a CV of 38%, as compared to 65% for the ocular inspection. The infectivity was completely lost after UV- and partially lost after heat inactivation of the virus, as seen with Q-RT-PCR, ocular inspection and IFA. The new approach is fast and easier to interpret; and since the results relay on molecular quantification of all cells in the well it gives a more exact and reproducible value.

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11-01 Oral The Challenge of Measuring the Clinical Impact of Acute HHV-6 Infections

H. Agut1([email protected])1Groupe Hospitalier Pitié-Salpêtrière, Paris, France

HHV-6 was first described in 1986 and for about 20 years it has been known that some antiherpetic drugs are efficient against this virus, both in vitro and, as suggested in a limited number of patients, in vivo. Acute HHV-6 infections are related to an active multiplication of the virus and are logically candidates for such therapy. They consist of primary infections, reactivations and exogenous reinfections. They may be congenital, neonatal or occur later after birth in different contexts of immune competence. Up to date, no firm guideline has been proposed for the treatment of these acute HHV-6 infections and no therapeutic trial has been designed to answer this question. Two major obstacles currently seem to prevent significant progress in that field. The first one is the diagnosis of active HHV-6 multiplication. Important improvements have emerged from the use of quantitative PCR-based techniques applied to body fluids. However, some confounding factors have been reported to obscure the interpretation of results, in particular chromosomal HHV-6 DNA integration, and existence of two distinct albeit closely related variants, HHV-6A and HHV-6B, whose epidemiology and reciprocal interactions are largely unknown. Thus the search for additional markers is needed. The second obstacle is the difficulty of establishing a causative relationship between the proven replication of the virus and the diseases observed, due to the ubiquitous nature of HHV-6 and its capability of inducing asymptomatic reactivations. Exanthem subitum in infants and limbic encephalitis in immunocompromised subjects are prototypic diseases clearly associated with primary infection and reactivation respectively. However, such a clear causative role of the virus is not the common rule for many diverse diseases which have been putatively related to HHV-6. Several strategies will be discussed to circumvent these obstacles and promote significant improvements in the medical management of acute HHV-6 infections.

11-02 Oral Identification of Bicyclic Sulfone Inhibitors of HHV-6 Targeting The HHV-6 U77 Helicase

L. Naesens1([email protected]), G. Andrei1, R. Snoeck1, D. Gerry2, J.E. Banning2, P.D. Wilkerson2, P.M. Johnson2, H. Shah2, Z.M. Renew2, C.E. Stephens2

1Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium; 2Department of Chemistry and Physics, Augusta State University, GA, USA

We previously reported on the promising anti-HHV-6 activity of bicyclic sulfone derivatives [Naesens et al., Antiviral Res. 2006, 72:60]. We have now examined the structure-activity relationship of a series of newly synthesized structural analogues. Their anti-HHV-6-activity was determined in HHV-6A (GS)-infected HSB-2 cells and HHV-6B (Z29)-infected MOLT-3 human T-lymphoblast cells, using a microscopic CPE reduction assay and real-time PCR quantitation of viral DNA. Some of the novel compounds were superior to the original compound, both in antiviral activity and selectivity. This strong inhibitory effect on HHV-6 replication was confirmed in HHV-6-infected fresh human cord blood lymphocytes. In order to identify the antiviral target, a resistance study was performed in which HHV-6A (GS) was serially passed in the presence of increasing concentrations of one bicyclic sulfone compound. After eight virus passages, a mutant virus was obtained with strong resistance to the bicyclic sulfones (antiviral EC50 values > 300 µM), while its sensitivity to foscarnet and cidofovir was the same as that of control virus. DNA sequencing on the resistant virus revealed an isoleucine to methionine substitution at position 318 of the HHV-6 U77 helicase. Protein alignment showed that the Ile-318 residue in HHV-6 U77 is identical in both HHV-6 A and B variants and lies adjacent to motif IV, which is highly conserved among the herpesviruses and is required for DNA helicase activity. The I318M substitution selected by the bicyclic sulfones lies in an HHV-6 U77 region that aligns with a sequence of HSV-1 UL5 helicase that contains most resistance mutations to herpes simplex virus (HSV) helicase-primase inhibitors, several of which are in (pre)clinical testing. The finding that these bicyclic sulfones act as HHV-6 helicase inhibitors opens new perspectives for the development of HHV-6 specific antiviral compounds.

Supported by a grant from the HHV-6 Foundation

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Oral 11-03Adoptive Immunotherapy to Prevent and Treat HHV-6 Reactivation Post Allogenic Stem Cell Transplant

U. Gerdemann1([email protected]), L. Keukens2, U.L. Katari1, J.M. Keirnan1, A.P. de Pagter2, H.E. Heslop1, C.M. Rooney1, A.M. Leen1

1Baylor College of Medicine, Houston, TX, USA; 2Department of Pediatric Immunology and Hematology, Utrecht, NetherlandsViral infections cause morbidity and mortality in allogeneic HSCT recipients. As an effective treatment option, we

and others have successfully infused in vitro expanded cytotoxic T-cell lines (CTLs) directed against EBV, CMV and Adenovirus. HHV6B is also detected in >50% of HSCT recipients and associated with severe clinical symptoms and increased overall mortality. However production of HHV6B-directed CTLs has been hindered by lack of information regarding immunogenic/protective target antigens. To identify immunodominant T-cell targets we assessed the cellular immune response directed against 5 HHV6 antigens (U71, U90, U11, U14 and U54), whose counterparts in the significantly homologous CMV or HHV7 viruses were found to be immunogenic. For T-cell stimulation we isolated PBMCs from HHV6 sero+ve, CMV sero-ve donors (thus eliminating the possibility of cross-reactive T-cell recognition), pulsed them with peptide libraries spanning each antigen, and then expanded them in vitro for 10 days. The resulting lines were polyclonal with a predominance of CD4+ T-cells (mean 62+/-6%, n=15). Using IFN-γ ELIspot we identified a hierarchy of immunodominance, with all donors responding to U90 (median 216; range 53-812 SFC/1x105 cells), U14 (164; 43-666), U11 (143; 25-209) and U54 (138; 44-247) whereas only 8/15 donors responded to U71 (50; 25-80). T-cells cultured with HHV6B Z29 wild-type virus-infected PBMCs as a stimulus showed comparable frequencies of antigen specificity, confirming that our chosen antigens are relevant targets. The CTLs were polyfunctional, producing multiple cytokines (IFN-γ, TNF-α, IL-2) and effector molecules (Granzyme B) upon stimulation with cognate antigen, as evaluated by intracellular cytokine staining and ELIspot, and killed HHV6B Z29 wild-type virus-infected autologous monocytes in a co-culture assay. In summary we generated and characterized T-cell immunity to 5 HHV-6 antigens which can now be used to assess endogenous T-cell recovery in patients after HSCT and to generate HHV6B-specific CTL for adoptive transfer.

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11-04 OralChromosomally Integrated Human Herpesvirus 6 in Patients with Acquired Cardiomyopathies and Heart Failure Symptoms

D. Lassner1([email protected]), M. Rhode¹, U. Kühl², U. M. Gross², G. R. F. Krüger³, B. Seeberg², F. Escher², H.-P. Schultheiss²1Institute Cardiac Diagnostics und Therapy (IKDT), Berlin, Germany; ²Department of Cardiology and Pneumology, Campus Benjamin Franklin, Charité Campus Benjamin Franklin Berlin, Berlin, Germany; ³Department of Pathology, Univ. Cologne, Germany

Human herpesvirus 6 (HHV-6) is a lymphotropic virus with life-long persistence after childhood infection and primarily associated with non-cardiac diseases. Recent studies have identified HHV-6 as a possible pathogenic cause of myocarditis and idiopathic cardiomyopathy. Its prevalence, subtype involvement and clinical course are unknown. We evaluated the prevalence and course of a HHV-6 reactivation in 3610 baseline and 197 follow-up endomyocardial biopsies (EMB) of consecutive patients with clinically suspected acquired cardiomyopathies or persisting heart failure symptoms by nested-PCR and following sequencing of genome fragments. Systemic HHV-6 reactivation was analyzed by nested PCR in blood samples of patients with proven viral genomes in EMB tissues. We identified 548 patients (14.39%) with cardiac HHV-6 infection/reactivation, including 300 tissue samples with multiple viral infections (54.7%). Sequence analysis confirmed HHV-6 subtype B as the major pathogen (92.7%). Immunohistochemistry analysis of myocardial tissue revealed intramyocardial inflammation in 28.8% of all HHV6 positive cases. 24 of the HHV-6 positive patients (4.9%) presented systemic HHV-6 infection, cardiac involvement and persisting high virus loads of >5x104 virion copies/µg isolated DNA indicating possible genomic integration of viral DNA (CIHHV-6). Suspected chromosomal integration of HHV-6 was seen in 58.4% of patients with HHV-6 genotype A infection but only in 4.10% of patients with detectable HHV-6 genotype B. While persistent or denovo HHV-6 infection/reactivation was associated with progression of left ventricular dysfunction (p<0.001), LV-EF improved in association with resolved HHV-6 infection/reactivation. Three CIHHV-6 patients with persisting heart failure symptoms were treated for six months with 900 mg valganciclovir (Valcyte) following a two week intravenous induction phase with twice 5 mg/kg bodyweight Ganciclovir daily. After 4 weeks of treatment, all three patients reported a reduction of angina pectoris, dyspnea, fatigue and an improvement of physical capacity. Although significantly reduced in severity, frequency and duration, these symptoms did not resolve completely but re-appeared for 2 to 3 days in intervals of 4 to 6 weeks.

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Oral 11-05 CMX001, a Novel Antiviral for the Treatment of dsDNA Viral Diseases

W.P. Painter1([email protected]), J. Kurtzberg2, H. Mommeja-Marin1, V. Prasad2

1Chimerix, Durham, NC, USA; 2Duke University Medical Center, USACMX001 is a novel nucleotide analogue that is active against dsDNA viruses. CMX001 is absorbed after oral dosing,

and is delivered to target cells where cidofovir diphosphate, the active antiviral, is generated through phosphorylation by anabolic kinases. Compared to cidofovir, CMX001 has increased oral bioavailability and delivers higher intracellular levels of active drug, while peak plasma concentrations of cidofovir remain low. In cell culture assays, CMX001 is significantly more active than cidofovir against dsDNA viruses including human herpesviruses (HHV), adenoviruses (AdV), orthopoxviruses, polyomaviruses, and papillomaviruses. Against HHV-6, CMX001 is 50 times more active than CDV (EC50 of 0.004 micromolar for CMX001). Animal models of herpes encephalitis have demonstrated that active levels of drug can be reached in the central nervous system. In several patients treated with CMX001 for CNS infections, cidofovir has been detected in the cerebrospinal fluid. CMX001 is in clinical development for the prophylaxis of cytomegalovirus as well as the pre-emptive treatment of AdV in stem cell transplant patients. Also, an open-label study of the treatment of 12 dsDNA viral diseases is underway. CMX001 has been used for emergency treatment of dsDNA viral diseases in more than 140 patients with no reasonable alternative treatment options. In one 19-year-old patient with relapsed T-cell ALL who developed fevers and oxygen requirement 9-11 days status-post his second allogeneic (double unrelated umbilical cord blood) transplant, CMX001 was used to treat HHV-6. The patient had ongoing renal insufficiency from prior therapies, and was not yet engrafted. HHV-6 plasma levels by PCR were 16,000 copies/ml prior to starting therapy, and it became undetectable after approximately 2 weeks. During the same time period, ANC increased from 0.22 to 1.48; platelet count and creatinine remained unchanged at 21K to 28K and 1.2 mg/dl, respectively. CMX001 has promise as a broad spectrum antiviral therapy.

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11-06 OralThe Challenges of Treating HHV-6 CNS Infections

J.G. Montoya¹, L. Merrihew¹, A. Ruiz¹, J. Norris¹, T. Watt¹, Stanford University, Department of Infectious Diseases & Internal Medicine

Abstract not available at press time.

11-07 Treatment of HHV6 infection in ME/CFS with Valganciclovir (Valcyte) and Immunoprop/ Immunoplus

D. Enlander1([email protected])1ME /CFS Clinic of New York, New York, NY, USAIntroduction: Patients diagnosed with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) often have elevated antibodies to HHV-6 and EBV. One group reported that this subset of patients improved with antiviral therapy for six months (Kogelnik 2007). Method: Forty patients diagnosed with chronic fatigue syndrome as well as elevated antibodies to HHV-6 (>IgG 640) agreed to participate in a double blind study. Group A was treated with oral antiviral therapy and two non-prescription immune stimulants and Group B received the antiviral therapy and a placebo. Antiviral therapy consisted of 1800 mg per day valganciclovir (Valcyte) for 3 weeks followed by 900 mg per day for six months. The immune stimulants were Immunoprop and ImmunoPlus (Immunoprop Pharma Inc) containing trace minerals and vitamins reported to act on the meythylation cycle and support immune response.Results: In Group A, 78% of patients showed an improved on the Karnofsky Performance Status Scale of 20% or more. In group B, 65% of the patients who were treated with Valcyte and placebo showed clinical improvement on the Karnofsky scale by at least 10%. IgG titers in group A dropped from an mean of 960 before to a mean of 226 after treatment. Group B IgG titers dropped from a median of 832 to a median of 350. Conclusion: Antiviral therapy combined with immune stimulants may be beneficial to ME/CFS patients with elevated antibodies to HHV-6 and warrants further study.

Disclosure: Immunoprop Inc supported the study.

11-08Diversity of T Cell Responses to Human Herpesvirus 6B

L. Martin1([email protected]), A. Schub1, A. Moosmann1

1Helmholtz-Zentrum München, Munich, GermanyControl of human herpesviruses such as EBV or CMV requires the maintenance of a diverse repertoire of virus-

specific T cells. Like these viruses, human herpesvirus 6B remains latent in healthy carriers, but may reactivate and cause severe disease in immunosuppressed patients. Thus, HHV-6 infection and maintenance of latency may be under continuous T cell control. However, little is known about the HHV6-specific T cell response. Earlier we identified the tegument protein U54 as a target of HHV6-specific T cells. Therefore, we used a peptide library covering the complete U54 protein sequence in order to characterize the U54-specific T cell repertoire. As we found U54-specific T cells to be of low frequency in peripheral blood, we enriched them by repeated stimulation with peptide library-loaded autologous B cell lines. T cell lines with distinct U54-specific reactivity in an interferon-gamma ELISPOT assay were obtained from 10 out of 13 donors. For closer analysis, single-cell cloning was performed for T cell lines from two donors. Of 486 clones and subcultures tested, 114 cultures specifically reacted to individual U54 peptides by secretion of interferon-gamma. At least 9 different non-overlapping 11-15 amino acid subsequences spread throughout the U54 sequence were recognized by T cells which were mostly CD8+; one epitope was recognized by CD4+ T cell clones. HLA restrictions of CD8+ T cells included HLA-B*3502 and HLA-B*4002. Recognition of HHV6B-infected target cells was tested for one HLA-B*4002-restricted T cell clone. This clone responded to infected targets with high specificity and intensity. Taken together, a majority of healthy carriers harbour T cells specific for U54, which might thus be one of the dominant T cell antigens of HHV-6B. Because they may react strongly against infected target cells, U54-specific T cells might be of interest for immunotherapy.

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12-01 Oral HHV-6 Reactivation and its Effect on Delirium and Cognitive Functioning in Hematopoietic Cell Transplant Recipients

D. Zerr1([email protected])1Seattle Children’s Hospital/University of Washington, Seattle, WA, USA

Human herpesvirus 6 (HHV-6) is detected in the plasma of approximately 40% of patients undergoing hematopoietic cell transplantation (HCT) and sporadically causes encephalitis in this population. The effect of HHV-6 reactivation on central nervous system (CNS) function has not been fully characterized. A prospective study was performed to evaluate associations between HHV-6 reactivation and CNS dysfunction after allogeneic HCT. Patients were enrolled prior to HCT. Plasma samples were tested for HHV-6 at baseline and twice-weekly after transplantation until day 84. Delirium was assessed at baseline, 3-times weekly until day 56, and weekly days 56-84 using a validated neuropsychiatric instrument. Neurocognitive testing was performed at baseline and at approximately day 84. HHV-6 was detected in 35% of the 315 included patients. Patients with HHV-6 were more likely to develop delirium and demonstrate neurocognitive decline in the first 84 days post HCT. Cord blood and unrelated transplantation increased risk of HHV-6 reactivation. In summary, we demonstrated an independent and quantitative association between HHV-6 reactivation and CNS dysfunction after HCT. A randomized antiviral trial is warranted to definitively establish causality and to provide rational approaches to treatment and/or prevention of HHV-6.

12-02 Oral Elevated Levels of HHV-6 Antibodies in Individuals with Psychiatric Disorders

R. Yolken1([email protected]), F. Dickerson2

1Johns Hopkins School of Medicine, Baltimore, MD, USA; 2Sheppard Pratt Hospital, USAHuman psychiatric disorders such as schizophrenia and bipolar disorder are major causes of morbidity and mortality

in young adults worldwide. The etiology of these disorders remains unknown. Clinical, pathological, and epidemiological studies point to infections and inflammation as contributing factors to some cases, perhaps in conjunction with genetic susceptibilities. Previous studies have indicated that the levels of antibodies to HHV-6 can be elevated in some populations of individuals with schizophrenia (Niebuhr et al, Schizophrenia Bulletin,34:2007). We measured antibodies to HHV-6 in 1087 samples obtained from 827 individuals. The study groups consisted of 265 individuals with established schizophrenia, 125 individuals with established bipolar disorder, 137 individuals with the recent onset of psychosis, 58 individuals hospitalized for mania, and 242 controls without a psychiatric disorder. Multiple samples obtained over a period of time were available for many of the individuals. Antibodies were measured by ELISA using commercially available kits (Advanced Biotechnology, Columbia Md.). Elevated levels of antibodies in the case individuals were defined as ones which were >=75th and >=90th percentile of control samples run on the same ELISA plate. Statistical analyses were performed using multinomial logistic regression controlling for age, gender, race, and level of maternal education. We found that individuals with established schizophrenia had elevated levels of antibodies to HHV6 as defined at the 75th (Odds ratio 1.95, 95% CI 1.26-3.02., p<.003) and at the 90th percentile (Odds Ratio 2.66, 95% CI 1.54-4.60, p<.0003). We also found that individuals with mania had increased levels of antibodies to HHV6 defined at the 90th percentile (Odds ratio 2.44, 95% CI 1.18-5.07, p<.018 ) Increased levels of antibodies to HHV-6 were not found in the other case populations. We did not detect significantly increased levels of antibodies to other human herpesviruses including HSV-1, HSV-2,CMV, EBV, VZV in any of the affected populations. These studies indicate that significantly increased levels of antibodies to HHV-6 are found in some populations of individuals with psychiatric disorders. These antibodies are not simply a reflection of an increased immune response since increased levels of antibodies to other herpesviruses were not detected. Ongoing studies are directed at determining the relationship of HHV-6 infection and the etiology and clinical course of these disorders.

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Oral 12-03Different Characteristics of Human Herpesvirus 6 Encephalitis Between Primary Infection and Viral Reactivation

Y. Kawamura1([email protected])1Fujita Health University, Toyoake, JapanBackground: Pathogenesis of human herpesvirus 6 (HHV-6) encephalitis, in particular difference between HHV-6 encephalitis at the time of primary infection and reactivation remains unclear.Objectives: To elucidate the mechanism of HHV-6 encephalitis at the time of primary infection and reactivation.Study design: Twenty-two HHV-6 encephalitis patients at the time of primary infection, 6 febrile convulsion (FC) patients caused by HHV-6 infection, and 14 FC patients without HHV-6 infection (non HHV-6 FC) were enrolled. Additionally, 7 stem cell transplant recipients with HHV-6 encephalitis and eight adult controls were also enrolled in this study. Cerebrospinal fluid (CSF) HHV-6 DNA copy numbers and biomarkers levels were compared.Results: Low copy number of CSF HHV-6 DNA was detected in 7 of the 22 patients with HHV-6 encephalitis in primary infection, whereas all seven CSF samples collected from post-transplant HHV-6 encephalitis patients contained high viral DNA copy numbers (P<0.001). CSF concentrations of IL-6 (P=0.032), IL-8 (P=0.014), MMP-9 (P=0.004), and TIMP-1 (P=0.002) were significantly higher in patients with HHV-6 encephalitis in primary infection than non-HHV-6 FC. CSF IL-6 (P=0.008), IL-8 (P=0.015), and IL-10 (P=0.019) concentrations were significantly higher in patients with post-transplant HHV-6 encephalitis than adult controls.Conclusion: The present study suggests that the characteristics of HHV-6 encephalitis are different between HHV-6 encephalitis at the time of primary infection and reactivation in transplant recipients.

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12-05 Oral Plasma HHV-6 DNA Loads and IL-6 Concentration as Factors in the Development of HHV-6 Encephalitis after Allogeneic Stem Cell Transplantation

M. Ogata1([email protected])1Oita University Hospital, Yufu City, Oita, JapanBackground: Reactivation of human herpesvirus (HHV)-6 is common among stem cell transplant (SCT) recipients, and a minority of patients with HHV-6 reactivation develop HHV-6-associated complications. A high incidence of HHV-6 encephalitis (3.6-12.2%) has recently been reported from several major Japanese transplant units, but little is known about conditions associated with the development of HHV-6 encephalitis.Methods: HHV-6 DNA load and concentrations of 17 cytokines and chemokines were measured in serially collected plasma from SCT recipients.Results: Among 178 SCT recipients, 55.6% displayed positive results for HHV-6 DNA with 23.3% of these displaying high-level HHV-6 reactivation (i.e., more than 104 copies/ml plasma) by 70 days after SCT. HHV-6 encephalitis or possible HHV-6 encephalitis developed in 9 patients. No patients developed central nerve system (CNS) dysfunction concomitant with HHV-6 reactivation if the peak HHV-6 DNA was less than 104 copies/ml, but 9 of 37 patients (24%) with high-level HHV-6 reactivation developed CNS dysfunction concomitant with the time of peak HHV-6 DNA. Analysis of cytokine kinetics (n=61) revealed peak IL-6 concentration by day 28 was higher in patients who developed HHV-6 encephalitis than in patients without encephalitis (median, 1215 pg/ml vs. 77 pg/ml, respectively; P=0.0008). All patients who developed HHV-6 encephalitis showed an IL-6 surge (more than 100 pg/ml) 1 week before the appearance of high-level HHV-6 reactivation (more than 104 copies/ml). Conclusion: Both high-level HHV-6 reactivation and increased levels of IL-6 levels may be essential for the development of HHV-6 encephalitis after SCT. As an IL-6 surge was observed before the development of encephalitis, a high IL-6 concentration before engraftment may predict at greater risk of developing HHV-6 encephalitis.

12-06HHV-6 and EBV Infections in Multiple Sclerosis: an Analysis by Calibrated Ultrasensitive QPCR Assays

F. Broccolo1([email protected]), S. Matà2, T. Biagioli2, G. Cassina3, L. Fusetti1, B. Matteoli4, L. Ceccherini-Nelli4, P. Lusso5, M. Malnati31University of Milano-Bicocca, Milan, Italy; 2Department of Neurological and Psychiatric Sciences, AOU Careggi, Italy; 3San Raffaele Scientific Institute, Italy; 4University of Pisa, Italy; 5Laboratory of Immunoregulation NIAID, NIH Bethesda, USA

Multiple sclerosis (MS) is predominantly regarded as an autoimmune disease in which viral infections could represent a trigger in genetically susceptible individuals. Amongst herpesviruses both human herpesvirus-6 (HHV-6) and Epstein-Barr virus (EBV) have been associated with an increased risk of MS. To elucidate the role of HHV-6 and EBV in the development of MS we examined paired serum and cerebral spinal fluid (CSF) samples for HHV-6 and EBV DNA load from 20 multiple sclerosis (MS) patients with definite, relapsing/remitting MS clinically active within one month of the onset of clinical symptoms (acute MS). In addition, a control group of 17 patients with other neurological diseases (OND) was studied. All the samples were tested by a ultrasensitive calibrated QPCR assays combining the detection of a synthetic DNA calibrator and the viral DNA target in a multiplex PCR format. The lower detection limit for both assays was of 10 genome equivalents/mL; CSF viral DNA for all except 4 samples was extract from a starting volume ≥ 900 µl by phenol/chloroform method. Overall, 11/20 MS CSFs (55%) contained HHV-6 (7 samples; 35%) or EBV DNA (4 samples; 20%).. By contrast none of 17 OND CSF resulted positive for HHV-6 or EBV DNA. None of the MS samples was positive for both viruses at the same time. HHV-6 Serum viremia was measured in 8/20 patients with MS (40%) and in 4/17 (23.5%) of patients with OND. EBV Serum viremia was detected only in 4/17 (23.5%) patients with OND.Currently, we are recruiting more patients analyzing the presence of these viruses also in cell free CSF and in CSF derived cellular pellets to exclude the contamination of HHV-6/EBV latently infected cells .

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12-07Recognition of Human Herpesvirus 6 Rhombencephalitis in Immunocompetent Children: Clinical and Neuroimaging Features

B. Lavenstein1([email protected]), J. Crawford2

1Children’s National Medical Center, Washington, DC, USA; 2University of California, San Diego, USAObjective:To describe the recognition and clinical aspects,neuroimmunology,neuroimaging, and outcome in immunocompetent children 9 months to 7 years affected with HHV-6 encephalitis.Background: Human herpesvirus 6 (HHV-6) is associated with CNS disease that includes encepahlitis, multiple sclerosis, mesial temporal sclerosis. Variant A and B have potential degreees of neuro tropism. Encephalitis includes seizures, ataxia, opsoclonus and MRI findings localizing in the cerebellum, basal ganglia/thalamus and cerebral hemispheres. Diagnosis by CSF PCR analysis is confirmatory.Design/Methods: Seven children 9 months to 7 years were evaluated for new onset encephalitis. Presentation included seizures, ataxia opsoclonus myoclonus, fever, mental status changes. Evaluation included serum and CSF studies and MRI with gadolinium acutely and in follow up. Patients had evaluation with CSF PCR for HHV 6 dduring the acute phase of the illness. A comprehensive encephalitis panel was performed. CSF HHV-6 PCR was performed by real time PCR using U 77 helicase gene-specfic primers. Patients were seen in follow up at 3 months to two years.Results: Immunocompetent children with HHV-6 encephalitis developed rhombencephalitis evidenced by MRI. Brainstem, cerebellum, and thalami were sites of predilection. Diagnosis was confirmed by PCR in CSF and by serum antibodies with both acute and convalescent sera obtined. Brain biopsy detected HHV-6 specific glycoproteins on cerebellar biopsy by immunoflourescence and immunochemistry consistent with acute viral infcetion. Electroencephalography normalized subsequently. A lack of correlation with disease severity or outcome was noted. Patients with abnormal MRI findings had involvement in five or more distinct locations. CSF oligoclonal bands and myelin basic protein were negative.Summary: HHV-6 encephalitis in childhood presents with rhombencephalitis frequently. MRI scanning shows a predilection for brainstem, pons, mesencephalon, cerebellum, and thalami. In immunocompetent children devastating CNS brain stem encephalitis may emerge. Experience using the drug ganciclovir is limited at this time.

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13-01 Oral Human Herpesvirus 6A Genes Interaction in Multiple Sclerosis Patients

R. Alvarez-Lafuente1([email protected])1Hospital Clinico San Carlos, Madrid, SpainBackground: Multiple sclerosis (MS) is an inflammatory disorder affecting the central nervous system, in which both genetic and environmental factors interact. Among these environmental contributors, human herpesvirus 6A (HHV-6A) has been proposed as an important etiologic factor.Objective: To review previously published data of our group and the last results about a possible HHV-6A gene interaction in MS patients.Materials and Methods: Serum samples of MS patients were analyzed for the presence of HHV-6A with a quantitative real-time PCR along two-years of follow-up. Furthermore, DNA was extracted from blood samples for genetic purposes: single nucleotide polymorphisms (SNPs) of genes that have been previously related with MS, as MHC2TA, IRF5, and HVEM were analyzed.Results: The results were as follows: i) MHC2TA: An association of the rs4774C allele with the HHV-6A-positive group was found when compared with the HHV-6A-negative (47.7% vs 18.8%, p=0.0001; OR=3.94) and also with the control group (47.7% vs 25.5%, p=0.001, OR=2.67). Furthermore, among those MS patients with HHV-6A and minor allele C, a significant number of them were not free of progression 2 years after the diagnosis (P=0.01), and only a third of them responded to IFN-beta treatment (P = 0.05). ii) IRF5: The T allele of rs3807306 unraveled as a promising marker for both, infection with HHV-6A [p=0.05, OR (95% CI)= 1.56 (1.00- 2.44)] and response to IFN-beta therapy [p=0.09, OR (95% CI)= 1.39 (0.95- 2.05)]. iii) HVEM: Both polymorphisms were associated with MS predisposition, with stronger effect in patients with HHV6A active replicationTNFRSF6B-rs4809330*A: P=0.028, OR=1.13; TNFRSF14-rs6684865*A: overall P=0.0008, OR=1.2; and HHV6-positive patients vs controls: P=0.017, OR=1.69.Conclusion: An interaction between HHV-6A and genes previously involved in MS has been suggested. Furthermore, a possible relation between HHV-6A and the clinical response to IFN-beta therapy in MS patients should be deeper studied.

13-02 Oral IFN-γ-Dependent Demyelinating Activity by Natural Killer Cells is Regulated by HLA-G Upregulation on Oligodendrocytes During HHV-6 Infection

P.P. Banerjee1([email protected]), S.S. Soldan2, S. Miah3, A. Esinberg1, S. Maru1, Y. Rosenberg-Hasson4, K.S. Campbell3, D. Ablashi5, J.S. Orange6

1The Children’s Hospital of Philadelphia, PA, USA; 2Department of Neurology, University of Pennsylvania School of Medicine, USA; 3Department of Immunology, Fox Chase Cancer Center, Philadelphia, USA; 4Human Immune Monitoring Center, Stanford School of Medicine, USA; 5Human Herpes Virus 6 Foundation, Santa Barbara, CA, USA; 6Department of Pediatrics, University of Pennsylvania School of Medicine, USA

Oligodendrocytes (OLs) generate myelin sheaths surrounding axons of the central nervous system. Destruction of these myelin sheaths occurs in demyelinating diseases like multiple sclerosis (MS). The Human Herpes virus six (HHV-6) has been implicated in the etiology of MS. However, the role for HHV-6 in immune-mediated pathogenesis of MS has yet to be specifically defined. Pro-inflammatory cytokines play a role in demyelination, and many of these are induced in response to viral infection. We examined the potential role of NK cells in pathogenesis of demyelination, both upon activation with IFN-α and IL-2, and in response to HHV-6A infection of oligodendrocytes. We determined that activated, but not resting NK cells were cytotoxic to uninfected OLs, and upon forming NK/OL conjugates, activated NK cells promoted reduction of myelin oligodendrocyte glycoprotein (MOG) in OLs. Resting NK cells, however, are cytotoxic to HHV-6A infected OLs. Cytokine profiling from supernatants of NK cells co-cultured with HHV-6A-infected OLs demonstrated that NK cells predominantly secreted type-1 cytokines in response to HHV-6A infection. Most of these cytokines are regulated by the killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4). Using biochemical analysis, FACS and confocal microscopy, we determined that HLA-G, the only reported ligand of KIR2DL4, is also expressed on human oligodendrocytes and is upregulated by HHV-6A infection. HLA-G on OLs interacts with KIR2DL4 of NK cells at the NK cell immunological synapse. This interaction between WT but not mutated KIR2DL4 and HLA-G rapidly generates IFN-γ, polarizes IFN-γ to the immunological synapse presumably resulting in the directed secretion of IFN-γ onto OLs enabling MOG decrease. IFN-γ siRNA or addition of neutralizing anti-IFN-γ antibody abrogated demyelinating activity of NK cells. These findings present a mechanism for NK cell-mediated damage in pathogenesis of demyelinating disease through activity against HHV-6A infected OLs.

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HHV-6 & Multiple Sclerosis

Oral 13-03Oligoclonal Antibody Response in Multiple Sclerosis Includes Antibodies to Herpesviruses

J.O. Virtanen1([email protected]), K. Fenton1, I. Cortese1, B. Bielekova1, S. Jacobson1

1NIH/NINDS, Bethesda, MD, USAThe clonal expansion of B-cells in central nervous system and intrathecal production of antibodies are characteristic

features of multiple sclerosis (MS). Intrathecally produced antibodies are the basis of the hallmark of MS, oligoclonal bands (OCBs). Still, 50 years after the discovery of OCBs in MS, their specificity remains unknown. Interestingly, in other inflammatory diseases of the CNS, OCBs are specific for the cause e.g. to measles in subacute sclerosing panencephalitis. Two herpesviruses, human herpesvirus 6 (HHV-6) and Epstein–Barr virus (EBV) have been associated with MS. Therefore, we studied the presence of herpesvirus-specific OCBs in patients with MS with isoelectric focusing and immunoblotting. HHV-6A or HHV-6B infected SupT1 cell lysates or EBV producing B95-8 cell lysate were used as antigens. Twenty-eight paired CSF and serum samples from MS patients were included in the study. Five patients were found to have HHV-6 specific OCBs and five patients EBV specific OCBs. OCBs were positive only for one or the other HHV-6 or EBV but never both. Collectively, these results demonstrate that as as much as 10 of 28 patients with MS (36%) had herpes-specific OCBs. Virus-specific OCBs in the CSF suggests active stimulation of humoral immune response in the CNS by human herpesviruses and support the hypothesis that these agents may play a role in the pathogenesis of this disorder. These observations also suggest strategies by which MS patients can be stratified with respect to herpesvirus specific OCBs for inclusion in antiviral clinical trials.

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HHV-6 & Multiple Sclerosis

14-01 Oral Effects of HHV-6A Infection in the Common Marmoset

J.E. Wohler1([email protected]), M.I. Gaitan2, E. Harberts1, A.C. Silva3, D.S. Reich2, S. Jacobson1

1Viral Immunology Section, Neuroimmunology Branch, NINDS/NIH, Bethesda, MD, USA; 2Translational Neuroradiology Unit, Neuroimmunology Branch, NINDS/NIH, USA; 3Cerebral Microcirculation Unit, Laboratory of Functional and Molecula, USA

HHV-6, a ubiquitous β-herpesvirus, is frequently associated with neurologic diseases including MS, mesial temporal lobe epilepsy (MTLE), encephalitis, and febrile illness. The two variants of HHV-6 include HHV-6B, the etiological agent of exanthema subitum (roseola) and HHV-6A. The pathogenesis and epidemiology of HHV-6A remain unclear, although it has been reported that this variant is more neurotropic than HHV-6B. Given the association with neurologic disease, the effects of a primary infection with HHV-6A are of interest. To examine this, we exposed C. jacchus marmosets to HHV-6A intravenously once a month for 4 months and monitored the resulting infection. In 3 of the 4 HHV-6A infected marmosets, we detected IgM responses one week post-inoculation. IgG responses were seen in 2 of the 4 infected animals one week post-inoculation, and by week 15 all the infected animals had IgG responses to HHV-6. In HHV-6A infected animals, the virus has not been consistently detected in the saliva, plasma, or PBMCs using nested PCR, suggesting that HHV-6A does not reside in the periphery at high frequencies. Infected animals began showing signs of neurologic disease after the second exposure to the virus. To date the disease has been monophasic in nature and mainly characterized by impairment of the sensory system. One marmoset developed a skin condition with blister like erythematous lesions, another marmoset developed a facial palsy, and another developed signs consistent with sensory ataxia. Currently, pathological studies are ongoing to determine the localization of the virus in tissue. Overall we find that primary HHV-6A infection is associated with rapid seroconversion and neurological symptoms in the marmoset. This infection will provide a model system to study the immune response during primary herpesvirus infection and the resulting neurological disease.

14-02 Oral Development of a Transgenic Murine Model for Human Herpesvirus-6 Infection

J. Reynaud1([email protected]), J. Jégou1, J. Welsch1, B. Horvat11INSERM/ENS Lyon, Human Virology laboratory, Lyon, France

Several clinical studies have correlated human herpesvirus 6 (HHV-6) infection to the pathogenesis of different diseases, including the neurological autoimmune disease multiple sclerosis (MS). However, a direct link of causality between HHV-6 and MS is still missing, and the lack of suitable small animal models for HHV-6 infection has considerably hampered the study of the viral infection in the central nervous system. Human CD46 molecule was identified as a receptor for HHV-6, opening new perspectives for experimental approaches. We have generated several lines of transgenic mice, expressing one or both isoforms of CD46 (Cyt1 and/or Cyt2), crossed or not into an interferon type 1 receptor deficient background, and analyzed their susceptibility to HHV-6 infection. We first generated primary brain cell cultures from these mice and infected them with HHV-6A (strain GS) or HHV-6B (strain Z29). We detected expression of viral transcripts only in cultures from CD46-expressing mice and not from wild type mice, after HHV-6A, but not HHV-6B infection. These mice were also infected in vivo with both variants of HHV-6 and were monitored for up to 3 months. In agreement with in vitro results, HHV-6A DNA persisted for up to 2 months in the brain of CD46-expressing mice but not in the non-transgenic littermates, whereas HHV-6B DNA levels decreased rapidly after infection in all mice. Although infected mice did not show clinical signs of disease, histological studies revealed the presence of demyelinated areas and infiltrating cells in the brain. Finally, preliminary experiments with experimental autoimmune encephalomyelitis (EAE), the animal model for MS, suggested that HHV-6 infection prior to EAE induction could significantly increase the severity of the disease, implying a possible effect of HHV-6 on MS-like disease development in vivo. Our results present the first murine model for HHV-6A infection, opening novel perspectives for the study of virus-associated pathologies.

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HHV-6 Animal Models

Oral 15-01Analysis of HHV-6 Presence in Brain Tissue from Well Defined Subgroups of Patients with Temporal Lobe Epilepsy

P. Niehusmann1([email protected]), T. Mittelstaedt1, C.G. Bien2, J.F. Drexler3, A. Grote3, S. Schoch1, A.J. Becker1

1Deptartment of Neuropathology, University of Bonn Medical Center, Bonn, Germany; 2Department of Epileptology, University of Bonn Medical Center, Germany; 3Deptartment of Virology, University of Bonn Medical Center, GermanyPurpose: Temporal lobe epilepsy (TLE) is the most frequent focal epilepsy and often associated with mesial temporal sclerosis (MTS). Numerous etiological aspects of TLE are still unresolved. Early age of initial precipitating injury is an important predictor of hippocampal pathology. Febrile seizures in children are frequently associated with human herpesvirus-6 (HHV-6) infections and more than 95% of children older than two years are HHV-6 seropositive. HHV-6 can establish lifelong latency in the CNS. A pathogenetic relationship of persistent HHV-6 infection and TLE has been suggested. Here, we analyzed HHV-6 presence in surgical tissue from well-defined subgroups of TLE-patients. Methods: Nested PCR, sequence analysis and immunohistochemistry were performed to identify HHV-6 DNA or antigen and to differentiate HHV-6 variants. The subgroups of pharmacoresistant TLE-patients were classified by clinical and neuropathological criteria as follows: Idiopathic MTS (n=10), lesion-associated (n=10), complex febrile seizures (CFS, n=6), history of encephalitis (n=9). Temporal autopsy samples without a history of neurological disorders served as controls (n=10). Results: HHV-6B DNA (but no HHV-6 antigen) was detected in 55.6% of specimens from patients with a history of encephalitis. In contrast, lesion-associated TLE and autopsy-control specimens were negative for HHV-6 DNA and antigen. Intriguingly, in non-lesional MTS (with or without history of febrile seizures) neither molecular nor immunohistochemical analyses revealed presence of HHV-6. Conclusion: Our data argue against HHV-6 as major local pathogenetic factor in MTS hippocampi after CFS. The high detection rate of HHV-6B DNA suggests a potential pathogenetic role of HHV-6B in TLE patients with a history of encephalitis.

Oral 15-02The Role of Human Herpesvirus 6 and 7 in Febrile Status Epilepticus: The FEBSTAT Study

L. Epstein1([email protected])1Northwestern University Feinberg School of Medicin.e, Chicago, IL, USA

In a prospective study of the consequences of prolonged febrile seizures (FEBSTAT), we determined the frequency of Human Herpesvirus (HHV)-6 and HHV-7 infection as a cause of febrile status epilepticus (FSE). Children ages 1 month to 5 years presenting with FSE were enrolled within 72 hours and received a comprehensive assessment including specimens for HHV-6 and HHV-7. The presence of HHV-6A, HHV-6B or HHV-7 DNA and RNA (amplified across a spliced junction) determined using quantitative polymerase chain reaction (qPCR) at baseline indicated viremia. Antibody titers to HHV-6 and HHV-7 were used in conjunction with the PCR results to distinguish primary infection from reactivated or prior infection. Of 198 children evaluated, HHV-6 or HHV-7 status could be determined in 168 (84.8%). HHV-6B viremia at baseline was found in 53 subjects (31.6%), including 37 with primary infection and 16 with reactivated infection. No HHV-6A infections were identified. HHV-7 viremia at baseline was observed in 12 (7.1%) subjects, including 8 with primary infection and 4 with reactivated infection. Two subjects had HHV-6/HHV-7 co-infection at baseline. HHV-6B infection is the most common cause of FSE. HHV-7 infection is a less frequent cause of FSE. Together, they account for one third of FSE, a condition associated with an increased risk of both hippocampal injury and subsequent temporal lobe epilepsy.

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HHV-6 & 7 in Epilepsy

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15-03 OralDetection of human herpes virus 6B in patients with mesial temporal lobe epilepsy in China and the possible association with elevated NF-KappaB expression

D. Zhou1([email protected]), JM. Li11Department of Neurology, West China Hospital/Sichuan University

There has been a long-standing suspicion that an association exists between mesial temporal lobe epilepsy (MTLE) and the herpes virus. Evidence for HHV-6B involvement were reported previously. However, there has been no similar investigation in China. We detected viral DNA of human herpes virus HHV-6B, HHV-6A, herpes simplex virus HSV-1 and HSV-2 in resected brain tissues from patients with MTLE and control subjects by nested PCR and immunohistochemistry. A principal transcription factor, NF-κB, that probably associates with inflammatory reaction was also investigated by real-time PCR, western blotting and immunohistochemistry.HHV-6B DNA was detected in hippocampal samples of 9 out of 32 (28.1%) patients with MTLE and in 1 of 12 (8.3%) control samples. Immunoreactivity for HHV-6B was consistently present in MTLE patients with positive HHV-6 detected by PCR. Significant staining for HHV-6B antigen distributed mainly around or in nucleus of cells that morphologically resemble astrocytes and microglial cells. Positive HHV-6B was related with febrile convulsion history of patients with MTLE. The expression of NF-κB was found up-regulated and distributed in the nucleus of glial cells in MTLE patient with positive HHV-6B. This study indicates that HHV-6B has a potential association with MTLE in West China. The data also suggests that the activation of NF-κB is related with HHV-6B positivity in MTLE patients. The detailed role of HHV-6B and its association with NF-κB in the development of chronic MTLE require further investigation.

HHV-6 & 7 in Epilepsy

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Author Index & Participant Roster

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AUTHOR INDEX

AAberle, S.W. . . . . . . . . . . . . . . . . . . . 4-11Ablashi, D. . . . . . . . . . 10-05, 4-06, 4-07, 4-08, 13-02Agut, H. . . . . . . . . . . . 10-05, 7-01, 7-03, 10-06, 110-05Ahlqvist, J. . . . . . . . . . . . . . . . . . . . . 4-11Ahmad, A. . . . . . . . . . . . . . . . . . . . . 2-07Alvarez-Lafuente, R. . . . . . . . . . . 13-01Andrei, G. . . . . . . . . . . . . . . . . . . . 11-02Aouni, M. . . . . . . . . . . . . . . . . . . . . . 7-03Arbuckle, J.H. . . . . . . . .4-05, 4-07, 4-08Aspinall, R. . . . . . . . . . . . . . . . . . . 10-04

BBak, R. . . . . . . . . . . . . . . . . . . . . . . . 3-08Banerjee, P.P. . . . . . . . . . . . . . . . . . 13-02Banning, J.E. . . . . . . . . . . . . . . . . . 11-02Barg-Hock, H. . . . . . . . . . . . . . . . . 10-09Bartoletti, M. . . . . . . . . . . . . . . . . . . 7-01Becerra-Artiles, A.R. . . . . . . 2-08, 9-04Becker, A.J. . . . . . . . . . . . . . . . . . . . 15-01Becker, T. . . . . . . . . . . . . . . . . . . . . 10-09Bell, A.J. . . . . . . . . . . . . . . . . . . . . . . 8-03Ben Fredj, N. . . . . . . . . . . . . . . . . . . 7-03Bhattacharjee, B. . . . . . . . . . . . . . . . 3-03Biagioli, T. . . . . . . . . . . . . . . . . . . . 12-06Bielekova, B. . . . . . . . . . . . . . . . . . 13-03Bien, C.G. . . . . . . . . . . . . . . . . . . . . 15-01Bigaillon, C. . . . . . . . . . . . . . . . . . . 10-06Böhme, L. . . . . . . . . . . . . . . . . . . . . . 2-06Bonnafous, P. . . . . . . . . . . . . 7-01, 10-06Borenstein, R. . . . . . . . . . . . . 3-05, 6-03Borges, N. . . . . . . . . . . . . . . . . . . . . 7-02Broccolo, F. . . . . 4-12, 8-02, 8-07, 12-06Bundgaard, B. . . . . . . . . . . . . . . . . . 3-08

CCalvo Calle, J.M. . . . . . . . . . . 2-08, 9-04Campbell, K.S. . . . . . . . . . . . . . . . 13-02Canfield, R.L. . . . . . . . . . . . . . . . . . 4-03Carrigan, D. . . . . . . . . . . . . . . . . . . . 8-04Caselli, E. . . . . . . . . . . . . . . . . . . . . . 8-01Caserta, M.T. . . . . . . . . . . . . 10-05, 4-03Cassina, G. . . . . . . . . . . . . . . 8-02, 12-06Ceccherini-Nelli, L. . . . . . . 8-02, 12-06Chang, S. . . . . . . . . . . . . . . . . . . . . . 8-05Chapenko, S. . . . . . . . . . . . . 8-06, 10-07Charrier, M. . . . . . . . . . . . . . . . . . . . 7-01Chistyakov, M. . . . . . . . . . . 8-06, 10-07Cho, Y. . . . . . . . . . . . . . . . . . . . . . . 12-04

Chung, W. . . . . . . . . . . . . . . . . . . . . 9-05Clark, D.J. . . . . . . . . . . . . . . . . . . . . 2-05Collot-Teixeira, S. . . . . . . . . . . . . . . 2-03Cortese, I. . . . . . . . . . . . . . . . . . . . . 13-03Coscoy, L. . . . . . . . . . . . . . . . . . . . . . 2-02Crawford, J. . . . . . . . . . . . . . 2-09, 12-07Cunskis, E. . . . . . . . . . . . . . . . . . . . . 8-06

DDanziger-Isakov, L. . . . . . . . . . . . . 7-02Davidson, P.W. . . . . . . . . . . . . . . . . 4-03De Pagter, A.P.. . . . . . . . . . . . . . . . 11-03Debbeche, O. . . . . . . . . . . . . . . . . . . 2-07Descamps, V. . . . . . . .10-05, 9-01, 10-06Desmonet, M. . . . . . . . . . . . . . . . . . 7-01Dewilde, A. . . . . . . . . . . . . . . . . . . . 4-09Dhédin, N. . . . . . . . . . . . . . . . . . . . 10-06Di Luca, D. . . . . . . . . . . . . . . . . . . . . 8-01Di Marco, E. . . . . . . . . . . . . . . . . . . . 4-12Dickerson, F. . . . . . . . . . . . . . . . . . 12-02Dillinger, S. . . . . . . . . . . . . . . . . . . . 9-03Dominguez-Amorocho, O.A. . . . 2-08, 9-04Donina, S. . . . . . . . . . . . . . . . . . . . 10-07Drago, F. . . . . . . . . . . . . . . . . . . . . . . 8-02Drexler, J.F. . . . . . . . . . . . . . . . . . . 15-01

EEgerer, A. . . . . . . . . . . . . . . . . . . . . . 4-04Engdahl, E.E. . . . . . . . . . . . . . 3-07, 4-11Engelmann, I. . . . . . . . . . . . . . . . . 10-09Enlander, D.. . . . . . . . . . . . . 8-05, 11-07Epstein, L. . . . . . . . . . . . . . . . . . . . 15-02Eriksson, K. . . . . . . . . . . . . . . . . . . . 9-02Escher, F. . . . . . . . . . . . . . . . . . . . . 11-06Esinberg, A. . . . . . . . . . . . . . . . . . . 13-02

FFava, A. . . . . . . . . . . . . . . . . . . . . . . 8-02Feki, S. . . . . . . . . . . . . . . . . . . . . . . . 7-03Fenton, K. . . . . . . . . . . . . . . . . . . . 13-03Flamand, L. . . . . . . . . . . . . .10-05, 3-02, 3-09, 4-10Fogdell-Hahn, A. . . . . . . . . . 3-07, 4-11Franchini, S. . . . . . . . . . . . . . . . . . . . 8-02Frenkel, N. . . . . . . . . . . . . . . . 3-05, 6-03Fusetti, L. . . . . . . . . . . . . . . . 8-02, 12-06

GGaitan, M.I. . . . . . . . . . . . . . . . . . . 14-01Gallagher, A. . . . . . . . . . . . . . . . . . . 8-03

Gatherer, D. . . . . . . . . . . . . . . . . . . . 8-03Gautheret-Dejean, A. . . . . .10-05, 4-09, 7-01, 7-03, 10-03, 10-06Géraudie, B. . . . . . . . . . . . . . . . . . . . 7-01Gerdemann, U. . . . . . . . . . . . . . . . 11-03Gerry, D.. . . . . . . . . . . . . . . . . . . . . 11-02Gibson, L. . . . . . . . . . . . . . . . . . . . . 9-04Giroux, M. . . . . . . . . . . . . . . . . . . . . 4-09Goldfarb, J. . . . . . . . . . . . . . . . . . . . 7-02Gompels, U. . . . . . . . . . . . . . 2-05, 3-01Gösling, J. . . . . . . . . . . . . . . . . . . . 10-09Govind, S. . . . . . . . . . . . . . . . . . . . 10-04Gowans, K. . . . . . . . . . . . . . . . . . . . 7-02Gravel, A. . . . . . . . . . . . .3-02, 3-09, 4-10Gravelsina, S. . . . . . . . . . . . 8-06, 10-07Grazia Marazzi, M. . . . . . . . . . . . . 4-12Grazia Sabbadini, M. . . . . . . . . . . . 8-02Gross, U.M. . . . . . . . . . . . . . . . . . . 11-06Grote, A. . . . . . . . . . . . . . . . . . . . . . 15-01Gubin-Jurgens, J. . . . . . . . . . . . . . . 4-05Gustafsson, R.K. . . . . . . . . . . . . . . 10-10

HHall, C.B. . . . . . . . . . . . . . . . 10-05, 4-03Harberts, E. . . . . . . . . . . . . . . . . . . 14-01Hayashi, E. . . . . . . . . . . . . . . . . . . . 3-06Hayashi, M. . . . . . . . . . . . . . . . . . . . 3-06Heim , A. . . . . . . . . . . . . . . . . . . . . 10-09Hendrickson, E. . . . . . . . . . . . . . . . 5-02Heslop, H.E. . . . . . . . . . . . . . . . . . 11-03Heurté, D. . . . . . . . . . . . . . . . . . . . . 7-01Höckerstedt, K. . . . . . . . . . . . . . . . 10-08Höllsberg, P. . . . . . . . . . . . . . 2-04, 3-08Horvat, B. . . . . . . . . . . . . . . . . . . . 14-02

IIannello, A. . . . . . . . . . . . . . . . . . . . 2-07

JJaccard, A. . . . . . . . . . . . . . . . . . . . . 2-03Jacobson, S. . . . . . . .12-04, 13-03, 14-01Jarosinski, K.W. . . . . . . . . . . . . . . . . 4-04Jarrett, R.F. . . . . . . . . . . . . . . . . . . . . 8-03Jaworska, J. . . . . . . . . . . . . . . . . . . . 3-02Jégou, J. . . . . . . . . . . . . . . . . . . . . . 14-02Johnson, P.M. . . . . . . . . . . . . . . . . 11-02

KKarlsson, T. . . . . . . . . . . . . . . . . . . 10-08Katari, U.L. . . . . . . . . . . . . . . . . . . 11-03

84

AUTHOR INDEX

Kaufer, B.B. . . . . . . . . . . . . . . . . . . . 4-04Kawabata, A. . . . . . . . . . . . . . . . . . . 3-06Kawamura, Y. . . . . . . . . . . . . . . . . 12-03Keirnan, J.M. . . . . . . . . . . . . . . . . . 11-03Keukens, L. . . . . . . . . . . . . . . . . . . 11-03Khlif, A. . . . . . . . . . . . . . . . . . . . . . . 7-03Knox, A. . . . . . . . . . . . . . . . . . . . . . . 8-04Knox, K. . . . . . . . . . . . . . . . . . . . . . . 8-04Kofod-Olsen, E. . . . . . . . . . . 2-04, 3-08Kohn, D. . . . . . . . . . . . . . . . . . . . . . . 7-02Kowalik, T. . . . . . . . . . . . . . . . . . . . 3-03Krueger, G. . . . . . . . . . . . . . . . . . . . 4-11Krüger, G.F. . . . . . . . . . . . . . . . . . . 11-06Kühl, U. . . . . . . . . . . . . . . . 10-05, 11-06Kurtzberg, J. . . . . . . . . . . . . . . . . . 11-05

LLacroix, A. . . . . . . . . . . . . . . . . . . . . 2-03Lassner, D. . . . . . . . . . . . . . 10-05, 11-04Lautenschlager, I. . . . . . . . . 10-05, 4-11, 10-02, 10-08Lavenstein, B. . . . . . . . . . . . . . . . . 12-07Leen, A.M. . . . . . . . . . . . . . . . . . . . 11-03Lejniece, S. . . . . . . . . . . . . . . . . . . . 10-07Lepage, C. . . . . . . . . . . . . . . . . . . . . 2-03Li, J.M. . . . . . . . . . . . . . . . . . . . . . . 15-03Lieberman, P. . . . . . . . . . . . . . . . . . . 5-01Loginov, R.. . . . . . . . . . . . . . . . . . . 10-08Loomis, K. . . . . . . . . . . . . . . . . . . . 10-05Luka, J. . . . . . . . . . . . . . .4-05, 4-07, 4-08Luppi, M. . . . . . . . . . . . . . . . 10-05, 4-01Lusso, P. . . . . . . . 2-01, 4-12, 8-02, 12-06

MMaeki, T. . . . . . . . . . . . . . . . . . . . . . 3-04Malnati, M. . . . . . . . . .4-12, 8-02, 12-06Mannonen, L. . . . . . . . . . . . . . . . . 10-08Manns, M. . . . . . . . . . . . . . . . . . . . 10-09Mardivirin, L. . . . . . . . . . . . . . . . . . 2-03Marie, M. . . . . . . . . . . . . . . . . . . . . . 2-03Martin, L. . . . . . . . . . . . . . . . 9-03, 11-08Maru, S. . . . . . . . . . . . . . . . . . . . . . 13-02Matà, S. . . . . . . . . . . . . . . . . . . . . . 12-06Matteoli, B. . . . . . . . . . . . . . 8-02, 12-06Maurage, C. . . . . . . . . . . . . . . . . . . . 4-09McCormick, M. . . . . . . . . . . . . . . . 12-04Medveczky, M.M. . . . . .4-06, 4-07, 4-08Medveczky, P.G. . . . . .10-05, 4-06, 4-08Merrihew, L. . . . . . . . . . . . . . . . . . 11-04Miah, S. . . . . . . . . . . . . . . . . . . . . . 13-02Mikkelsen, J.G. . . . . . . . . . . . . . . . . 3-08Milovanovitch, A. . . . . . . . . . . . . . 10-06

Mitchell, W. . . . . . . . . . . . . . . . . . . 10-04Mittelstaedt, T. . . . . . . . . . . . . . . . 15-01Møller, J.M. . . . . . . . . . . . . . . . . . . . 3-08Mommeja-Marin, H. . . . . . . . . . . 11-05Montoya, J. . . . . . . . . . . . . 10-05, 11-06Moosmann, A. . . . . . . . . . . . 9-03, 11-08Mori, Y. . . . . . . . . 3-04, 3-06, 6-02, 10-05Murovska, M. . . . . . . . . . . . 8-06, 10-07

NNacheva, E.P. . . . . . . . . . . . . . . . . . . 4-11Naesens, L. . . . . . . . . . . . . . . . . . . 11-02Nastke, M.D. . . . . . . . . . . . . . . . . . . 9-04Nefsi, F. . . . . . . . . . . . . . . . . . . . . . . 7-03Nguyen, S. . . . . . . . . . . . . . . . . . . . 10-06Niehusmann, P. . . . . . . . . . . . . . . . 15-01Nora-Krukle, Z. . . . . . . . . . 8-06, 10-07Norris, J. . . . . . . . . . . . . . . . . . . . . . 11-04

OOgata, M. . . . . . . . . . . . . . . 10-05, 12-05Onraed, B. . . . . . . . . . . . . . . . . . . . . 4-09Orange, J.S. . . . . . . . . . . . . . . . . . . 13-02Osterrieder, N. . . . . . . . . . . . . . . . . 4-04Outteryck, O.. . . . . . . . . . . . . . . . . . 4-09Oyaizu, H. . . . . . . . . . . . . . . . 3-04, 3-06

PPainter, W.P. . . . . . . . . . . . . . . . . . . 11-05Pantry, S.N. . . . . . . . . . .4-06, 4-07, 4-08Paolino, S. . . . . . . . . . . . . . . . . . . . . 8-02Paredes, A. . . . . . . . . . . . . . . . . . . . . 6-01Parodi, A. . . . . . . . . . . . . . . . . . . . . . 8-02Pellett, P.E. . . . . . . . . .10-01, 7-02, 10-05Penasse, C. . . . . . . . . . . . . . . . . . . . . 7-01Peterson, D. . . . . . . . . . . . . . . . . . . . 8-04Pinon, A. . . . . . . . . . . . . . . . . . . . . . 2-03Pischke, S. . . . . . . . . . . . . . . . . . . . 10-09Popow-Kraupp, T. . . . . . . . . . . . . . 4-11Prasad, V. . . . . . . . . . . . . . . . . . . . . 11-05Prusty, B.K. . . . . . . . . . . . . . . . . . . . 2-06

RRazonable, R. . . . . . . . . . . . . . . . . 10-05Reich, D.S. . . . . . . . . . . . . . . . . . . . 14-01Renew, Z.M. . . . . . . . . . . . . . . . . . 11-02Renne, R. . . . . . . . . . . . . . . . . . . . . . 4-07Renzette, N. . . . . . . . . . . . . . . . . . . . 3-03Reynaud, J. . . . . . . . . . . . . . . . . . . 14-02Rhode, M. . . . . . . . . . . . . . . . . . . . 11-06Richter, S. . . . . . . . . . . . . . . . . . . . . . 4-11

Riethman, H. . . . . . . . . . . . . . . . . . . 5-03Rogez, S. . . . . . . . . . . . . . . . . . . . . . 2-03Rooney, C.M. . . . . . . . . . . . . . . . . . 11-03Rosenberg-Hasson, Y. . . . . . . . . . 13-02Rudel, T. . . . . . . . . . . . . . . . . . . . . . . 2-06Ruiz, A. . . . . . . . . . . . . . . . . . . . . . 11-04

SSamarani, S. . . . . . . . . . . . . . . . . . . . 2-07Schlue, J.. . . . . . . . . . . . . . . . . . . . . 10-09Schmitt, C. . . . . . . . . . . . . . . . . . . . 10-09Schoch, S. . . . . . . . . . . . . . . . . . . . . 15-01Schub, A. . . . . . . . . . . . . . . . 9-03, 11-08Schultheiss, H.P. . . . . . . . . . . . . . . 11-06Schulz, T. . . . . . . . . . . . . . . . . . . . . 10-09Schwinger, W. . . . . . . . . . . . . . . . . . 4-11Seeberg, B. . . . . . . . . . . . . . . . . . . . 11-06Seto, E. . . . . . . . . . . . . . . . . . . . . . . 10-05Shah, H. . . . . . . . . . . . . . . . . . . . . . 11-02Sharon, E. . . . . . . . . . . . . . . . . . . . . . 6-03Shield, L. . . . . . . . . . . . . . . . . . . . . . 8-03Silva, A.C. . . . . . . . . . . . . . . . . . . . 14-01Sinnett, D. . . . . . . . . . . . . . . . . . . . . 4-10Snoeck, R. . . . . . . . . . . . . . . . . . . . 11-02Soldan, S.S. . . . . . . . . . . . . . . . . . . 13-02Stephens, C.E. . . . . . . . . . . . . . . . . 11-02Stern, L.J. . . . . . . . . . . . . . . . . 2-08, 9-04Stern, M.. . . . . . . . . . . . . . . . . . . . . 10-06Strassburg, C. . . . . . . . . . . . . . . . . 10-09Strenger, V. . . . . . . . . . . . . . . . . . . . . 4-11Sultanova, A. . . . . . . . . . . . . 8-06, 10-07

TTang, H. . . . . . . . . . . . . . . . . . 3-04, 3-06Terriou, L. . . . . . . . . . . . . . . . . . . . . 4-09Trocjukas, I. . . . . . . . . . . . . . . . . . . 10-07

UUrban, C. . . . . . . . . . . . . . . . . . . . . . 4-11

VVermersch, P. . . . . . . . . . . . . . . . . . . 4-09Virtanen, J.O. . . . . . . . . . . . . . . . . . 13-03

WWang, H. . . . . . . . . . . . . . . . . . . . . . 4-03Ward, K. . . . . . . . . . . . . . . . . . . . . . . 4-02Watt, T. . . . . . . . . . . . . . . . . . . . . . . 11-04Wedemeyer, H. . . . . . . . . . . . . . . . 10-09Weismann, J. . . . . . . . . . . . . . . . . . . 8-04Welsch, J. . . . . . . . . . . . . . . . . . . . . 14-02

85

AUTHOR INDEX

Wilkerson, P.D. . . . . . . . . . . . . . . . 11-02Wohler, J.E. . . . . . . . . . . . . . . . . . . 14-01Wölk, B. . . . . . . . . . . . . . . . . . . . . . 10-09Woodard, C. . . . . . . . . . . . . . . . . . . 7-02

YYamanishi, K. . . . . . . . . . . . . 3-04, 3-06Yao, K. . . . . . . . . . . . . . . . . . . . . . . 12-04Yen-Lieberman, B. . . . . . . . . . . . . . 7-02Yin, L. . . . . . . . . . . . . . . . . . . . . . . . . 9-04Yolken, R. . . . . . . . . . . . . . . . . . . . . 12-02Yoshikawa, T. . . . . . . . . . . 10-05, 10-01Yu, D. . . . . . . . . . . . . . . . . . . . . . . . . 6-01

ZZaccaria, E. . . . . . . . . . . . . . . . . . . . 8-02Zeigerman, H. . . . . . . . . . . . . 3-05, 6-03Zerr, D. . . . . . . . . . . . . . . . . . . . . . . 12-01

86

PARTICIPANT ROSTER

Dharam AblashiScientific DirectorHHV-6 Foundation277 San Ysidro Rd.Santa Barbara CA 93108 United StatesPhone: 805-969-1174Email: [email protected]

Henri AgutHead of Virology DepartmentGroupe Hospitalier Pitié-SalpêtrièreService de Virologie du Cervi83 Blvd. de l’HôpitalParis 75013 FrancePhone: 33142177400Email: [email protected]

Roberto Alvarez-LafuenteHospital Clinico San CarlosCiudad UniversitariaLaboratorio de Investigación de Esclerosis Múltiple (4ª planta)Pabellón 8 - Hospital Clinico San CarlosMadrid 28040 SpainPhone: 349133030002970Email: [email protected]

Jesse ArbucklePh.D Graduate StudentUniversity of South Florida College of Medicine12901 Bruce B. Downs Blvd., MDC Box 7Tampa FL 33612 United StatesPhone: 727-688-4890Email: [email protected]

Yoshizo AsanoProfessorHokkaido UniversityResearch Center for Zoonosis ControlKita 20 Nishi 10Kita-ku, Sapporo 470-1192 JapanPhone: 81117069500Email: [email protected]

Pinaki BanerjeeThe Children’s Hospital of Philadelphia3615 Civic Center Blvd., ARC 912 GPhiladelphia PA 19104 United StatesPhone: 267-237-7378Email: [email protected]

Aniuska Becerra-ArtilesUniversity of Massachusetts Medical School55 Lake Avenue NorthWorcester MA 1655 United StatesPhone: 508-856-1023Email: [email protected]

Abbas Behzad BehbahaniLaboratory Sciences Research CenterFaculty of Paramedical SciencesMeshkinfam StreetShiraz 7143914693 IranPhone: 987116234944Email: [email protected]

Adam BellGraduate StudentUniversity of GlasgowBotham Building, Garscube EstateGlasgow G61 1QH United KingdomPhone: 441413305774Email: [email protected]

Pascale BonnafousEngineerUPMC Paris 06 ER1 DETIVFaculté de Médecine Pierre et Marie Curie - Paris VI

CHU Pitié-Salpêtrière Bâtiment CERVI83, Bd de l’Hôpital 75651Paris 75013 FrancePhone: 33145826298Email: [email protected]

Francesco BroccoloResearcher and Adjunct ProfessorUniversità Milano-BicoccaDepartment of Clinical Medicine and PreventionVia Cadore 48Monza (MB) 20052 ItalyPhone: 393358093301Email: [email protected]

J. Mauricio Calvo-CalleInstructorUniversity of MassachusettsSchool of Medicine55 Lake Avenue North, Lab S2-8-8Worcester MA 01655 United StatesPhone: 508-856-8806Email: [email protected]

Wen-Hung ChungSchool of MedicineNational Yang-Ming University405, Institute of PharmacologyNo.155, Sec.2, Linong StreetTaipei 112 TaiwanPhone: 886-2-28267201Email: [email protected]

87

PARTICIPANT ROSTER

David ClarkPhD StudentLondon School of Hygiene and Tropical MedicineRoom 280, Keppel StreetLondon WC1E7HT United KingdomPhone: 4402079588179Email: [email protected]

Laurent CoscoyAssociate Professor of ImmunologyUniversity of California, BerkeleyDept. of Molecular and Cell Biology, Coscoy LabBerkeley CA 94720-3200 United StatesPhone: 510-643-4128Email: [email protected]

John CrawfordDirector of Pediatric Neuro-OncologyUniversity of California, San DiegoAssistant Professor of Neurosciences and Pediatrics8010 Frost St., Suite 400, NeurologySan Diego CA 92123 United StatesPhone: 858-966-5819Email: [email protected]

Olfa DebbechePhD StudentCentre de Recherche de l’Hopital St-Justine3175, Chemin de la Côte-Sainte-CatherineMontreal QC H3T1C5 CanadaPhone: 5149611263Email: [email protected]

Vincent DescampsProfessorBichat Claude Bernard HospitalDepartment of Dermatology46 rue Henri HuchardParis F-75018 FrancePhone: 33140258730Email: [email protected]

Dario Di LucaProfessor of MicrobiologyUniversity of FerraraDepartment of Experimental and Diagnostic MedicineVia Borsari 46Ferrara 44100 ItalyPhone: 390532455318Email: [email protected]

Derek EnlanderClinic DirectorME /CFS Clinic of New York860 Fifth AvenueNew York NY 10065 United StatesPhone: 212-794-2000Email: [email protected]

Leon EpsteinHead of NeurologyNorthwestern University Feinberg School of MedicinePediatrics and Neurology2300 Children’s Plaza - Box 51Chicago IL 60614 United StatesPhone: 773-880-4150 ext.7738804909Email: [email protected]

Kristina ErikssonProfessorUniversity of GothenburgDepartment of Rheumatology andInflammation ResearchBox 480Göteborg 40530 SwedenPhone: 46313424761Email: [email protected]

Louis FlamandProfessorUniversité LavalCentre de Recherche en Rhumatologie et Immunologie2705 Boulevard LaurierQuebec QC G1V 4G2 CanadaPhone: 418-656-4141 ext.46164Email: [email protected]

Niza FrenkelProfessor, Dept. of Cell Research and ImmunologyTel-Aviv UniversityBritania Bldg., Room 203Tel-Aviv 69978 IsraelPhone: 972544573892Email: [email protected]

Agnès Gautheret-DejeanLaboratoire de VirologieHôpital Pitié-Salpêtrière83 Boulevard de l’HôpitalParis 75013 FrancePhone: 33142177401Email: [email protected]

88

PARTICIPANT ROSTER

Ulrike GerdemannPost-doctoral FellowBaylor College of MedicineCenter for Cell and Gene Therapy1102 Bates Street, Suite 1770Houston TX 77030 United StatesPhone: 832-660-5284Email: [email protected]

Johanna GoldfarbPhysicianChildren’s Hospital Cleveland Clinic9500 Euclid Avenue, S25Cleveland OH 44124 United StatesPhone: 216-445-6863Email: [email protected]

Ursula GompelsReader in Molecular VirologyLondon School of Hygiene & Tropical MedicineDepartment of Infectious DiseasesUniversity of LondonLondon WC1E 7HT United KingdomPhone: 4402079272315Email: [email protected]

Andrew GoodmanProfessor of NeurologyUniversity of Rochester601 Elmwood AvenueRochester NY 14642 United StatesPhone: 585-273-1184Email: [email protected]

Sheila GovindPost-doctoral Research FellowCranfield UniversityCranfield Health. Vincent Building 52a,Wharley End, CranfieldMilton Keynes MK43 0AL United KingdomPhone: 441234758363Email: [email protected]

Annie GravelResearch ProfessionnalRheumatology and ImmunologyResearch Center2705 Laurier Blvd., Room T1-49Quebec QC G1V 4G2 CanadaPhone: 418-654-2772Email: [email protected]

Rasmus GustafssonPhD StudentKarolinska InstitutetClinical NeuroscienceThe Multiple Sclerosis Research GroupCMM L8:00, Karolinska University Hospital SolnaStockholm 17176 SwedenPhone: 46851770258Email: [email protected]

Caroline HallProfessorUniversity of Rochester601 Elmwood Avenue, Box 689Rochester NY 14642 United StatesPhone: 585-275-5242Email: [email protected]

Eric HendricksonProfessorUniversity of MinnesotaDept. of Biochemistry6-115 Jackson Hall, 321 Church St., SEMinneapolis MN 55455 United StatesPhone: 612-624-5988Email: [email protected]

Steven JacobsonNIH/NINDS/NIB10 Center Drive, Building 10, Room 5C103Bethesda MD 20892-1400 United StatesPhone: 301-496-1801Email: [email protected]

Jaideep KapurProfessor of NeurologyUniversity of Virginia Health Sciences CenterPO Box 800394Hospital Drive, McKim Hall, Rm. 2027Charlottesville VA 22908 United StatesPhone: 434-924-5312Email: [email protected]

Teemu KarlssonMicrobiologist (M.Sc.)Dept. of Virology, Helsinki University Hospital

University of HelsinkiHaartmaninkatu 3, FI-PL400 00029 HUSHelsinki FI-PL400 00029 HUS FinlandPhone: 358405127035Email: [email protected]

Benedikt B. KauferFreie Universtiät BerlinInstitut für VirologiePhilippstrasse 13, Haus 18Berlin 10555 GermanyPhone: 4915787602878Email: [email protected]

89

PARTICIPANT ROSTER

Akiko KawabataKobe University7-5-1, Kusunoki-cho, Chuo-kuKobe 650-0017 JapanPhone: 81783826272Email: [email protected]

Yoshiki KawamuraAssistantFujita Health UniversityToyoake 4701192 JapanPhone: 81562939251Email: [email protected]

Konstance KnoxWisconsin Viral Research Group10437 Innovation Drive, Suite 321Milwaukee WI 53226 United StatesPhone: 414-774-8612Email: [email protected]

Emil Kofod-OlsenPhD StudentAarhus UniversityWilhelm Meyers Allé 4Aarhus 8000 DenmarkPhone: 4589421773Email: [email protected]

Anthony L. KomaroffProfessor of MedicineHarvard Medical SchoolDepartment of MedicineHarvard Health Pubs. Countway, 2nd FloorBoston MA 2115 United StatesPhone: 617-432-4712Email: [email protected]

Timothy KowalikUMass Medical SchoolDept. of Microbiology and Physiological Systems55 Lake Ave. NorthWorcester MA 01655 United StatesPhone: 508-856-6035Email: [email protected]

Dirk LassnerLaboratory DirectorInstitute for Cardiac Diagnosis & TherapyMoltkestrasse 31Berlin D-12203 GermanyPhone: 493084415540Email: [email protected]

Irmeli LautenschlagerAsst. Professor of Clinical VirologyHelsinki University HospitalDepartment of VirologyHaartmaninkatu 3, PL 400Helsinki 00029 HUS FinlandPhone: 358919126346Email: [email protected]

Bennet LavensteinPhysicianChildren’s National Medical Center111 Michigan AvenueWashington DC 20010 United StatesPhone: 571-226-8368Email: [email protected]

Pul LiebermanProfessorThe Wistar InstituteGene Expression and Regulation3601 Spruce StreetPhiladelphia PA 19096 United StatesPhone: 215-898-9491Email: [email protected]

Janos LukaBioworld Consulting Laboratories, LLC3700 Franklinville Rd.New Windsor MD 21776 United StatesPhone: 410-635-8089Email: [email protected]

Mario LuppiUniversity of Modena and Reggio EmiliaDepartment of Oncology Hematology and Respiratory DiseasesVia del Pozzo, 71Modena I-41100 ItalyPhone: 39594225570Email: [email protected]

Paolo LussoSenior InvestigatorNIAID/NIHViral Pathogenesis Laboratory of ImmunoregulationBldg. 10, Rm. 6A11Bethesda MD 20892 United StatesPhone: 301-451-7495Email: [email protected]

Laurent MardivirinFaculté de Pharmacie de Limoges2 rue du Dr MarclandLimoges 87000 FrancePhone: 33675386065Email: [email protected]

90

PARTICIPANT ROSTER

Larissa MartinHelmholtz-Zentrum MünchenMarchioninistr. 25Munich 81377 GermanyPhone: 49897099236Email: [email protected]

Peter MedveczkyUniversity of South Florida12901 Bruce B. Downs Blvd., MDC 7Tampa FL 33612 United StatesPhone: 813-974-2372Email: [email protected]

Jose G. MontoyaAssociate ProfessorStanford University School of MedicineDepartment of Medicine300 Pasteur Drive, Room S-101, M/C 5107Stanford CA 94305 United StatesPhone: 650-853-4824Email: [email protected]

Andreas MoosmannUniversity of Munich / Helmholtz CenterMarchioninistr. 25Munich 81377 GermanyPhone: 49897099202Email: [email protected]

Yasuko MoriProfessorKobe University Graduate School of Medicine7-5-1, Kusunoki-cho, Chuo-kuKobe 650-0017 JapanPhone: 81783826272Email: [email protected]

Modra MurovskaAugust Kirchenstein Institute of Microbiology and

Virology, Riga Stradins UniversityRatsupites St. 5Riga LV-1067 LatviaPhone: 37167426197Email: [email protected]

Lieve NaesensLecturerRega Institute, Katholieke Universiteit

LeuvenDepartment of Microbiology and ImmunologyMinderroedersstraat 10Leuven B-3000 BelgiumPhone: 3216337345Email: [email protected]

Pitt NiehusmannResident PhysicianDept. of NeuropathologyUniversity of Bonn Medical CenterSigmund-Freud-Str. 25Bonn 53105 GermanyPhone: 4922828719027Email: [email protected]

Zaiga Nora-KrukleSenior ScientistAugust Kirchenstein Institute of Microbiology and

Virology, Riga Stradins UniversityRatsupites 5Riga LV-1067 LatviaPhone: 37167427920Email: [email protected]

Masao OgataAssistant ProfessorOita University Faculty of MedicineDepartment of HematologyHasama-machi Yufu-shiOita 879-5593 JapanPhone: 81975865804Email: [email protected]

Gwendolyn PainterChief Medical OfficerChimerix, Inc.2505 Meridian Parkway, Suite 340Durham NC 27713 United StatesPhone: 919-806-1074Email: [email protected]

Shara PantryUniversity of South Florida12901 Bruce B. Downs Blvd., MDC Box 7Tampa FL 33612 United StatesPhone: 813-974-5541Email: [email protected]

Philip E. PellettProfessorWayne State University, School of MedicineDepartment of Immunology and Microbiology6225 Scott HallDetroit MI 48201 United StatesPhone: 313-577-6494 ext.3135771595Email: [email protected]

Daniel PetersonPrincipal InvestigatorSierra Internal MedicineInternal Medicine865 Tahoe Boulevard, Suite 306Incline Village NV 89451 United StatesPhone: 775-832-0989Email: [email protected]

91

PARTICIPANT ROSTER

Bhupesh PrustyPost Doctoral FellowUniversity of Würzburg, GermanyDepartment of MicrobiologyBiozentrum Am HublandWürzburg 97074 GermanyPhone: 499313188067Email: [email protected]

Sylvie Ranger-RogezCHRU DupuytrenDepartment of Virology, EA3175, MENRLimoges cedex 87042 FrancePhone: 33555056721Email: [email protected]

Abdur RazzaqueSenior Regulatory ScientistCBER/ FDAOBRR, 1401 Rockville Pike, HFM-394Rockville MD 20852 United StatesPhone: 301-827-5355Email: [email protected]

Joséphine ReynaudStudentINSERM21, Avenue Tony GarnierLyon 69007 FrancePhone: 33437282397Email: [email protected]

Harold RiethmanAssociate ProfessorThe Wistar Institute3601 Spruce St.Philadelphia PA 19104 United StatesPhone: 215-898-3872Email: [email protected]

Corinna SchmittHannover Medical School, Institute of VirologyCarl-Neuberg-Str. 1Hannover 30625 GermanyEmail: [email protected]

Shlomo ShinnarProfessor of NeurologyMontefiore Medical CenterAlbert Einstein College of MedicineEpilepsy Center111 East 210th StreetBronx NY 10467 United StatesPhone: 718-920-4378Email: [email protected]

Volker StrengerDept. of Pediatrics and Adolescent Medicine Medical

University of GrazAuenbruggerplatz 30Graz 8036 AustriaPhone: 4369919488250Email: [email protected]

Huamin TangNational Institute of Biomedical Innovation7-6-8, Saito-AsagiIbaraki 567-0085 JapanPhone: 81726419012Email: [email protected]

William TheodoreNIHNIH Building 10, Room 7C-103Bethesda MD 20892 United StatesPhone: 301-496-1505Email: [email protected]

Jussi VirtanenPost-docNIH/NINDS10 Center Drive, Building 10, Room 5C103Bethesda MD 20892 United StatesPhone: 301-443-7409Email: [email protected]

Katherine WardConsultant Virologist and Honorary Senior LecturerRoyal Free University College Medical SchoolDepartment of Infection46 Cleveland StreetLondon W1T4JF United KingdomPhone: 4420767994909130Email: [email protected]

Jillian WohlerNIH10 Center Dr., Bldg. 10, Room 5C103, MSC 1400Bethesda MD 20892 United StatesPhone: 301-496-0519Email: [email protected]

Koichi YamanishiDirector GeneralNational Institute of Biomedical InnovationDepartment of Microbiology7-6-8, Saito-Asagi, IbarakiOsaka 567-0085 JapanPhone: 81726419810Email: [email protected]

92

PARTICIPANT ROSTER

Robert H. YolkenDirectorJohns Hopkins University School of MedicineStanley Neurovirology LaboratoryBlalock 1111600 Norh Wolfe StreetBaltimore MD 21287-4933 United StatesPhone: 410-955-3271Email: [email protected]

Tetsushi YoshikawaProfessor and ChairFujita Health UniversityDepartment of Pediatrics1-98 Dengakugakubo,Kutsukake-choToyoake Achi-Ken 470-1192 JapanPhone: 81562939251Email: [email protected]

Dong YuAssistant ProfessorWashington University School of MedicineDepartment of Molecular Microbiology,

Campus Box 8230660 South Euclid AvenueSt. Louis MO 63110-1093United StatesPhone: 314-362-7367Email: [email protected]

Haim ZeigermanTel Aviv UniversityRamat Aviv, PO Box 39040Tel Aviv 69978 IsraelPhone: 97236407166Email: [email protected]

Danielle ZerrAssociate ProfessorSeattle Children’s HospitalUniversity of WashingtonPediatric Infectious DiseasesSeattle Children’s HospitalSeattle WA 98105 United StatesPhone: 206-987-2653Email: [email protected]

Dong ZhouWest China Hospital / Sichuan UniversityDepartment of NeurologyNo. 37, Guoxue AlleyChengdu 610041 ChinaPhone: 86-028-85422114Email: [email protected]

93

Notes

94

Notes

95

Notes

96

Notes

97

HHV-6 Foundation Repository of Regents

98

99

HHV-6 Foundation Repository of HHV-6 & HHV-7 ReagentsHHV-6 Foundation Scientific Director and Co-Discoverer of HHV-6, Dharam Ablashi, asked the Foundation board to start a repository in 2005 to share reagents because he knew that it would be a great way to facilitate research. Six years later, we have over 760 vials of material stored in our repository at KamTek, Inc. in Gaithersburg, MD. The repository has truly been a cooperative effort that continues to grow. In the last few years we have acquired new cell lines, serum and monoclonal antibodies for the use of researchers. We send out over a hundred reagents to scientists around the world every year with the hopes that HHV-6 & 7 research will continue to push the boundaries and provide us with more answers about these viruses and their associated diseases.

The HHV-6 Foundation Repository offers infected and uninfected cell lines to HHV-6 & 7, monoclonal antibodies, limited human serum, plasma and tissue slides, and plasmid & cosmid clones. A complete list of repository items is available at the HHV-6 Foundation website (www.hhv-6foundation.org) or by contacting the Foundation. We ask an interested researcher to send an email with your research goals and a list of your requested reagents. We offer the reagents free of charge, but ask for a small processing fee to cover the cost of handling and packaging the reagents, as well as a Fedex account number for payment of the shipment. For more information, please contact Dharam Ablashi at 805-969-1174 or contact Jill Chase, Repository Manager: [email protected].

We would like to thank the scientists who so generously provided HHV-6 & 7 monoclonal antibodies, hybridomas, HHV-6 isolates and cells lines. They include Drs. Henri Agut, Pinaki Banerjee, Donald Carrigan, Bala Chandran, Kenny De Meirleir, Stephen Dewhurst, Louis Flamand, Jacqueline Friedman, Steven Jacobson, Konnie Knox, Gerhard Krueger, Susan Levine, Janos Luka, Mario Luppi, Yasuki Mori, Gary Pearson, Phil Pellett, Koichi Yamanishi, and Tetsushi Yoshikawa. Stocks to propagate HHV-6 and HHV-7 cell lines such as HSB2, SUPT1, and MOLT-3, were also contributed from Dharam Ablashi and Janos Luka. We ask scientists in the HHV-6 research community to please think about what you might be able to contribute to help others in the field.

We would also like to thank Sharan VedBrat and the staff at KamTek for their commitment to the HHV-6 Repository and for the professional care they give to our valuable materials.

The following two pages provide a list of our current inventory of monoclonal antibodies and infected cell lines available to scientists. A complete list of our inventory can be found at:http://hhv-6foundation.webs.com/repositoryinventory.htm

Thank you!

HHV-6 FOUNDATION REPOSITORY OF REAGENTS

100

HHV-6 & 7 INVENTORY OF INFECTED CELLS


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101

HHV-6 & 7 INVENTORY OF MONOCLONAL ANTIBODIES


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The HHV-6 Foundation Board’s Past

DHARAM ABLASHILIFETIME ACHIEVEMENT AWARD

WINNERS

Dharam Ablashi, 2006HHV-6 Foundation

USA

Koichi Yamanishi, 2008National Institute of Biomedical Innovation

Japan

Yoshizo Asano, 2011The Zambia Project, Hokkaido Univeristy

Fujita Health University (1979-2010)Japan

www.HHV-6Foundation.org

7th INTERNATIONAL Conference on HHV-6 & 7

Documents