Pneumo- and neurotropism of avian origin Italian highly pathogenic avian influenza H7N1 isolates in experimentally infected mice

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Virology xx (200

Pneumo- and neurotropism of avian origin Italian highly pathogenic avianinf luenza H7N1 isolates in experimentally infected mice

Michela Rigoni a, Kyoko Shinya b,c,d,e, Anna Toffan a, Adelaide Milani a, Francesca Bettini a,Yoshihiro Kawaoka d,e,f,g, Giovanni Cattoli a, Ilaria Capua a,⁎

a OIE, FAO and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie,Viale dell’Università 10, 35020 Legnaro, Padova, Italy

b Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japanc Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

d Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku,Tokyo 108-8639, Japan

e International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japanf Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

g Department of Pathological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA

Received 29 September 2006; returned to author for revision 10 November 2006; accepted 9 February 2007

Abstract

An experimental infection of mice was performed in order to investigate the potential for interspecies transmission in mammals of Italian HPAIviruses of the H7N1 subtype. Three avian origin isolates were selected, two strains obtained from ostrich (one of which contained a PB2-627Lysine residue) and one from a chicken. Following intranasal infection of mice, clinical signs and mortality were recorded in the experimentalgroups challenged with the two ostrich isolates, while only weight loss was observed in those receiving the chicken strain. Viruses were recoveredto a varying extent from respiratory and nervous tissues of infected animals. These results suggest that HPAI viruses, other than H5N1 and H7N7,may have zoonotic implications, and support the consensus that AI infections in poultry are to be eradicated rather than contained.© 2007 Elsevier Inc. All rights reserved.

Keywords: Highly pathogenic avian influenza; H7N1; Mouse model; Pneumotropism; Neurotropism; PB2

Introduction

Highly Pathogenic Avian influenza (HPAI) viruses pose aconcern both for public and animal health. Since 1999, there hasbeen a marked increase of the number of outbreaks in poultry,resulting in death or culling of millions of birds worldwide.Major poultry outbreaks include the Italian 1999–2000 H7N1(13 million birds), the Dutch 2003 H7N7 (30 million birds) theCanadian 2004 H7N3 (17 million birds) and the ongoing H5N1(unknown, over 100 million birds). From the public healthperspective, several of these viruses have crossed the speciesbarrier and infected human beings. In some instances this has

⁎ Corresponding author. Fax: +39 049 8084360.E-mail address: [email protected] (I. Capua).

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2007.02.031

Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian oriinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

resulted in a self-limiting conjunctivitis or influenza-like illnessand in others with a more severe condition, resulting in death ofthe individual (Capua and Alexander, 2004).

The Italian 1999–2000 H7N1 HPAI epidemic affected avariety of avian species over approximately 5 months, but therewas no evidence of infection of any non-avian species (Capuaand Mutinelli, 2001; Puzelli et al., 2005). The highly pathogenicvirus emerged from mutation of a low pathogenic strain of thesame subtype which had circulated in poultry for approximately9 months (Capua and Marangon, 2000). The HPAI virus waseradicated through stamping out of approximately 13 millioninfected and 3 million uninfected birds.

Sequence analysis of a selection of viruses from the Italianepidemic indicated that a natural isolate from an ostrich(Struthio camelus) had a lysine (K) residue at position 627 of

gin Italian highly pathogenic avian inf luenza H7N1 isolates in experimentally

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the PB2 protein (PB2-627K). This mutation is very rare in avianorigin isolates, the vast majority of which have a glutamic acid(E) residue in this position.

Several studies (Hatta et al., 2001; Subbarao et al., 1993)have established the crucial role of an amino acid residue atposition 627 of the viral protein PB2 (PB2-627), in determiningthe host range of influenza A virus of the H5N1 subtype. Inmice, although mutations other than that PB2-627 can enhanceviral replication and contribute to virus adaptation to thisanimal, the glutamic acid to lysine mutation at position 627 ofPB2 appears to be one of the most critical. Indeed, natural H5N1viruses isolated from felines and other mammals also possessPB2-627K, suggesting that lysine at this position provides agrowth advantage to the virus in mammalian hosts (Amonsin etal., 2006; Hatta et al., 2001). However, for ferrets, although themajority of lethal H5N1 viruses possess lysine, it has beenshown that some of the H5N1 viruses with glutamic acid at thisposition may also cause lethal infection (Maines et al., 2005;Salomon et al., 2006). Thus, the precise role of this mutationremains to be established, although the general consensus is thatit is acquired only after adaptation of avian viruses to themammalian hosts (Subbarao et al., 1993). In fact most humaninfluenza A viruses possess PB2-627K, including more thanhalf of the H5N1 viruses isolated from humans. In contrast,most highly pathogenic avian viruses possess PB2-627E, withthe notable exception of the H5N1 viruses isolated during anoutbreak among wild waterfowl in Qinghai Lake, China, in2005 (Chen et al., 2005) and the viral descendants of thoseresponsible for the outbreak (Chen et al., 2006).

The aim of this investigation was to evaluate thepathogenicity of selected Italian HP H7N1 isolates in amouse model. In detail, we examined whether the ostrichisolate with PB2-627K would exhibit enhanced virulence inmice, in comparison to ostrich and chicken isolates obtainedduring the same epidemic, possessing glutamic acid at 627 ofPB2 (PB2-627E).

Results

Clinical observations

The most severe clinical signs were observed in the group ofmice infected with OS/2332/00. On day 4 post-infection (p.i.),all the animals started presenting signs of depression, a ruffledcoat and anorexia. These non-specific signs of illness werefollowed by dyspnoea which became progressively more severeuntil day 7 p.i., day bywhichmice in this group started dying. Asplanned in the experimental protocol, two mice were euthanizedon days 3, 5, and 7 p.i. On day 7 two mice were found dead. Thetwo remaining animals of this group died on day 8 p.i. A meanweight loss of 40% was recorded in the group.

All mice infected with OS/984/00 started to show slightlyruffled coats from day 4 p.i., but were active with no additionalclinical signs until day 7 p.i. On this day, mice appeareddepressed, and started to show nervous signs such asincoordination and tremors. On day 8 p.i. one mouse diedwhereas the others, although displaying nervous signs, inappe-

Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian oriinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

tence and poor general condition, survived until day 10 p.i., bywhich time they were euthanized. At the end of the experimentthe group had undergone an average weight loss of 7.9%.

Mice infected with CK/5093/99 did not show any clinicalsigns throughout the duration of the experiment. None of themdied although they exhibited an average weight loss of 8%.

The uninfected control mice remained healthy throughoutthe duration of the experiment and did not register any weightloss (Fig. 1).

RRT–PCR results and virus isolation

Among the organs collected, viral RNA was detected onlyfrom lung/trachea and brain in all three experimental groups byRRT–PCR. Viable viruses were also isolated from lung/tracheaand brain (Table 1). Although viral replication was detected inthe same organs for all three viruses, replication of OS/2332/00was more prevalent than OS/984/00 and CK/5093/99 in thebrain. Thus, the former virus appeared to be more neuroinvasivethan the latter.

Histological examination

Although all viruses tested caused bronchitis, tracheitis,alveolitis, and brain lesions of various grades, they differed inthe timing of viral spread and the extent of viral replication andtropism in brain and lung. The extent of viral spread andreplication was as follows: (fast/severe) OS/2332/00≫OS/984/00>CK/5093/99 (late/mild) (Table 2 and Figs. 2 and 3). OS/2332/00 caused the most rapid lung and brain invasion.Antigens in the lung were detectable on day 3 p.i., but not onday 8 p.i. The brain lesions started to appear at day 3 p.i., andwere widespread at the time of death (8 days p.i.). For OS/984/00 lung and brain lesions started to be detected from day 5 p.i.In mice infected with CK/5093/99 lesions in lung were detectedonly from day 7 p.i. and in the brain only from one of the twomice on day 10 p.i.

Although all three viruses tested were pneumo- and neuro-pathogenic in mice, the strain possessing PB2-627K (OS/2332/00)spread more rapidly and caused more severe lesions than theothers.

Sequence analysis of isolates

The complete open reading frame (ORF) of the original strainsand of post-infection isolates were sequenced. The comparison ofthe full sequences indicates a high degree of homology for allgenes (Table 4). Amino acid sequencing of post-infection isolatesrevealed that no change occurred in OS/2332/00 whereas onlyone change at position 97 of PA (T→ I) was detected in OS/984/00. The significance of this mutation is unclear but sequenceanalysis of 2418 influenza viruses of various origins suggests thatthis mutation is not associated with host adaptation (data notshown). The post-infection isolate of CK/5093/99 obtained fromthe brain of one animal euthanized on day 10 p.i., displayed fouramino acid changes compared to the original isolate. Amino acidsequence of M1 gene showed one substitution R→K at position

gin Italian highly pathogenic avian inf luenza H7N1 isolates in experimentally

Fig. 1. Mean body weight of mice infected with avian H7N1 influenza viruses. Mice (n=10) were infected intranasally with 50 μl (105 EID50) of A/chicken/Italy/5093/1999, A/ostrich/Italy/984/2000, or A/ostrich/Italy/2332/2000. Control mice (n=6) were mock-infected. The mean body weight was calculated compared to bodyweight at the time of infection. Standard deviation error bars are shown.

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95; inPB2 gene, 3 changes occurred at positions 172 (V→ I), 489(S→P) and 627 (E→K) (Table 3). Notably the change at position627, fromE→K is in keepingwith other studies, which report theoccurrence of the same mutation in the brain followingexperimental infection of mice with avian-origin HP H5N1influenza viruses (Lipatov et al., 2003).

Discussion

In contrast to most other HPAI outbreaks, the Italian HPAIH7N1 virus affected a wide variety of avian species includingchickens, turkeys, quails, guinea fowl, Pekin ducks, Muscovy

Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian oriinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

ducks, geese and ostriches. Infection was never detected in anywild or domestic mammals and no evidence of seroconversionwas found in humans (Puzelli et al., 2005). In our experimenthowever, all the three H7N1 viruses were pathogenic for mice,although varying degrees of pathogenicity and neuroinvasive-ness were observed.

The most severe clinical and pathological findings wererecorded, as expected, as a result of infection with OS/2332/00,which is a natural avian isolate with a PB2-627K. The virus wasisolated and passaged only in SPF chicken embryonated eggs, andthus, did not have the opportunity to acquire the mutationfollowing multiplication in mammalian cells.

gin Italian highly pathogenic avian inf luenza H7N1 isolates in experimentally

Table 1Virus titre and real-time PCR results in organs of mice infected with avian H7N1HPAI viruses

Virus Day a Trachea and lung Brain

Viral isolation(EID50

log10/ml) b

RRT–PCRanalysis

Viral isolation(EID50

log10/ml) b

RRT–PCRanalysis

A/ostrich/Italy/2332/2000(PB2-627K)

3 + (<1) + − +5 + (1) + + (2.5) +7/a c + (1.5) + + (2.17) +7/b − + + (1) +8 − + + (3.17) +

A/ostrich/Italy/984/2000(PB2-627E)

3 − + − −5 + (1) + + (<1) +7 − + − +10/a c − − − +10/b − + − +

A/chicken/Italy/5093/1999(PB2-627E)

3 − + − +5 − + − −7 + (1.5) + − +10/a c + (2.4) + + (1.6) +10/b − − − −

a Mice were killed humanely and sampled on days 3, 5, 7, and 10 p.i. Amongthe mice infected with OS/984/00, one died on day 8 p.i. and was processed onlyfor histopathological examination. Mice infected with OS/2332/00 died allbetween 7 and 8 days p.i. None of the mice infected with CK/5093/99 died.b Calculated by the method of Reed and Muench from the results of serial

titrations of viruses in embryonated eggs, from lung and brain collected from asingle mouse in each experimental point.c Two mice were found dead or killed humanely and processed separately.

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The PB2-627K mutation is thought to occur following anadaptation of avian influenza A viruses to mammalian hosts.For example, this mutation was detected in an H7N7 virusisolated from a lethal case in a human, but not from non-lethal

Table 2Pathological findings of mice infected with H7N1 highly pathogenic influenzaviruses

Virus Day Trachea Lung Brain Liver Others a

A/ostrich/Italy/2332/2000(PB2-627K)

3 NAb ++/++ c −/+ −/− −/−5 NA ++/+ ++/++ −/− −/−7/a d ++/+ +/+ ++/+++ −/− Nerve

ganglion(−/+)

7/b +/+ −/− +++/+++ −/− −/−8 ++/++ −/− +/++ −/− −/−

A/ostrich/Italy/984/2000(PB2-627E)

3 −/− −/− −/− −/− −/−5 +/− +/+ +/+ −/− −/−7 −/− −/− +/+ −/− −/−8 +++/+ +/+ +/+++ −/− Fatty tissue

(+/+)10 −/− −/− ++/± −/− −/−

A/chicken/Italy/5093/1999(PB2-627E)

3 −/− −/− −/− −/− −/−5 ++/+ −/− −/− −/− −/−7 NA +/± −/− −/− −/−10/a d −/− +/+ ++/++ +/− −/−10/b −/− −/− −/− −/− −/−

a Including spleen, kidney, small intestine and large intestine.b Not analyzed.c Degree of inflammatory lesion/viral antigen distribution: +++, severe/global

to wide; ++, moderate/focal; +, mild/some; ±, sporadic/sparse; −, not detected.d Two mice were found dead or euthanized.

Fig. 2. Viral antigen detection by immunostaining using rabbit polyclonalantiserum to an H5N1 virus in lungs. (A) Viral antigen in the lungs of a mouseinfected with the OS/2332/00 virus was first detected 3 days after infection.Most of the bronchial epithelia were desquamated with significant amounts ofviral antigen. Peribronchitis was also apparent. (B) The lungs of a mouseinfected with the OS/984/00 virus 5 days after infection. A small cluster ofbronchial epithelium was positive for viral antigen (arrow). Peribronchiolitiswith lymphocyte recruitment was detected. (C) The lung of a mouse infectedwith the CK/5093/99 virus 7 days after infection. Mild thickening of the alveolarwall and peribronchitis was observed with scattered viral antigen (arrows).Bar=50 μm.

Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian oriinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

human cases or avian isolates obtained from chickens, duringthe HPAI outbreak in The Netherlands in 2003 (Fouchier et al.,2004). The same mutation was identified upon repeatedpassages of an H7N7 equine influenza virus in mice (Shinya

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et al., 2005; Shinya and Kawaoka, unpublished). Withreference to viruses of the H5N1 subtype, this mutation ispresent in isolates obtained from mammals including humans(Hatta et al., 2001; Maines et al., 2005), tigers (Amonsin et al.,2006), experimentally infected mice (Gao et al., 1999; Shinyaet al., 2004) and ferrets (Govorkova et al., 2005; Zitzow et al.,2002). As mentioned earlier, among avian origin HPAIisolates, only certain H5N1 viruses, isolated from wild birdsduring the Qinghai Lake outbreak in China (Chen et al., 2006;Zhou et al., 2006), also possess this mutation. In this case, it isunclear whether the presence of PB2-627K mutation occurredduring replication in the wild birds or whether wild birds havebeen infected with strains that had previously acquired themutation in mammalian hosts. Similarly, it is not possible toestablish with certainty whether OS/2332/00 acquired themutation in ostriches or whether it could have infectedostriches following undetected infection of mammals. How-ever, a support for this former possibility exists; another ostrichisolate, A/ostrich/South Africa/9508103/95(H9N2) possessesPB2-627K (GenBank accession number AF508640). It shouldbe noted that ostriches are phylogenetically very distant fromgallinaceous birds as they belong to a different superorder, andit is possible that this evolutionary distance could require anadaptive mutation of the virus.

In this respect, it is of interest that ostriches have been shownto have unique clinical behaviour to AI infections. Naturaloutbreaks with H7N1 LPAI (Allwright et al., 1993), H5N2HPAI in the Republic of South Africa (Olivier, 2006), andfollowing natural infection with an HPAI H7N1 in Italy (Capuaet al., 2000) have resulted in the development of a very similarclinical condition regardless of the virulence of the viruses forchickens. In all these episodes, mortality ranged between 20%and 30% and was restricted to juveniles. In contrast, aspreviously reported (Manvell et al., 2003; Clavijo et al., 2003)neither a H5N2 LPAI nor a H5N2 HPAI were able to causeclinical signs in experimentally infected ostrich chicks, despiteextensive viral replication in these species. Thus, in ostriches,both HPAI and LPAI viruses as tested in chickens causerelatively mild infection, which may in some cases be clinicallyunapparent. The reason for this peculiar behaviour of AIinfections in ostriches remains unclear.

The pathogenicity of the second ostrich isolate OS/984/00for mice appears to be of intermediate level compared to theother two isolates. Although this isolate has a PB2-627Eresidue, it replicated in the lung and brain and caused death ofone subject. The molecular basis for this neuro- and pneumo-invasiveness remains to be established and should be inves-tigated further.

Chicken isolate CK/5093/99 was the least pathogenic of theisolates and all mice survived infection. However one of theisolates recovered from the brain of one of the two miceeuthanized on day 10 p.i., revealed that a mutation of the PB2-627K variant had occurred. Histological results, indicatingreplication in the brain, are in-keeping with this finding.Experimental infections are in progress to assess whether thepathogenicity of this passage variant has increased. Furthermoresequencing analysis of post-infection isolates revealed three

Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian oriinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

other amino acid changes in PB2 and M1 protein (Table 3). Therole of these mutations remains unclear and will be furtherinvestigated by means of reverse genetics technique.

The potential of acquiring an E to K mutation at PB2-627does not appear to be an isolated event among Italian H7N1HPAI viruses. Among the three viruses tested, one of the ostrichisolates had naturally acquired it, while the chicken strainacquired the mutation after one passage in mice. This evidencesuggests that the genetic predisposition to the acquisition ofvirulence for mammals in HPAI viruses, other than H5N1 andH7N7, may be much more widespread than expected.Furthermore, the findings suggest that the PB2-627 E to Kmutation may occur naturally in certain avian species such asostriches, supporting the general consensus that HPAI infectionsmust be eradicated from poultry rather than only contained.

Materials and methods

Viruses

Highly pathogenic avian influenza A viruses of the H7N1subtype, isolated in Italy during the 1999–2000 epidemic, wereused in this study.

A/ostrich/Italy/2332/00 (H7N1)(OS/2332/00) virus was iso-lated from a natural outbreak in an ostrich farm, and has PB2-627K. A/ostrich/Italy/984/00 (H7N1)(OS/984/00) was isolatedfrom an unrelated ostrich farm and has PB2-627E. A/chicken/Italy/5093/99(H7N1)(CK/5093/99) virus was isolated from achicken farm and also has PB2-627E.

Stocks for each of these viruses were propagated in theallantoic cavity of 9- to 11-day-old embryonated-specificpathogen-free eggs. Allantoic fluid was harvested, clarified bycentrifugation, aliquoted and stored at −80 °C. The doserequired to infect 50% of eggs (EID50) for each strain wascalculated by the formula of Reed and Muench (1938).

Laboratory facilities

All experiments using live viruses were conducted in BSL3containment facilities.

Animal experiment

Balb/C mice (6- to 8-week-old females; Charles RiverLaboratories) were divided in three different groups of ten each,while a fourth group of six animals was used as a control. Theanimals were infected intranasally with 50 μl of a suspensioncontaining 105 EID50 of each of the three viruses. The controlgroup received 50 μl of uninfected allantoic fluid. On days 3, 5and 7 p.i. two mice from each group were euthanized in order tocollect organs, namely lung and trachea (examined together),brain, spleen, liver, kidney and intestine. On day 10 p.i. all thesurviving animals were sacrificed. On the sampling days, theorgans collected from one mouse were processed for virologicalassay and real-time reverse transcriptase polymerase chainreaction (RRT–PCR) analysis, whereas the organs from theother animal were collected for histological investigations. In case

gin Italian highly pathogenic avian inf luenza H7N1 isolates in experimentally

Fig. 3. Antigen distribution pattern in the brain detected by immunostaining using rabbit polyclonal antiserum to an H5N1 virus. (A) The brain of a mouse infected withthe CK/5093/99 virus 10 days after infection. Viral antigen was limited to the area around the ventricles (V). (B) The brain of a mouse infected with the OS/984/00virus 8 days after infection. Scattered viral antigen was observed around the ventricles and parenchyma. (C) Another area of the brain of the mouse infected with theOS/984/00 virus 8 days after infection. Viral antigen was widely distributed in the parenchyma. (D) Wide parenchymal and periventricular distribution of viral antigenin the brain of a mouse infected with the OS/2332/00 strain 7 days after infection. Bar=400 μm.

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Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian origin Italian highly pathogenic avian inf luenza H7N1 isolates in experimentallyinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

Table 4GenBank accession numbers

Gene Accession # Virus

PB2 DQ991301 A/ostrich/Italy/2332/00(H7N1) p.i. in mousePB1 DQ991302 A/ostrich/Italy/2332/00(H7N1) p.i. in mousePA DQ991303 A/ostrich/Italy/2332/00(H7N1) p.i. in mouseHA DQ991304 A/ostrich/Italy/2332/00(H7N1) p.i. in mouseNP DQ991305 A/ostrich/Italy/2332/00(H7N1) p.i. in mouseNA DQ991306 A/ostrich/Italy/2332/00(H7N1) p.i. in mouseMA DQ991307 A/ostrich/Italy/2332/00(H7N1) p.i. in mouseNS DQ991308 A/ostrich/Italy/2332/00(H7N1) p.i. in mousePB2 DQ991309 A/ostrich/Italy/2332/00(H7N1)PB1 DQ991310 A/ostrich/Italy/2332/00(H7N1)PA DQ991311 A/ostrich/Italy/2332/00(H7N1)HA DQ991312 A/ostrich/Italy/2332/00(H7N1)NP DQ991313 A/ostrich/Italy/2332/00(H7N1)NA DQ991314 A/ostrich/Italy/2332/00(H7N1)MA DQ991315 A/ostrich/Italy/2332/00(H7N1)NS DQ991316 A/ostrich/Italy/2332/00(H7N1)PB2 DQ991317 A/chicken/Italy/5093/99(H7N1)PB1 DQ991318 A/chicken/Italy/5093/99(H7N1)PA DQ991319 A/chicken/Italy/5093/99(H7N1)HA DQ991320 A/chicken/Italy/5093/99(H7N1)NP DQ991321 A/chicken/Italy/5093/99(H7N1)NA DQ991322 A/chicken/Italy/5093/99(H7N1)MA DQ991323 A/chicken/Italy/5093/99(H7N1)NS DQ991324 A/chicken/Italy/5093/99(H7N1)PB2 DQ991325 A/chicken/Italy/5093/99(H7N1) p.i. in mousePB1 DQ991326 A/chicken/Italy/5093/99(H7N1) p.i. in mousePA DQ991327 A/chicken/Italy/5093/99(H7N1) p.i. in mouseHA DQ991328 A/chicken/Italy/5093/99(H7N1) p.i. in mouseNP DQ991329 A/chicken/Italy/5093/99(H7N1) p.i. in mouseNA DQ991330 A/chicken/Italy/5093/99(H7N1) p.i. in mouseMA DQ991331 A/chicken/Italy/5093/99(H7N1) p.i. in mouseNS DQ991332 A/chicken/Italy/5093/99(H7N1) p.i. in mousePB2 DQ991333 A/ostrich/Italy/984/00(H7N1)PB1 DQ991334 A/ostrich/Italy/984/00(H7N1)PA DQ991335 A/ostrich/Italy/984/00(H7N1)HA DQ991336 A/ostrich/Italy/984/00(H7N1)NA DQ991337 A/ostrich/Italy/984/00(H7N1)MA DQ991338 A/ostrich/Italy/984/00(H7N1)NS DQ991339 A/ostrich/Italy/984/00(H7N1)NP EF376184 A/ostrich/Italy/984/00(H7N1)PB2 DQ991340 A/ostrich/Italy/984/00(H7N1) p.i. in mousePB1 DQ991341 A/ostrich/Italy/984/00(H7N1) p.i. in mousePA DQ991342 A/ostrich/Italy/984/00(H7N1) p.i. in mouseHA DQ991343 A/ostrich/Italy/984/00(H7N1) p.i. in mouseNP DQ991344 A/ostrich/Italy/984/00(H7N1) p.i. in mouseNA DQ991345 A/ostrich/Italy/984/00(H7N1) p.i. in mouseMA DQ991346 A/ostrich/Italy/984/00(H7N1) p.i. in mouseNS DQ991347 A/ostrich/Italy/984/00(H7N1) p.i. in mouse

p.i.: post-infection.

Table 3Amino acid differences between HP H7N1 inoculum and post-infection isolates

Virus Amino acid position

PB2 PA M

172 286 489 591 627 97 95

A/ostrich/Italy/2332/2000 (pre-infection) V S S Q K T RA/ostrich/Italy/2332/2000 (8 days

post-infection)V S S Q K T R

A/chicken/Italy/5093/1999 (pre-infection) V G S Q E T RA/chicken/Italy/5093/1999 (10 days

post-infection)I G P Q K T K

A/ostrich/Italy/984/2000 (pre-infection) V G S K E T RA/ostrich/Italy/984/2000 (5 days

post-infection)V G S K E I R

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of mortality the organs were collected within 8 h of death andprocessed as described above. All mice were weighed daily.

Virological assay

Samples for virological examination were homogenized toobtain a 10% w/v suspension in phosphate-buffered saline(0.05 M to pH 7.0–7.4) with antibiotics (penicillin: 10000 IU/ml,streptomycin: 10 mg/ml, nystatin: 5000 UI/ml, gentamycinsulphate: 250 μg/ml) and clarified by centrifugation. Theundiluted and tenfold serially diluted supernatants were usedfor the titration of virus infectivity in embryonated eggsaccording to EU guidelines (CEC, 1992).

Pathological examination

Tissues (liver, spleen, kidney, lung, trachea, intestine, andbrain) were harvested from animals and fixed in 10% phosphate-buffered formalin. They were dehydrated, embedded in paraffin,and cut into 5-μm-thick sections. Sections were stained withhematoxylin-and-eosin using a standard method. For viralantigen detection, sections were processed for immunostainingby using a two-step dextran polymer method (DAKO Japan Inc.,Kyoto) and a rabbit polyclonal antibody to A/Vietnam/1203/04(H5N1) as the primary antibody.

RRT–PCR analysis

Samples for genetic analyses were homogenized in 0.5 ml ofphosphate-buffered saline. Total RNA was prepared using thecommercial kit (High Pure™ RNA extraction kit; RocheDiagnostics GmbH, Mannheim, Germany) and cDNA wassynthesized with “High capacity cDNA archive kit” (AppliedBiosystems) according to the manufacturer's instructions.

An influenza virus matrix gene-specific PCR primer set –M+25 and M−124 – and hydrolysis probe M+64 (Spackman etal., 2002) were used in real-time PCR to detect virus. cDNAwasamplified in a final volume of 25 μl using TaqMan UniversalPCR Master Mix 1X (Applied Biosystems), 300 nM of eachprimer and 100 nm of the probe.

The real-time PCR reaction was performed in a 7300 Real-Time PCR System (Applied Biosystem) with the following

Please cite this article as: Rigoni, M., et al., Pneumo- and neurotropism of avian oriinfected mice, Virol. (2007), doi:10.1016/j.virol.2007.02.031

protocol: 2 min at 50 °C and 10 min at 95 °C followed by40 cycles at 95 °C for 15 s and 60 °C for 1 min.

Sequence analysis

The ORF regions of the eight genome segments of theoriginal strains and of post-infection isolates were fullysequenced essentially as described (Hoffman et al., 2001).DNA sequencing was performed using a 3130xl GeneticAnalyzer and BigDye Terminator Mix v3.1 (Applied Biosys-tems). All sequences of pre- and post-infection isolates havebeen deposited in GenBank (Table 4).

gin Italian highly pathogenic avian inf luenza H7N1 isolates in experimentally

8 M. Rigoni et al. / Virology xx (2007) xxx–xxx

ARTICLE IN PRESS

Acknowledgments

This work was funded by the Italian Ministry of Health(RC IZSVE 03-02), by a project coordinated by the PoliclinicoSan Matteo of Pavia (Italy) (ICSO3O.4RF2004/118), by theEU funded project FLUPATH (contract CT 044220), byCREST (Japan Science and Technology Agency) and bygrants-in-aid from the Ministries of Education, Culture, Sports,Science, and Technology and of Health, Labor, and Welfare ofJapan and by National Institute of Allergy and InfectiousDisease Public Health Service research grants. The authorsgratefully acknowledge Dr F. Mutinelli for carrying out post-mortem examinations, Dr M. Mancin for performing statisticalanalysis and Drs D.J. Alexander and G. Gerna for the criticalrevision of the final manuscript.

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