The Antiviral Activity of The Antiviral Activity of Oseltamivir against H5N1 Human Oseltamivir against H5N1 Human A/Vietnam/1203/04 and A/Turkey/15/06 A/Vietnam/1203/04 and A/Turkey/15/06 Influenza Viruses in Ferrets Influenza Viruses in Ferrets Elena Govorkova, Elena Govorkova, N.A. Ilyushina, D.A. Boltz, J.L. N.A. Ilyushina, D.A. Boltz, J.L. McClaren, McClaren, A. Douglas, N. Yilmaz, and R.G. Webster A. Douglas, N. Yilmaz, and R.G. Webster
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The Antiviral Activity of Oseltamivir against H5N1 Human A/Vietnam/1203/04 and A/Turkey/15/06 Influenza Viruses in Ferrets Elena Govorkova, N.A. Ilyushina,
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The Antiviral Activity of Oseltamivir against The Antiviral Activity of Oseltamivir against
H5N1 Human A/Vietnam/1203/04 and H5N1 Human A/Vietnam/1203/04 and
A/Turkey/15/06 Influenza Viruses in FerretsA/Turkey/15/06 Influenza Viruses in Ferrets
A. Douglas, N. Yilmaz, and R.G. WebsterA. Douglas, N. Yilmaz, and R.G. Webster
Recommendations for Tamiflu prophylaxis Recommendations for Tamiflu prophylaxis and treatment are based on the data from and treatment are based on the data from seasonal influenza viruses (H1N1, H3N2, B)seasonal influenza viruses (H1N1, H3N2, B)
H5N1 influenza viruses have a potential for H5N1 influenza viruses have a potential for systemic spread and involvement of multiple systemic spread and involvement of multiple organsorgans
Limited information is available on the Limited information is available on the efficacy of oseltamivir against H5N1 infection efficacy of oseltamivir against H5N1 infection in the fieldin the field
Initial mouse studies suggest that prolonged Initial mouse studies suggest that prolonged oseltamivir treatment is required for most oseltamivir treatment is required for most beneficial protectionbeneficial protection
Oseltamivir Therapy for H5N1 Virus InfectionOseltamivir Therapy for H5N1 Virus Infection
Clade 1: A/Vietnam/1203/04 Fatal human case Lethal infection in ferrets
Clade 2 Subclade 2: A/Turkey/15/06 Fatal human caseNon- Lethal infection in ferrets
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 21 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 21
Oseltamivir 2x daily for 5 daysOseltamivir 2x daily for 5 days
Infect withInfect with10 or 100 EID10 or 100 EID5050
A/VN/1203/04 A/VN/1203/04
5 mg/kg/d (equiv. to half approved human dose of 75 mg bid)5 mg/kg/d (equiv. to half approved human dose of 75 mg bid)
↔↔4 hrs4 hrs
Early Post-exposure Treatment: SurvivalEarly Post-exposure Treatment: Survival
VirusVirus DoseDose
Experimental Experimental GroupGroup
No. dead/No. dead/Total no.Total no.
Day of Day of DeathDeath
10 EID10 EID
5050
Treatment Treatment 0/30/3 NoNo
ControlControl 2/32/3 10, 1010, 10
100 EID100 EID5050
TreatmentTreatment 0/30/3 NoNo
ControlControl 3/33/3 7, 7, 97, 7, 9
0
1
2
3
4
5
6
7
Treatment ControlTreatment Control
Vir
us
tite
r, l
og
10E
ID50
/ml
Day 3 Day 5 Day 7Day 3 Day 5 Day 7 Day 3 Day 5 Day 7Day 3 Day 5 Day 7 Day 3 Day 5 Day 7 Day 3 Day 5 Day 7
Virus Replication in Upper Respiratory TractVirus Replication in Upper Respiratory Tract
Treatment ControlTreatment Control
10 EID10 EID5050 100 EID100 EID5050
* P<0.05* P<0.05
* * ** * * * * ** * *
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
Vir
us
tite
r, lo
gV
iru
s ti
ter,
log
1010E
IDE
ID505
0/g/g
10 EID10 EID5050100 EID50
Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.
1/2 1/2 1/21/2
Note: In treatment groups, virus was detected in 1/2 ferretsNote: In treatment groups, virus was detected in 1/2 ferrets In control groups, virus was detected in 2/2 ferretsIn control groups, virus was detected in 2/2 ferrets* P<0.05
TreatmentTreatment
ControlControl
TreatmentTreatment
ControlControl
Virus Replication in Internal OrgansVirus Replication in Internal Organs
** ** ** ** **
Detection of Resistant VariantsDetection of Resistant Variants
VirusVirus
DoseDose
Origin ofOrigin of
SampleSample
Amino acid changeAmino acid change
NANA HA1HA1
10 10 EIDEID5050 BrainBrain I418M*I418M* ____
100 100 EIDEID5050 BrainBrain
LiverLiver
SpleenSpleen
____ ____
− − no mutations; * confirmed by TOPO TA cloningno mutations; * confirmed by TOPO TA cloning
I418MI418M
N2 NA numbering (Colman et al, J. Virol.,1993)N2 NA numbering (Colman et al, J. Virol.,1993)
No changes in Oseltamivir susceptibility No changes in Oseltamivir susceptibility in vitroin vitro
Immunostaining: Virus Detection in the BrainImmunostaining: Virus Detection in the Brain
ControlControl TreatmentTreatment
Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Cells positive for viral antigen have a dark-brown granular appearance; Cells positive for viral antigen have a dark-brown granular appearance; viral distribution is shown by red shading (insets).viral distribution is shown by red shading (insets).
50 microns
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21
Oseltamivir 2x daily for 5 daysOseltamivir 2x daily for 5 days
Infect withInfect with100 EID100 EID5050
A/VN/1203/04 A/VN/1203/04
25 mg/kg/d 25 mg/kg/d 24 hours24 hoursDelayDelay
Re-infection with Lethal H5N1 Virus DoseRe-infection with Lethal H5N1 Virus Dose
All animalsAll animalssurvived lethalsurvived lethal
challengechallenge
Detection of Resistant VariantsDetection of Resistant Variants
DoseDose Origin ofOrigin of
SampleSample
Amino acid changeAmino acid change
NANA HA1HA1
10 mg/kg/d10 mg/kg/d
Nasal washNasal wash
Lungs Lungs
V116AV116A(1/20)(1/20)
H274YH274Y(1/10)(1/10)
V178IV178I(1/20)(1/20)
____
Nasal washNasal wash ____ NDND
25 mg/kg/d25 mg/kg/d
BrainBrain H274RH274R(1/10)(1/10)
E277QE277Q(1/10)(1/10)
NDND
− − no mutations; ND – not doneno mutations; ND – not doneDetected by TOPO TA cloning and analysis of individual plaquesDetected by TOPO TA cloning and analysis of individual plaques
H274YH274Y
E277QE277QE277QE277Q
N2 NA numbering (Colman et al, J. Virol.,1993)N2 NA numbering (Colman et al, J. Virol.,1993)
No changes in Oseltamivir susceptibility No changes in Oseltamivir susceptibility in vitroin vitro
Note. 10 mg/kg/d in a ferret model equiv. to approved human dose of 75 mg bid Note. 10 mg/kg/d in a ferret model equiv. to approved human dose of 75 mg bid
Virus Replication in Virus Replication in Upper Respiratory TractUpper Respiratory Tract
0
1
2
3
4
5
10 mg/kg/day
Control
# of Inflammatory cells# of Inflammatory cells
Inflammatory Responses in Upper Inflammatory Responses in Upper Respiratory TractRespiratory Tract
3 5 73 5 7Days post-challengeDays post-challenge
0
2
4
6
8
1010 mg/kd/day
Control
Cel
l co
un
t (n
um
ber
x 1
0C
ell c
ou
nt
(nu
mb
er x
1066 /
ml)
/ml)
3 5 73 5 7Days post-challengeDays post-challenge
Protein concentrationProtein concentration
****
**
**
**
* P<0.05
Virus Replication in Internal OrgansVirus Replication in Internal Organs
Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.0
0.5
1
1.5
2
2.5
3
TreatmentTreatment
ControlControl
2/22/2 1/21/2
** **
* P<0.05
Immunostaining: Virus Detection in the BrainImmunostaining: Virus Detection in the Brain
ControlControl TreatmentTreatment
Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Cells positive for viral antigen have a dark-brown granular appearance; Cells positive for viral antigen have a dark-brown granular appearance; viral distribution is shown by red shading (insets). viral distribution is shown by red shading (insets).
50 microns
Detection of Resistant VariantsDetection of Resistant Variants
DoseDose Origin ofOrigin of
SampleSample
Amino acid changeAmino acid change
NANA HA1HA1
10 mg/kg/d10 mg/kg/d Nasal wash Nasal wash
── R193KR193K− − no mutationsno mutations
No changes in Oseltamivir susceptibility No changes in Oseltamivir susceptibility in vitroin vitro
R193KR193K
SummarySummary
Oseltamivir treatment (25 mg/kg/d) protects ferrets against lethal challenge and further re-infection with A/VN/1203/04 (H5N1) virus;
Oseltamivir (10 mg/kg/d) reduces lethargy of animals, inhibits inflammation and blocks A/Turkey/15/06 (H5N1) virus spread to internal organs;
Virulence may affect the antiviral treatment schedule and higher oseltamivir dosages may be required against more pathogenic virus;
Early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 influenza viruses;
Oseltamivir-resistant variants were not detected by direct Oseltamivir-resistant variants were not detected by direct sequencing. Analysis of individual viral clones detected a sequencing. Analysis of individual viral clones detected a minor population of clones carrying NA and/or HA mutations, minor population of clones carrying NA and/or HA mutations, although changes in drug susceptibility were not determined.although changes in drug susceptibility were not determined.
Support: NIAID, NIH (Grants AI-95357, AI-70005 and AI-57570); ALSAC ; F. Hoffmann-La Roche
St. Jude Children’s Research Hospital:
Robert G. Webster Natalia IlyushinaNatalia Ilyushina
David BoltzDavid BoltzLana McClarenLana McClaren
Oseltamivir Expert Working GroupOseltamivir Expert Working Group Frederick G. Hayden Noel RobertsFrederick G. Hayden Noel RobertsArnold S. Monto James SmithArnold S. Monto James Smith
Albert D.M.E. Osterhaus Ron A.M. FouchierAlbert D.M.E. Osterhaus Ron A.M. Fouchier
T.D. Nguyen ((Vietnamese Ministry of Agric. and Rural Health Develop.)Neziha Yilmaz (Virology and NIC of Turkey Refic Saydam Hygiene Inst.)