PHYLOGENETICANALYSISOFHA1 PROTEINOFINFLUENZAA (H1N1)
ISOLATESOBTAINEDINKENYAIN2007-2008
Meshack Wadegu
USAMRU-KENYA
Introduction
•
Type A influenza viruses are unique among influenza viruses:
1.
-Show the greatest genetic diversity
2.
-Infect widest range of host species
3.
-Cause most of the severe disease in humans including the great
pandemics.
Introduction cont’
•
Among influenza A viruses, influenza A/H1N1 viruses have been
responsible for the pandemics of 1918 and 2009.
•
Amino acid variants of the HA1 domain enable virus to evade immunity,
replicate and infect more hosts.
•
Anecdotal reports of a mismatch in the H1N1 component in the 2008
vaccine highlighted need to analyze Kenyan isolates.
USAMRU-K Kenya Influenza Sentinel Surveillance Site Network
•
Mbagathi District Hospital (DH)
•
Malindi DH
•
Kondele Children’s Hospital
•
Isiolo DH
•
Port Reitz DH
•
Alupe Sub DH
•
Kericho DH
•
Kisii DH
4
Laboratory Analysis
•
ILI specimens delivered in Liquid Nitrogen once a week and initially
screened and subtyped by real time RT-PCR
•
PCR Machine provided by CDC
•
Isolations performed in MDCK cells followed by HAI assays using guinea
pig RBCs and reference antisera
•
Nucleotide sequencing by US Air Force School of Aerospace Medicine or
commercially
5
1 2 4 8 16 32 64 128 256 512 1024 C
A/H1N1
A/H3N2
Bs
Bm
Results (epidemiologic)
•
6044 influenza like illness(ILI) specimen were collected
•
1643 were positive for influenza by Real Time PCR
•
Using RT-PCR assay, 21.9%(1322/6044) of ILI cases were caused by
influenza A
•
140 influenza A isolates were obtained
•
37.1%(52/140) of the influenza A isolates were H1N1 subtype
Strategy of Phylogenetic analysis
•
Nucleotide (nt) sequencing of PCR amplified HA1 gene segment was
carried out.
•
Amino acid sequence from conceptual translation of ntwere used for
phylogeneticanalysis
•
The comparison with WHO-recommended vaccine strains.
•
Multiple sequence alignment done using Muscle 3.6
•
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Phylogeny carried out using MrBayes
0.0040A_Mbagathi_4825_2007H1N1A_Mbagathi_4844_2007H1N1A_Kericho_4831_2007H1N1A_Malindi_4836_2007H1N1A_Mbagathi_4834_2007H1N1A_Kisii_4839_2007H1N1A_Solomon_Islands_3_2006H1N1A_Brisbane_59_2007H1N1A_Kisumu_4830_2007H1N1A_Mbagathi_4826_2007H1N1A_Mbagathi_4840_2007H1N1A_Malindi_4837_2007H1N1A_Kisii_4823_2007H1N1A_New_Caledonia_20_99H1N1A_Kericho_4822_2007H1N1A_Malindi_4835_2007H1N1A_Mbagathi_4827_2007H1N155%99%58%100%100%70%
2007 Season
K273E
D221G
Q104H
S121N
0.0040A_Mbagathi_4825_2007H1N1A_Mbagathi_4844_2007H1N1A_Kericho_4831_2007H1N1A_Malindi_4836_2007H1N1A_Mbagathi_4834_2007H1N1A_Kisii_4839_2007H1N1A_Solomon_Islands_3_2006H1N1A_Brisbane_59_2007H1N1A_Kisumu_4830_2007H1N1A_Mbagathi_4826_2007H1N1A_Mbagathi_4840_2007H1N1A_Malindi_4837_2007H1N1A_Kisii_4823_2007H1N1A_New_Caledonia_20_99H1N1A_Kericho_4822_2007H1N1A_Malindi_4835_2007H1N1A_Mbagathi_4827_2007H1N155%99%58%100%100%70%
2007 Season
K273E
D221G
Q104H
S121N
0.0040A_Mbagathi_4825_2007H1N1A_Mbagathi_4844_2007H1N1A_Kericho_4831_2007H1N1A_Malindi_4836_2007H1N1A_Mbagathi_4834_2007H1N1A_Kisii_4839_2007H1N1A_Solomon_Islands_3_2006H1N1A_Brisbane_59_2007H1N1A_Kisumu_4830_2007H1N1A_Mbagathi_4826_2007H1N1A_Mbagathi_4840_2007H1N1A_Malindi_4837_2007H1N1A_Kisii_4823_2007H1N1A_New_Caledonia_20_99H1N1A_Kericho_4822_2007H1N1A_Malindi_4835_2007H1N1A_Mbagathi_4827_2007H1N155%99%58%100%100%70%
2007 Season
K273E
D221G
Q104H
S121N
0.0040A_Mbagathi_4825_2007H1N1A_Mbagathi_4844_2007H1N1A_Kericho_4831_2007H1N1A_Malindi_4836_2007H1N1A_Mbagathi_4834_2007H1N1A_Kisii_4839_2007H1N1A_Solomon_Islands_3_2006H1N1A_Brisbane_59_2007H1N1A_Kisumu_4830_2007H1N1A_Mbagathi_4826_2007H1N1A_Mbagathi_4840_2007H1N1A_Malindi_4837_2007H1N1A_Kisii_4823_2007H1N1A_New_Caledonia_20_99H1N1A_Kericho_4822_2007H1N1A_Malindi_4835_2007H1N1A_Mbagathi_4827_2007H1N155%99%58%100%100%70%
2007 Season
K273E
D221G
Q104H
S121N
0.0040A_Kisii_7543_2008A_Kisumu_5928_2008A_Kericho_7530_2008A_Mbagathi_7596_2008A_Kericho_7535_2008A_Kisii_7565_2008A_Kisii_7577_2008A_Kisii_5886_2008A_Kisii_7559_2008A_Kisumu_5933_2008A_Kisii_7541_2008A_Kisumu_7611_2008A_Solomon_Islands_3_2006A_Kisii_7576_2008A_Kisii_7562_2008A_Kisii_7554_2008A_Kisii_7570_2008A_Kisumu_5923_2008A_Kisii_7547_2008A_Kenya_6901_2008A_Kericho_7529_2008A_Brisbane_59_2007A_Kericho_7525_2008A_Kisii_5896_2008A_New_Caledonia_20_99A_Kisumu_5934_2008A_Kisumu_6543_200898%84%99%100%100%94%
2008 Season
0.0040A_Kisii_7543_2008A_Kisumu_5928_2008A_Kericho_7530_2008A_Mbagathi_7596_2008A_Kericho_7535_2008A_Kisii_7565_2008A_Kisii_7577_2008A_Kisii_5886_2008A_Kisii_7559_2008A_Kisumu_5933_2008A_Kisii_7541_2008A_Kisumu_7611_2008A_Solomon_Islands_3_2006A_Kisii_7576_2008A_Kisii_7562_2008A_Kisii_7554_2008A_Kisii_7570_2008A_Kisumu_5923_2008A_Kisii_7547_2008A_Kenya_6901_2008A_Kericho_7529_2008A_Brisbane_59_2007A_Kericho_7525_2008A_Kisii_5896_2008A_New_Caledonia_20_99A_Kisumu_5934_2008A_Kisumu_6543_200898%84%99%100%100%94%
2008 Season
0.0040A_Kisii_7543_2008A_Kisumu_5928_2008A_Kericho_7530_2008A_Mbagathi_7596_2008A_Kericho_7535_2008A_Kisii_7565_2008A_Kisii_7577_2008A_Kisii_5886_2008A_Kisii_7559_2008A_Kisumu_5933_2008A_Kisii_7541_2008A_Kisumu_7611_2008A_Solomon_Islands_3_2006A_Kisii_7576_2008A_Kisii_7562_2008A_Kisii_7554_2008A_Kisii_7570_2008A_Kisumu_5923_2008A_Kisii_7547_2008A_Kenya_6901_2008A_Kericho_7529_2008A_Brisbane_59_2007A_Kericho_7525_2008A_Kisii_5896_2008A_New_Caledonia_20_99A_Kisumu_5934_2008A_Kisumu_6543_200898%84%99%100%100%94%
2008 Season
0.0040A_Kisii_7543_2008A_Kisumu_5928_2008A_Kericho_7530_2008A_Mbagathi_7596_2008A_Kericho_7535_2008A_Kisii_7565_2008A_Kisii_7577_2008A_Kisii_5886_2008A_Kisii_7559_2008A_Kisumu_5933_2008A_Kisii_7541_2008A_Kisumu_7611_2008A_Solomon_Islands_3_2006A_Kisii_7576_2008A_Kisii_7562_2008A_Kisii_7554_2008A_Kisii_7570_2008A_Kisumu_5923_2008A_Kisii_7547_2008A_Kenya_6901_2008A_Kericho_7529_2008A_Brisbane_59_2007A_Kericho_7525_2008A_Kisii_5896_2008A_New_Caledonia_20_99A_Kisumu_5934_2008A_Kisumu_6543_200898%84%99%100%100%94%
2008 Season
#G185N Sb
E273K
0.0040A_Kisii_7543_2008A_Kisumu_5928_2008A_Kericho_7530_2008A_Mbagathi_7596_2008A_Kericho_7535_2008A_Kisii_7565_2008A_Kisii_7577_2008A_Kisii_5886_2008A_Kisii_7559_2008A_Kisumu_5933_2008A_Kisii_7541_2008A_Kisumu_7611_2008A_Solomon_Islands_3_2006A_Kisii_7576_2008A_Kisii_7562_2008A_Kisii_7554_2008A_Kisii_7570_2008A_Kisumu_5923_2008A_Kisii_7547_2008A_Kenya_6901_2008A_Kericho_7529_2008A_Brisbane_59_2007A_Kericho_7525_2008A_Kisii_5896_2008A_New_Caledonia_20_99A_Kisumu_5934_2008A_Kisumu_6543_200898%84%99%100%100%94%
2008 Season
A189T Sb
#G185N Sb
E273K
0.0040A_Kisii_7543_2008A_Kisumu_5928_2008A_Kericho_7530_2008A_Mbagathi_7596_2008A_Kericho_7535_2008A_Kisii_7565_2008A_Kisii_7577_2008A_Kisii_5886_2008A_Kisii_7559_2008A_Kisumu_5933_2008A_Kisii_7541_2008A_Kisumu_7611_2008A_Solomon_Islands_3_2006A_Kisii_7576_2008A_Kisii_7562_2008A_Kisii_7554_2008A_Kisii_7570_2008A_Kisumu_5923_2008A_Kisii_7547_2008A_Kenya_6901_2008A_Kericho_7529_2008A_Brisbane_59_2007A_Kericho_7525_2008A_Kisii_5896_2008A_New_Caledonia_20_99A_Kisumu_5934_2008A_Kisumu_6543_200898%84%99%100%100%94%
2008 Season
#N183S
G185A
A189T Sb
T210N
#G185N Sb
E273K
0.0040A_Mbagathi_4825_2007_H1N1A_Kericho_7530_2008_H1N1A_Kericho_7529_2008_H1N1A_Kisii_4839_2007_H1N1A_Kisii_7554_2008_H1N1A_Kisii_7565_2008_H1N1A_Kenya_6901_2008_H1N1A_Kisii_5886_2008_H1N1A_Solomon_Islands_3_2006_H1N1A_Mbagathi_4834_2007_H1N1A_Kisii_7541_2008_H1N1A_Kisii_7543_2008_H1N1A_Mbagathi_4827_2007_H1N1A_Kisumu_4830_2007_H1N1A_Kisumu_5933_2008_H1N1A_Kisii_7576_2008_H1N1A_Kisumu_5934_2008_H1N1A_Kisii_5896_2008_H1N1A_Kericho_7535_2008_H1N1A_Kisumu_5923_2008_H1N1A_Kisii_7547_2008_H1N1A_Mbagathi_4840_2007_H1N1A_Kisii_7570_2008_H1N1A_Kisii_7562_2008_H1N1A_Kisii_7577_2008_H1N1A_Malindi_4835_2007_H1N1A_Kericho_7525_2008_H1N1A_Kisumu_6543_2008_H1N1A_Kericho_4831_2007_H1N1A_Malindi_4837_2007_H1N1A_Kisii_7559_2008_H1N1A_Mbagathi_4844_2007_H1N1A_Kisumu_5928_2008_H1N1A_Kisumu_7611_2008_H1N1A_Kericho_4822_2007_H1N1A_Brisbane_59_2007_H1N1A_New_Caledonia_20_99_H1N1A_Mbagathi_7596_2008_H1N1A_Mbagathi_4826_2007_H1N1A_Kisii_4823_2007_H1N1A_Malindi_4836_2007_H1N199%97%61%99%72%99%98%98%93%
2007-2008 seasons
#N183S
A189T Sb
G185A
T210N
#185N Sb
S121N
K273E
0.0040A_Mbagathi_4825_2007_H1N1A_Kericho_7530_2008_H1N1A_Kericho_7529_2008_H1N1A_Kisii_4839_2007_H1N1A_Kisii_7554_2008_H1N1A_Kisii_7565_2008_H1N1A_Kenya_6901_2008_H1N1A_Kisii_5886_2008_H1N1A_Solomon_Islands_3_2006_H1N1A_Mbagathi_4834_2007_H1N1A_Kisii_7541_2008_H1N1A_Kisii_7543_2008_H1N1A_Mbagathi_4827_2007_H1N1A_Kisumu_4830_2007_H1N1A_Kisumu_5933_2008_H1N1A_Kisii_7576_2008_H1N1A_Kisumu_5934_2008_H1N1A_Kisii_5896_2008_H1N1A_Kericho_7535_2008_H1N1A_Kisumu_5923_2008_H1N1A_Kisii_7547_2008_H1N1A_Mbagathi_4840_2007_H1N1A_Kisii_7570_2008_H1N1A_Kisii_7562_2008_H1N1A_Kisii_7577_2008_H1N1A_Malindi_4835_2007_H1N1A_Kericho_7525_2008_H1N1A_Kisumu_6543_2008_H1N1A_Kericho_4831_2007_H1N1A_Malindi_4837_2007_H1N1A_Kisii_7559_2008_H1N1A_Mbagathi_4844_2007_H1N1A_Kisumu_5928_2008_H1N1A_Kisumu_7611_2008_H1N1A_Kericho_4822_2007_H1N1A_Brisbane_59_2007_H1N1A_New_Caledonia_20_99_H1N1A_Mbagathi_7596_2008_H1N1A_Mbagathi_4826_2007_H1N1A_Kisii_4823_2007_H1N1A_Malindi_4836_2007_H1N199%97%61%99%72%99%98%98%93%
2007-2008 seasons
#N183S
A189T Sb
G185A
T210N
#185N Sb
S121N
K273E
Discussion
•
The 2007 viruses were genetically distinct from A/Solomon
Islands/3/2006(H1N1), but similar to A/Brisbane/59/2007(H1N1)
•
The 2008 viruses were also genetically divergent compared to
A/Brisbane/59/2007(H1N1)
–
The 2008 Kenyan isolates contained G185N and A189T mutations compared
to A/Brisbane/59/2007(H1N1);
–
Both mutations are at the antigenic site Sb
–
Position 185 is involved at the sialic acid receptor binding site
–
Most mutations(G185N, A189T, N183S) occurred within the receptor
binding domain and at site Sb
Conclusion
•
A/H1N1 viruses circulating in Kenya during years 2007 to 2008 were
genetically distinct from recommended vaccine strains
•
Genetic evidence of the mismatch reported elsewhere in 2008, appeared
early in Kenya.
•
Characterization of genetic variation reinforces the importance of
continuing surveillance and molecular characterization of influenza
viruses from Africa.
22
Disclaimer
•
The views expressed are those of the author and should not be construed
to represent the positions of the US Department of the Army or
Department of Defense
Acknowledgments
USAMRU-K
Dr. Wallace Bulimo
Berhane Assefa
COL Scott Gordon
MAJ Mike Walter
COL Sam Martin
MAJ Sheryl Bedno
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Finnley Osunna
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Director KEMRI
Director CVR
Director CPHR
Dr. Walter Ochieng
James Magana
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SITE COs AND LAB TECHSDuke OmaribaMartha NderituAlfred OdindoCharles
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DOD-GEIS
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CAPT Kevin Russell
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CDR David Blazes
Dr. Toti Sanchez
CDC –Kenya, GDD
Dr. Robert Breiman
Dr. Kariuki Njenga
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