The PB2 Polymerase Host Adaptation Substitutions Prime Avian Indonesia Sub Clade 2.1 H5N1 Viruses for Infecting Humans
Identifieur interne : 000F72 ( Ncbi/Merge ); précédent : 000F71; suivant : 000F73The PB2 Polymerase Host Adaptation Substitutions Prime Avian Indonesia Sub Clade 2.1 H5N1 Viruses for Infecting Humans
Auteurs : Pui Wang ; Wenjun Song [République populaire de Chine] ; Bobo Wing-Yee Mok ; Min Zheng ; Siu-Ying Lau ; Siwen Liu ; Pin Chen ; Xiaofeng Huang ; Honglian Liu ; Conor J. Cremin ; Honglin ChenSource :
- Viruses [ 1999-4915 ] ; 2019.
Abstract
Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2–3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection.
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DOI: 10.3390/v11030292
PubMed: 30909490
PubMed Central: 6480796
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The PB2 Polymerase Host Adaptation Substitutions Prime Avian Indonesia Sub Clade 2.1 H5N1 Viruses for Infecting Humans</title><author><name sortKey="Wang, Pui" sort="Wang, Pui" uniqKey="Wang P" first="Pui" last="Wang">Pui Wang</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Song, Wenjun" sort="Song, Wenjun" uniqKey="Song W" first="Wenjun" last="Song">Wenjun Song</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-viruses-11-00292">State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, Guangzhou Medical University, Guangzhou 510180, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, Guangzhou Medical University, Guangzhou 510180</wicri:regionArea><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Mok, Bobo Wing Yee" sort="Mok, Bobo Wing Yee" uniqKey="Mok B" first="Bobo Wing-Yee" last="Mok">Bobo Wing-Yee Mok</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Zheng, Min" sort="Zheng, Min" uniqKey="Zheng M" first="Min" last="Zheng">Min Zheng</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Lau, Siu Ying" sort="Lau, Siu Ying" uniqKey="Lau S" first="Siu-Ying" last="Lau">Siu-Ying Lau</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Liu, Siwen" sort="Liu, Siwen" uniqKey="Liu S" first="Siwen" last="Liu">Siwen Liu</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Chen, Pin" sort="Chen, Pin" uniqKey="Chen P" first="Pin" last="Chen">Pin Chen</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Huang, Xiaofeng" sort="Huang, Xiaofeng" uniqKey="Huang X" first="Xiaofeng" last="Huang">Xiaofeng Huang</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Liu, Honglian" sort="Liu, Honglian" uniqKey="Liu H" first="Honglian" last="Liu">Honglian Liu</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Cremin, Conor J" sort="Cremin, Conor J" uniqKey="Cremin C" first="Conor J." last="Cremin">Conor J. Cremin</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Chen, Honglin" sort="Chen, Honglin" uniqKey="Chen H" first="Honglin" last="Chen">Honglin Chen</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">30909490</idno><idno type="pmc">6480796</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480796</idno><idno type="RBID">PMC:6480796</idno><idno type="doi">10.3390/v11030292</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000C48</idno><idno type="wicri:explorRef" 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China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Song, Wenjun" sort="Song, Wenjun" uniqKey="Song W" first="Wenjun" last="Song">Wenjun Song</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-viruses-11-00292">State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, Guangzhou Medical University, Guangzhou 510180, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, Guangzhou Medical University, Guangzhou 510180</wicri:regionArea><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Mok, Bobo Wing Yee" sort="Mok, Bobo Wing Yee" uniqKey="Mok B" first="Bobo Wing-Yee" last="Mok">Bobo Wing-Yee Mok</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Zheng, Min" sort="Zheng, Min" uniqKey="Zheng M" first="Min" last="Zheng">Min Zheng</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Lau, Siu Ying" sort="Lau, Siu Ying" uniqKey="Lau S" first="Siu-Ying" last="Lau">Siu-Ying Lau</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Liu, Siwen" sort="Liu, Siwen" uniqKey="Liu S" first="Siwen" last="Liu">Siwen Liu</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Chen, Pin" sort="Chen, Pin" uniqKey="Chen P" first="Pin" last="Chen">Pin Chen</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Huang, Xiaofeng" sort="Huang, Xiaofeng" uniqKey="Huang X" first="Xiaofeng" last="Huang">Xiaofeng Huang</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Liu, Honglian" sort="Liu, Honglian" uniqKey="Liu H" first="Honglian" last="Liu">Honglian Liu</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Cremin, Conor J" sort="Cremin, Conor J" uniqKey="Cremin C" first="Conor J." last="Cremin">Conor J. Cremin</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author><author><name sortKey="Chen, Honglin" sort="Chen, Honglin" uniqKey="Chen H" first="Honglin" last="Chen">Honglin Chen</name><affiliation><nlm:aff id="af1-viruses-11-00292">State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</nlm:aff></affiliation></author></analytic><series><title level="j">Viruses</title><idno type="eISSN">1999-4915</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2–3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K.Y. Yuen</name></author><author><name sortKey="Chan, P K" uniqKey="Chan P">P.K. Chan</name></author><author><name sortKey="Peiris, M" uniqKey="Peiris M">M. Peiris</name></author><author><name sortKey="Tsang, D N" uniqKey="Tsang D">D.N. Tsang</name></author><author><name sortKey="Que, T L" uniqKey="Que T">T.L. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Viruses</journal-id><journal-id journal-id-type="iso-abbrev">Viruses</journal-id><journal-id journal-id-type="publisher-id">viruses</journal-id><journal-title-group><journal-title>Viruses</journal-title></journal-title-group><issn pub-type="epub">1999-4915</issn><publisher><publisher-name>MDPI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">30909490</article-id><article-id pub-id-type="pmc">6480796</article-id><article-id pub-id-type="doi">10.3390/v11030292</article-id><article-id pub-id-type="publisher-id">viruses-11-00292</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>The PB2 Polymerase Host Adaptation Substitutions Prime Avian Indonesia Sub Clade 2.1 H5N1 Viruses for Infecting Humans</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Pui</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref><xref ref-type="author-notes" rid="fn1-viruses-11-00292">†</xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Wenjun</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref><xref ref-type="aff" rid="af2-viruses-11-00292">2</xref><xref ref-type="author-notes" rid="fn1-viruses-11-00292">†</xref></contrib><contrib contrib-type="author"><name><surname>Mok</surname><given-names>Bobo Wing-Yee</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Zheng</surname><given-names>Min</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Lau</surname><given-names>Siu-Ying</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Siwen</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Pin</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Xiaofeng</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Honglian</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><name><surname>Cremin</surname><given-names>Conor J.</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-5108-8338</contrib-id><name><surname>Chen</surname><given-names>Honglin</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00292">1</xref><xref rid="c1-viruses-11-00292" ref-type="corresp">*</xref></contrib></contrib-group><aff id="af1-viruses-11-00292"><label>1</label>State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Hong Kong SAR, China;<email>puiwang@hku.hk</email> (P.W.);<email>wjsong@hku.hk</email> (W.S.);<email>bobomok@hku.hk</email> (B.W.-Y.M.);<email>min.zheng@stjude.org</email> (M.Z.);<email>sylau926@hku.hk</email> (S.-Y.L.);<email>siwen531@CONNECT.HKU.HK</email> (S.L.);<email>u3508816@connect.hku.hk</email> (P.C.);<email>stevehxf@connect.hku.hk</email> (X.H.);<email>lhlotus@connect.hku.hk</email> (H.L.);<email>conor93@connect.hku.hk</email> (C.J.C.)</aff><aff id="af2-viruses-11-00292"><label>2</label>State Key Laboratory of Respiratory Disease, Institute of Integration of Traditional and Western Medicine, Guangzhou Medical University, Guangzhou 510180, China</aff><author-notes><corresp id="c1-viruses-11-00292"><label>*</label>Correspondence: <email>hlchen@hku.hk</email>; Tel.: +852-39179941 or +852-39179830; Fax: +852-28551241</corresp><fn id="fn1-viruses-11-00292"><label>†</label><p>These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="epub"><day>22</day><month>3</month><year>2019</year></pub-date><pub-date pub-type="collection"><month>3</month><year>2019</year></pub-date><volume>11</volume><issue>3</issue><elocation-id>292</elocation-id><history><date date-type="received"><day>15</day><month>2</month><year>2019</year></date><date date-type="accepted"><day>22</day><month>3</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 by the authors.</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2–3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection.</p></abstract><kwd-group><kwd>Influenza virus</kwd><kwd>H5N1</kwd><kwd>PB2</kwd><kwd>host adaptation</kwd><kwd>RNP</kwd><kwd>cross species transmission</kwd></kwd-group></article-meta></front><floats-group><fig id="viruses-11-00292-f001" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Phylogenetic tree of the PB2 gene of Indonesian H5N1 virus. The tree was constructed using the neighbor-joining method <styled-content style="color:#212121">with the Tajima-Nei model of nucleotide substitution using MEGA X</styled-content>. Blue indicates avian strains carrying Arg at position 526 of the PB2 gene (526R). Human strains are highlighted in boldface. Numbers represent the bootstrap values (percentages) from 1000 replicates: only bootstrap values greater than 50 are shown. Strains in red are the avian isolate, A/Chicken/Indonesia/2A/2003 (CK2A), and the human isolate, A/Indonesia/5/2005 (IND5), used as backbones for constructing reverse genetic versions of viruses in this study.</p></caption><graphic xlink:href="viruses-11-00292-g001"></graphic></fig><fig id="viruses-11-00292-f002" orientation="portrait" position="float"><label>Figure 2</label><caption><p>RNP polymerase activity in HEK293T and DF-1 cells. RNP expression plasmids containing NP, PB1, PA and wild type PB2, or the corresponding PB2 526 mutant derived from CK2A or IND5, along with a firefly luciferase RNP reporter plasmid and the Renilla luciferase expressing plasmid, pRL-TK (as an internal control), were transfected into HEK293T cells. The transfected cells were incubated for 24 h at: 37 °C (<bold>A</bold>); or 33 °C (<bold>B</bold>). DF-1 cells were transfected with the same sets of RNP complex plasmids but with an avian specific RNP firefly reporter and Renilla luciferase control (<bold>C</bold>). DF-1 cells were incubated at 39 °C. Luciferase activities were measured at 24 h post transfection using a Dual-Luciferase Reporter Assay System (Promega). Firefly RNP activity was normalized against Renilla activity. RNP without PB2 was used as a negative control. Data represent mean normalized luciferase activity from three independent experiments. Error bars represent standard deviation calculated from three separate experiments. Statistical significance was analyzed by Student’s <italic>t</italic>-test. *** <italic>p</italic> < 0.001, ** <italic>p</italic> < 0.01.</p></caption><graphic xlink:href="viruses-11-00292-g002"></graphic></fig><fig id="viruses-11-00292-f003" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Growth kinetics of H5N1 viruses in mammalian and avian cells. Reverse genetic (RG) versions of the human H5N1 isolate, A/Indonesia/5/05 (IND5), and a mutant virus containing 526K-PB2 (IND5-526K) were used to infect MDCK and DF-1 cells at an MOI of 0.001, or A549 cells at an MOI of 0.01 (<bold>A</bold>). Similarly, RG versions of the avian H5N1 isolate, A/Chicken/Indonesia/2A/03 (CK2A), and a mutant virus containing 526R-PB2 (CK2A-526R) were used to infect MDCK and DF-1 cells at an MOI of 0.001, or A549 cells at an MOI of 0.01 (<bold>B</bold>). Growth of IND5 (<bold>C</bold>) and CK2A (<bold>D</bold>) viruses at 33 °C was estimated in MDCK and A549 cells. After adsorption for 1 h, the cells were washed and overlaid with infection medium containing 1 µg/mL of TPCK-trypsin. At the indicated time points, culture supernatants were collected for virus titration by plaque assay in MDCK cells. Data represent mean viral titers from three independent experiments. Error bars represent standard deviation calculated from three separate experiments. Statistical significance was analyzed by one-way ANOVA, corrected by the Bonferroni post test: *** <italic>p</italic> < 0.001., h.p.i., h post infection.</p></caption><graphic xlink:href="viruses-11-00292-g003"></graphic></fig><fig id="viruses-11-00292-f004" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Effect of PB2 526R on H5N1 viral RNA and protein synthesis. (<bold>A</bold>) A549 cells were infected with reverse genetic wild type IND5 or CK2A H5N1 viruses or mutant versions of these viruses containing either 526K or 526R PB2, respectively, at an MOI of 1. Total RNA was extracted at 4 or 8 h post infection and reverse transcription (RT) performed using uni-12 or oligo dT primers, to detect vRNA or mRNA, respectively. Expression of these two RNA species of NP was quantified by relative quantitative real time RT-PCR and normalized against the β-actin gene. Data represent mean relative NP expression levels from three independent experiments. Statistical significance was analyzed by one-way ANOVA, corrected by the Bonferroni post test: *** <italic>p</italic> < 0.001and * <italic>p</italic> < 0.05. (<bold>B</bold>) A549 cells were infected as above. Cells were lysed at 8 h post infection and expression levels of NP and PB2 estimated by Western blot using anti-NP (1:5000) and anti-PB2 (1:1000) antibodies. Expression levels of β-tubulin were detected using anti-β-tubulin antibody, and served as a loading control.</p></caption><graphic xlink:href="viruses-11-00292-g004"></graphic></fig><fig id="viruses-11-00292-f005" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Effect of 526R PB2 on RNP activity in the presence of NEP and NP-PB2 interaction. Increasing amounts of NEP expression vector, or empty vector, were co-transfected with expression plasmids for the RNP complexes of IND5 and IND5-526K PB2 (<bold>A</bold>), or CK2A and CK2A-526R PB2 (<bold>B</bold>), together with RNP luciferase reporter and pRL-TK control plasmids, into HEK293T cells, followed by culture at 37 °C. Similarly, RNP polymerase activity with the RNP complexes of CK2A and CK2A-526R and increasing amounts of NEP expression was examined in DF-1 cells, cultured at 39 °C (<bold>C</bold>). Luciferase activities were measured at 24 h post transfection. Firefly RNP polymerase activity was normalized against Renilla activity, and RNP without PB2 was used as a negative control. Data represent mean normalized luciferase activity from three independent experiments. Error bars represent standard deviation from three separate experiments. Statistical significance was analyzed by Student’s <italic>t</italic>-test. *** <italic>p</italic> < 0.001. (<bold>D</bold>) Interaction between NP and PB2 in RNP complexes. Different sets of plasmids expressing PB1, PA, NP and Flag-tagged PB2 from IND5-526K or CK2A-526R, were transfected into HEK293T cells. At 48 h post transfection, cell lysates were prepared and rabbit polyclonal NP antibody used to co-precipitate PB2. Proteins from co-precipitated complexes were resolved using SDS-PAGE and detected by Western blot using anti-NP (1:5000) and anti-Flag (Sigma) (1:5000) antibodies.</p></caption><graphic xlink:href="viruses-11-00292-g005"></graphic></fig><fig id="viruses-11-00292-f006" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Evaluation of H5N1 virus infection and replication in mice. Groups of four or six BALB/c mice, aged 4–6 weeks, were intranasally inoculated with 10<sup>3</sup> (<bold>A</bold>) or 10<sup>2</sup> (<bold>B</bold>) PFU of virus containing wild type IND5 or CK2A, or the 526K (IND5-526K) or 526R (CK2A-526R) mutant viruses, in 25ul PBS. Body weight and survival were monitored daily for 14 days post infection. Results of infection with high doses of virus (10<sup>4</sup>, 10<sup>5</sup> and 10<sup>6</sup> PFU) are shown in the <xref ref-type="app" rid="app1-viruses-11-00292">Supplementary Materials (Figure S1)</xref>. The MLD<sub>50</sub> was calculated by the method of Reed and Muench [<xref rid="B30-viruses-11-00292" ref-type="bibr">30</xref>]. (<bold>C</bold>) Replication efficiency of viruses in lung tissues of infected mice. To determine virus titers in mouse lung tissues, groups of three mice were infected with 10<sup>3</sup> PFU of the respective viruses described above and then euthanized at 72 h post infection, with lung tissues from each mouse being collected and homogenized for virus titration by plaque assay using MDCK cells. Error bars represent standard deviation from virus-infected mice mouse in the group. Statistical significance was analyzed by one-way ANOVA or Student’s <italic>t</italic>-test *** <italic>p</italic> < 0.001, ** <italic>p</italic> < 0.01 and * <italic>p</italic> < 0.05.</p></caption><graphic xlink:href="viruses-11-00292-g006"></graphic></fig><fig id="viruses-11-00292-f007" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Comparison of effects of PB2 526R and 627K on Indonesian H5N1 virus replication. (<bold>A</bold>) Growth kinetics of reverse genetic versions of CK2A virus containing PB2 526R or 627K in avian cells. DF-1 cells were infected separately with CK2A, CK2A-526R or CK2A-627K RG virus and cultured at 39 °C. Culture supernatant was collected at the indicated time points and virus titrated by plaque assay in MDCK cells. (<bold>B</bold>) Growth competition assay: CK2A wild type versus CK2A-526R virus in avian cells. DF-1 cells were infected at an MOI of 0.001 with CK2A and CK2A-526R viruses, mixed at a ratio of 1:1. Continuous cultures were performed for four passages. Viral RNA was isolated from culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. Representative sequencing chromatograms from each passage are displayed; the genetic code for PB2 526R is AGA, whereas 526K is AAA. (<bold>C</bold>) Growth competition assay: CK2A wild type versus CK2A-627K virus in avian cells. The assay was conducted similarly to (<bold>B</bold>). Representative sequencing chromatograms are shown: the genetic code for PB2 627E is GAG, whereas 627K is AAG. (<bold>D</bold>) Growth kinetics of reverse genetic versions of CK2A virus containing 526R or 627K. A549 cells were infected separately with RG CK2A-526R or CK2A-627K viruses and cultured at 37 °C. Culture supernatant was collected at the indicated time points and virus titrated by plaque assay in MDCK cells. (<bold>E</bold>) Growth competition assay: CK2A-526R versus CK2A-627K virus in A549 cells. The procedure was performed as in (<bold>B</bold>). Sequencing chromatograms representing mixed populations of 526R/K and 627E/K are shown. h.p.i., hours post infection. Error bars represent standard deviation from three separate experiments. Statistical significance was analyzed by one-way ANOVA or Student’s <italic>t</italic>-test *** <italic>p</italic> < 0.001.</p></caption><graphic xlink:href="viruses-11-00292-g007"></graphic></fig><fig id="viruses-11-00292-f008" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Effect of PB2 R288Q on viral replication and RNP activity in Indonesian H5N1 viruses. R288Q, alone and in combination with K526R, was introduced into the PB2 segment of the H5N1 avian isolate, A/Chicken/Indonesia/2A/2003, and alternatively, the back mutations Q288R and R526K, separately and in combination, were introduced into the PB2 of the H5N1 human isolate, A/Indonesia/5/2005. Plasmids of the minigenome system, consisting of NP, PB1, PA and wild type PB2, or the corresponding PB2 mutants derived from CK2A or IND5, together with a firefly RNP luciferase reporter plasmid and the Renilla luciferase expressing plasmid pRL-TK (as an internal control) were transfected into HEK293T cells. (<bold>A</bold>) Effect of R288Q, alone or in conjunction with K526R, on RNP polymerase activity in the background of other RNP proteins from A/Chicken/Indonesia/2A/2003. (<bold>B</bold>) Effect of Q288R, alone or in conjunction with R526K, on RNP polymerase activity, with other RNP proteins derived from A/Indonesia/5/2005. Luciferase activities were measured at 24 h post transfection using a Dual Luciferase Reporter Assay System (Promega). Firefly RNP activity was normalized against Renilla activity. RNP without PB2 was used as a negative control. Data represent mean normalized luciferase activity from three independent experiments. Statistical significance was analyzed by Student’s <italic>t</italic>-test. *** <italic>p</italic> < 0.001. (<bold>C</bold>) Growth kinetics of wild type CK2A virus and mutant viruses containing R288Q PB2 or R288Q/K526R PB2. A549 cells were infected with CK2A, CK2A-R288Q or CK2A-K526R/R288Q viruses at an MOI of 0.01 and cultured at 37 °C. Culture supernatants were collected at the indicated time points and virus titrated by plaque assay in MDCK cells. Error bars represent standard deviation from three separate experiments.</p></caption><graphic xlink:href="viruses-11-00292-g008"></graphic></fig><table-wrap id="viruses-11-00292-t001" orientation="portrait" position="float"><object-id pub-id-type="pii">viruses-11-00292-t001_Table 1</object-id><label>Table 1</label><caption><p>Determination of the MLD<sub>50</sub> of RG viruses*.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Viral Strain</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">MLD<sub>50</sub> (PFU)</th></tr></thead><tbody><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">IND5 WT</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">1.5</td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">IND5-526K</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">3.2 × 10<sup>2</sup></td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">CK2A</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">2.2 × 10<sup>3</sup></td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">CK2A-526R</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">2.2 × 10<sup>3</sup></td></tr></tbody></table><table-wrap-foot><fn><p>* Groups of four mice were inoculated with different doses of virus, ranging from 1 to 10<sup>6</sup> PFU, and mouse survival monitored for 14 days post infection. The MLD<sub>50</sub> was calculated by the method of Reed and Muench [<xref rid="B30-viruses-11-00292" ref-type="bibr">30</xref>].</p></fn></table-wrap-foot></table-wrap><table-wrap id="viruses-11-00292-t002" orientation="portrait" position="float"><object-id pub-id-type="pii">viruses-11-00292-t002_Table 2</object-id><label>Table 2</label><caption><p>E627K adaptive mutations in mice fatally infected with H5N1 virus *.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Viral Strain</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Number of Mice</th><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Mouse Group (Infective Viral Dose, PFU) in Which E627K Mutation Emerged</th></tr></thead><tbody><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">IND5 WT</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">0/7</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">None detected</td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">IND5-526K</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">2/4</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">10<sup>3</sup></td></tr><tr><td rowspan="2" align="center" valign="middle" style="border-bottom:solid thin" colspan="1">CK2A WT</td><td rowspan="2" align="center" valign="middle" style="border-bottom:solid thin" colspan="1">3/4</td><td align="center" valign="middle" rowspan="1" colspan="1">One from 10<sup>2</sup></td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">Two from 10<sup>3</sup></td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">CK2A-526R</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">1/5 <sup>&</sup></td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">10<sup>4</sup></td></tr></tbody></table><table-wrap-foot><fn><p><bold>*</bold> Virus isolated from fatally infected mice died or euthanized from Day 4 post infection was characterized for adaptation markers in the PB2 gene by sequencing analysis. <sup>&</sup> Among the five fatally infected mice in this group, only one mouse was found to contain E627K PB2 and K526R remained unchanged in this mouse.</p></fn></table-wrap-foot></table-wrap><table-wrap id="viruses-11-00292-t003" orientation="portrait" position="float"><object-id pub-id-type="pii">viruses-11-00292-t003_Table 3</object-id><label>Table 3</label><caption><p>Comparison of amino acid variations in the PB2 segments of the IND5 and CK2A strains.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1" colspan="1">Viral Strain</th><th colspan="8" align="center" valign="middle" style="border-top:solid thin;border-bottom:solid thin" rowspan="1">Position in PB2 Gene</th></tr></thead><tbody><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1"></td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">288</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">323</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">368</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">389</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">524</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">526</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">658</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">756</td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">IND5</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1"><bold>Q</bold></td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">L</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">R</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">R</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">I</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">R</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">Y</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">M</td></tr><tr><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">CK2A</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1"><bold>R</bold></td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">F</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">Q</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">K</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">T</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">K</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">H</td><td align="center" valign="middle" style="border-bottom:solid thin" rowspan="1" colspan="1">T</td></tr></tbody></table></table-wrap></floats-group></pmc><affiliations><list><country><li>République populaire de Chine</li></country><region><li>Guangdong</li></region><settlement><li>Jiangmen</li></settlement></list><tree><noCountry><name sortKey="Chen, Honglin" sort="Chen, Honglin" uniqKey="Chen H" first="Honglin" last="Chen">Honglin Chen</name><name sortKey="Chen, Pin" sort="Chen, Pin" uniqKey="Chen P" first="Pin" last="Chen">Pin Chen</name><name sortKey="Cremin, Conor J" sort="Cremin, Conor J" uniqKey="Cremin C" first="Conor J." last="Cremin">Conor J. Cremin</name><name sortKey="Huang, Xiaofeng" sort="Huang, Xiaofeng" uniqKey="Huang X" first="Xiaofeng" last="Huang">Xiaofeng Huang</name><name sortKey="Lau, Siu Ying" sort="Lau, Siu Ying" uniqKey="Lau S" first="Siu-Ying" last="Lau">Siu-Ying Lau</name><name sortKey="Liu, Honglian" sort="Liu, Honglian" uniqKey="Liu H" first="Honglian" last="Liu">Honglian Liu</name><name sortKey="Liu, Siwen" sort="Liu, Siwen" uniqKey="Liu S" first="Siwen" last="Liu">Siwen Liu</name><name sortKey="Mok, Bobo Wing Yee" sort="Mok, Bobo Wing Yee" uniqKey="Mok B" first="Bobo Wing-Yee" last="Mok">Bobo Wing-Yee Mok</name><name sortKey="Wang, Pui" sort="Wang, Pui" uniqKey="Wang P" first="Pui" last="Wang">Pui Wang</name><name sortKey="Zheng, Min" sort="Zheng, Min" uniqKey="Zheng M" first="Min" last="Zheng">Min Zheng</name></noCountry><country name="République populaire de Chine"><region name="Guangdong"><name sortKey="Song, Wenjun" sort="Song, Wenjun" uniqKey="Song W" first="Wenjun" last="Song">Wenjun Song</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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