tetano
Editor, Senior Moderator
Viruses
. 2023 Nov 18;15(11):2274.
doi: 10.3390/v15112274. Challenges for Precise Subtyping and Sequencing of a H5N1 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Virus Isolated in Japan in the 2022-2023 Season Using Classical Serological and Molecular Methods
James G Komu[SUP] 1 2 [/SUP], Hiep Dinh Nguyen[SUP] 1 [/SUP], Yohei Takeda[SUP] 3 4 [/SUP], Shinya Fukumoto[SUP] 3 5 [/SUP], Kunitoshi Imai[SUP] 3 [/SUP], Hitoshi Takemae[SUP] 6 [/SUP], Tetsuya Mizutani[SUP] 6 [/SUP], Haruko Ogawa[SUP] 3 [/SUP]
Affiliations
The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from a dead white-tailed eagle (Haliaeetus albicilla albicilla) were screened via real-time RT-PCR targeting the influenza A virus matrix (M) gene, followed by virus isolation. The hemagglutination inhibition test was applied in order to subtype and antigenically characterize the isolate using anti-A/duck/Hong Kong/820/80 (H5N3) reference serum or anti-H5N1 cross-clade monoclonal antibodies (mAbs). Sequencing using previously reported universal primers was attempted in order to analyze the full-length hemagglutinin (HA) gene. Oropharyngeal and conjunctival samples were positive for the M gene, and high hemagglutination titers were detected in inoculated eggs. However, its hemagglutination activity was not inhibited by the reference serum or mAbs. The antiserum to a recently isolated H5N1 clade 2.3.4.4b strain inhibited our isolate but not older strains. A homologous sequence in the previously reported forward primer and HA2 region in our isolate led to partial HA gene amplification. Finally, next-generation sequencing confirmed the isolate as H5N1 clade 2.3.4.4b HPAIV, with genetic similarity to H5N1 strains circulating in Japan since November 2021.
Keywords: H5N1; antigenic and genetic evolution; classical diagnostic methods; highly pathogenic avian influenza.
. 2023 Nov 18;15(11):2274.
doi: 10.3390/v15112274. Challenges for Precise Subtyping and Sequencing of a H5N1 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Virus Isolated in Japan in the 2022-2023 Season Using Classical Serological and Molecular Methods
James G Komu[SUP] 1 2 [/SUP], Hiep Dinh Nguyen[SUP] 1 [/SUP], Yohei Takeda[SUP] 3 4 [/SUP], Shinya Fukumoto[SUP] 3 5 [/SUP], Kunitoshi Imai[SUP] 3 [/SUP], Hitoshi Takemae[SUP] 6 [/SUP], Tetsuya Mizutani[SUP] 6 [/SUP], Haruko Ogawa[SUP] 3 [/SUP]
Affiliations
- PMID: 38005950
- PMCID: PMC10675786
- DOI: 10.3390/v15112274
The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from a dead white-tailed eagle (Haliaeetus albicilla albicilla) were screened via real-time RT-PCR targeting the influenza A virus matrix (M) gene, followed by virus isolation. The hemagglutination inhibition test was applied in order to subtype and antigenically characterize the isolate using anti-A/duck/Hong Kong/820/80 (H5N3) reference serum or anti-H5N1 cross-clade monoclonal antibodies (mAbs). Sequencing using previously reported universal primers was attempted in order to analyze the full-length hemagglutinin (HA) gene. Oropharyngeal and conjunctival samples were positive for the M gene, and high hemagglutination titers were detected in inoculated eggs. However, its hemagglutination activity was not inhibited by the reference serum or mAbs. The antiserum to a recently isolated H5N1 clade 2.3.4.4b strain inhibited our isolate but not older strains. A homologous sequence in the previously reported forward primer and HA2 region in our isolate led to partial HA gene amplification. Finally, next-generation sequencing confirmed the isolate as H5N1 clade 2.3.4.4b HPAIV, with genetic similarity to H5N1 strains circulating in Japan since November 2021.
Keywords: H5N1; antigenic and genetic evolution; classical diagnostic methods; highly pathogenic avian influenza.