RESUMO
We developed a method to determine the sequences of hemagglutinin (HA) and neuraminidase (NA) from RNA extracted directly from wild bird fecal samples, using Nanopore Flongle. We determined the nucleotide sequences and subtypes of HA and NA in 16 and 15 samples respectively, using Flongle. The results of HA and NA subtyping determined by the conventional method were consistent with their subtypes determined by our method, thereby the applicability of this method in the identification of HA and NA subtypes. In addition, the homology between the HA fragments in this and the Sanger methods ranged from 98.5 % to 100 %. Compared with conventional PCR with the Sanger method, this method can easily determine HA and NA subtypes and sequences directly from the fecal samples. It is easier to implement and has lower running costs (USD100$) than other NGS-based methods, making it a useful tool for avian influenza surveillance in wild birds.
Assuntos
Vírus da Influenza A , Influenza Aviária , Sequenciamento por Nanoporos , Animais , Hemaglutininas , Neuraminidase/genética , Sequenciamento por Nanoporos/veterinária , Animais Selvagens , Vírus da Influenza A/genética , Aves , Influenza Aviária/epidemiologia , Fezes , FilogeniaRESUMO
On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs.
Assuntos
Surtos de Doenças/veterinária , Vírus da Influenza A/classificação , Vírus da Influenza A/imunologia , Influenza Aviária/epidemiologia , Animais , Animais de Zoológico/virologia , Antígenos Virais/imunologia , Aves , Galinhas/virologia , Patos/virologia , Variação Genética , Hemaglutininas/genética , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/transmissão , Influenza Aviária/virologia , Japão/epidemiologia , Doenças das Aves Domésticas/virologia , República da Coreia/epidemiologia , Taiwan/epidemiologiaRESUMO
Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.