Sequence analysis of HA and NA genes of human infected H9N2 avian influenza virus in Yunnan province, 2019 / 中国热带医学

@#Abstract: Objective To investigate the molecular characteristics of the H9N2 avian influenza virus (AIV) causing human infection in Yunnan Province in 2019, and to provide the scientific basis for the prevention and control of avian influenza in Yunnan Province. Methods Influenza virus typing was performed by real-time RT-PCR in two influenza-like illness samples, and the Illumina Miseq high-throughput sequencer was used to determine the viral genome sequence. HA and NA gene sequence alignment and phylogenetic tree construction were performed using Mega7.0 software. Results Real-time RT-PCR results showed that two influenza-like illness samples were positive for H9N2 subtype. The full length of HA and NA were obtained by genomic sequencing. Sequence system evolution analysis showed that the HA and NA of the two AIVs in Yunnan Province were in the same evolutionary clade as A/Chicken/Zhejiang/HJ/2007 and belonged to the G57 type. The HA nucleotide and amino acid homology of the two AIVs were 93.92% and 95.00%, respectively, and the NA nucleotide and amino acid homology was 93.31% and 82.03%, respectively. The nucleotide (amino acid) homology of HA was 92.29%-96.94% (93.77%-98.43%) and 92.84%-94.92% (94.18%-96.23%), respectively, and NA nucleotide homology (amino acid) were 91.81%-97.60% (77.82%-94.83%), 94.38%-97.22% (85.47%-94.55%), respectively, compared with that of human infected H9N2 epidemic strains obtained in China from 2015 to 2020. Both AIVs HA protein cleavage site sequences were PSRSSR↓GLF, which was in line with the characteristics of low pathogenic influenza. The analysis of HA protein receptor binding site showed that amino acids at positions 109, 161, 163, 191, 202, 203 and 234 were consistent with the reference strains, while amino acids at position 198 were mutated to T. N166D and 168N mutations were also found in HA protein, and both AIVs had 7 potential glycosylation sites. Analysis of the erythrocyte binding site of NA gene found that there were amino acid mutations at positions 369, 402, 403, and 432, and amino acid deletion at positions 63-65 was found in the NA genes. There were 4 and 5 potential glycosylation sites in the two AIVs, respectively, and no drug resistance site mutations were found. Conclusions　The receptor binding sites, erythrocyte binding sites and glycosylation sites of HA and NA genes of H9N2 AIV in Yunnan Province have different degrees of variation, and monitoring and prevention and control should be strengthened.

Molecular characteristic analysis of neuraminidase genes of avian influenza virus H9N2 in environments in Weining, Guizhou Province during 2015-2017 / 中华传染病杂志

Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining,Guizhou Province,and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM)environments in north of Weining Yi and Hui and Miao autonomous county (Weining),Guizhou Province during 2015 to 2017.Then the homology,genetic evolution,and sites of stalk deletion areas,potential N-glycosylation,receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software.Results Homology analysis revealed that there were 93.0％-100.0％ and 92.1％-100.0％ similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene,respectively.All strains belonged to DK/HK/Y280/97 sub-lineage,but their genetic sources were complex and diverse.Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40.The potential N-glycosylation sites at amino acid residues 86,146,200,and 234 of the NA protein of all strains were highly conserved,while other N-glycosylation sites had quantity and site mutations.There were different mutation types at the three sialic acid binding site areas,especially at 399-404 area.All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs.Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining,Guizhou Province during 2015-2017,and their genetic sources are complex and diverse.The mutations on sites of stalk areas,potential N-glycosylation and sialic acid binding site areas are presented at different degrees.Hence,enhancing surveillance and controlling H9N2 avian influenza virus is necessary.

Development and verification of reference nucleic acid materials of H9N2 influenza viruses by real-time RT-PCR / 生物工程学报

The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 10⁹ copies/μL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.

Genetic evolution of HA and NA genes of H9N2 influenza viruses isolated in regions of Hunan Province, China, in 2015 / 生物工程学报

The high prevalence of influenza A virus is identified in Hunan Province because of the high density of poultry farms. To survey the variations of H9N2 subtype avian influenza virus in Hunan province, we analyzed HA and NA genes of 10 virus strains isolated from different areas of Hunan Province. All these strains belong to the Eurasian lineage, Y280-like sub-lineage. The cleavage sites in their HA genes were all RSSR↓GLT, corresponding to the feature of low pathogenic AIV. All strains had an L (Leu) at the site 234 in the HA genes, indicating the ability of binding with the SAα-2,6 receptor. NA gene stalk deletions at aa 63-65 were also detected from all the isolates, indicating a possibility of increased virus replication in mammals. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.

Molecular epidemiological characteristics of avian influenza A(H9N2) viruses from environment in poultry markets in Changsha, China, 2014 / 中国人兽共患病学报

We analyzed the evolutional and molecular characteristics of Hemagglutinin(HA),Neuraminidase(NA) and nonstructural(NS) genes of avian influenza A(H9n2) viruses from environment in poultry markets in Changsha,China,2014,providing laboratory data for prevention and control of human infection with avian influenza A(H9N2) virus.Five hundred and one specimens (263 poultry drinking water specimens,226 poultry sewage specimens and 17 others specimens) were collected from environment in poultry markets in Changsha,2014,and real-time RTPCR was used for influenza A typing and subtyping (H5,H7 and H9) detection.HA and NA universal primer sets for conventional RT-PCR and sequencing were used for the positivity of single H9.The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST).The ClustalW multiple alignments of amino acids and the phylogenetic trees for HA,NA and NS genes were constructed using the BioEdit and MEGA 5 software,respectively.Results showed that among 501 environmental samples,350 samples were positive for influenza A virus,191 (38.12％) for H9 subtype,177 (35.33％) for H5 subtype,11 (2.20％) for H7 subtype and 68 (13.57％) for H5 and H9 subtypes co-detection.Twenty-three H9N2 subtype AIV were confirmed by conventional RT-PCR and sequencing from the samples of the positivity of single H9.Phylogenetic analysis revealed that most of HA,NA and NS genes of the H9N2 subtype AIV isolated in Changsha City had gene constellations of genotype S,and these virues might have acquired their HA,NA and NS from A/Chicken/Shanghai/F/1998-like (H9N2).L235 (correspond to H3 numbering 226) of the HA protein of the receptor binding site (RBS) were found in these H9N2 viruses,and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of human influenza virus,and the low pathogenicity molecular characteristics of HA,NA and NS genes were showed in these viruses.The positive rate of nucleic acid of the H9 subtype of avian influenza virus from environment was the highest in poultry markets in Changsha,2014,and molecular characteristics of the HA,NA and NS of these H9N2 subtype AIV showed low pathogenicity,but that may facilitate human infection.So,the prevalence and genetic evolution of this virus should be closely monitored.

Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSRdownward arrowGLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSRdownward arrowGLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.

In vivo Inhibition of NAS Preparation on H9N2 Subtype AIV / 中国病毒学·英文版

NAS preparation, a kind of Chinese herbal medicine found by the Yunnan Eco-agricultural Research Institute, has potential antiviral activity. In this paper, the inhibiting effect of NAS preparation on H9N2 subtype Avian influenza virus (AIV) was investigated in vivo. Chickens infected with H9N2 virus were treated with NAS preparation for 4 days. The virus was then detected by hemoagglutination (HA) test and reverse transcription polymerase chain reaction (RT-PCR). The results showed that no H9N2 virus could be detected at the 7th day when the chickens were treated with 0.2g/kg/d or 0.1g/kg/d of NAS preparation. However the virus could be detected in other chickens without NAS preparation treatment. This result suggested that NAS preparation may be a potential drug candidate to control infection of H9N2 subtype AIV in chickens.

Analysis on the non-structural protein genes of avian influenza viruses subtype H5N1 isolated from chicken in China / 中国人兽共患病学报

Fourteen H9N2 avian influenza viruses (AIV) were isolated from sick chickens in China from 1998 to 2008. The sequences of the Non-structural(NS) gene of these isolates were determined by RT-PCR and sequencing, and the entire ORF sequences of NS1 and NS2 protein were obtained.-The homology of these nucleotide sequences and the putative amino acid sequences were compared with several classic reference viruses of H9N2. These isolates were proved to be highly homologous in NS gene (92.9%-99.9% identity) and all belonged to A/Chicken/Beijing/1/1994-like group in the Asia bird-swine branch of allele A of HS gene phylogenetic tree.-According to this study and previous reports of other researchers, NS gene of H9N2 subtype AIV in chickens of China is genetically stable and there is no enough evidence to support the establishment of other sub-lineages in chickens.

Research on multiplication of the H9N2 subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system / 中国人兽共患病学报

To explore the regularity for the multiplication of avian influenza virus subtype H9N2 in large-scale microcarrier-based MDCK cell culture system, and to determine the optimal proliferation conditions. H9N2 subtype of avian influenza virus was inoculated into the MDCK cell growing on 24 well plate, and the HA titers of virus at different time were detected in the conditions of different infectious doses,different concentrations of TPCK- trypsin and different pH. The optimal conditions were determined. Then the H9N2 subtype avian influenza virus was grown in microcarrier-based MDCK cell in 250mL and 5L roller bottles. It was demonstrated that high viruse yield with a hemagglutination unit of 9 log2(1:512) could be obtained under the optimal conditions of multiplication . The result indicated the H9N2 subtype avian influenza virus could be produced in microcarrier-based MDCK cell in a large-scale culture system with a high virus yield and demonstrates the feasibility of the development of mammalian cell-based in influenza vaccine in microcarrier culture systems.