Distribution of avian influenza viruses according to environmental surveillance during 2014-2018, China.

BACKGROUND: Recurrent infections of animal hosts with avian influenza viruses (AIVs) have posted a persistent threat. It is very important to understand the avian influenza virus distribution and characteristics in environment associated with poultry and wild bird. The aim of this study was to analyze the geographic and seasonal distributions of AIVs in the 31 provinces, municipalities and autonomous region (PMA) of China, compare the AIVs prevalence in different collecting sites and sampling types, analyze the diversity of AIVs subtypes in environment. METHODS: A total of 742 005 environmental samples were collected from environmental samples related to poultry and wild birds in different locations in the mainland of China during 2014-2018. Viral RNA was extracted from the environmental samples. Real-time RT-PCR assays for influenza A, H5, H7 and H9 subtypes were performed on all the samples to identify subtypes of influenza virus. The nucleic acid of influenza A-positive samples were inoculated into embryonated chicken eggs for virus isolation. Whole-genome sequencing was then performed on Illumina platform. SPSS software was used to paired t test for the statistical analysis. ArcGIS was used for drawing map. Graphpad Prism was used to make graph. RESULTS: The nucleic acid positivity rate of influenza A, H5, H7 and H9 subtypes displayed the different characteristics of geographic distribution. The nucleic acid positivity rates of influenza A were particularly high (25.96%-45.51%) in eleven provinces covered the Central, Eastern, Southern, Southwest and Northwest of China. The nucleic acid positivity rates of H5 were significantly high (11.42%-13.79%) in two provinces and one municipality covered the Southwest and Central of China. The nucleic acid positivity rates of H7 were up to 4% in five provinces covered the Eastern and Central of China. The nucleic acid positivity rates of H9 were higher (13.07%-2.07%) in eleven PMA covered the Southern, Eastern, Central, Southwest and Northwest of China. The nucleic acid positivity rate of influenza A, H5, H7 and H9 showed the same seasonality. The highest nucleic acid positivity rates of influenza A, H5, H7, H9 subtypes were detected in December and January and lowest from May to September. Significant higher nucleic acid positivity rate of influenza A, H5, H7 and H9 were detected in samples collected from live poultry markets (LPM) (30.42%, 5.59%, 4.26%, 17.78%) and poultry slaughterhouses (22.96%, 4.2%, 2.08%, 12.63%). Environmental samples that were collected from sewage and chopping boards had significantly higher nucleic acid positivity rates for influenza A (36.58% and 33.1%), H5 (10.22% and 7.29%), H7(4.24% and 5.69%)and H9(21.62% and 18.75%). Multiple subtypes of AIVs including nine hemagglutinin (HA) and seven neuraminidase (NA) subtypes were isolated form the environmental samples. The H5, H7, and H9 subtypes accounted for the majority of AIVs in environment. CONCLUSIONS: In this study, we found the avian influenza viruses characteristics of geographic distribution, seasonality, location, samples types, proved that multiple subtypes of AIVs continuously coexisted in the environment associated with poultry and wild bird, highlighted the need for environmental surveillance in China.

Detection of reassortant avian influenza A (H11N9) virus in environmental samples from live poultry markets in China.

BACKGROUND: Avian influenza viruses have caused human infection and posed the pandemic potential. Live poultry markets are considered as a source of human infection with avian influenza viruses. Avian influenza routine surveillance of live poultry markets is taken annually in China. We isolated the 2 H11N9 influenza virus from the surveillance program. To better understand the risk caused by these new viruses, we characterize the genetic and pathogenicity of the two viruses. METHODS: Viral isolation was conducted with specific pathogen-free (SPF) embryonated chicken eggs. Whole genome was sequenced, and phylogenetic analysis was conducted. RESULTS: Two H11N9 viruses were identified, with all 8 segments belonging to the Eurasian lineage. The HA, NA, M, NS and PA genes were similar to virus isolates from ducks, and the NP, PB2 and PB1 gene segments were most similar to those viruses from wild birds, indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds. The HA receptor binding preference was avian-like, and the cleavage site sequence of HA showed low pathogenic. The NA gene showed 94.6 % identity with the novel H7N9 virus that emerged in 2013. There was no drug resistance mutation in the M2 protein. The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice. Both viruses were low-pathogenic strains, as assessed by the standards of intravenous pathogenicity index (IVPI) tests. CONCLUSION: Two reassortant H11N9 avian influenza viruses were detected. These viruses showed low pathogenicity to chickens in the IVPI test. Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance.

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China.

OBJECTIVE: To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. METHODS: Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses. RESULTS: The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. CONCLUSION: The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

A novel reassortant H3N8 influenza virus isolated from drinking water for duck in a domestic duck farm in Poyang Lake area.

OBJECTIVE: To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011. METHODS: The virus was cultivated by specific pathogen free (SPF) chicken embryo eggs and was subtyped into hemagglutinin (HA) and neuraminidase (NA) by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted. RESULTS: The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB1 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr215Ala of the M1 protein implied the potential of pathogenicity increase in mice. CONCLUSION: The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.

[Molecular characterization of highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province, 2006 - 2009].

OBJECTIVE: To understand the possible origins, genetic re-assortment and molecular characterization of 4 highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province, between 2006 and 2009. METHODS: H5N1 PCR test-positive specimens were inoculated in embryonated eggs while H5N1 virus was isolated and genomes sequenced. Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 4.0. RESULTS: All gene segments of the 4 viruses were avian in origin. No re-assortment was found between avian influenza A (H5N1) viruses and human seasonal influenza viruses. Viruses that isolated from domestic poultry shared high similarity with the 4 human viruses in gene homology. Data from the whole genome phylogenetic analysis showed that the 4 viruses were in clade 2.3.4, while 2 viruses belonged to genotype V, and another 2 were new genotypes. Results from molecular characterization showed that amino acid sequences of HA cleavage site of the 4 viruses were PLRERRKR/G. All 4 viruses had A160T mutation in HA, a 20 amino acid deletion in the neuraminidase (NA) stalk at position 49 - 68, and a 5 amino acid deletion in the non-structural protein 1 (NS1). Most sites in the HA molecules showed that the viruses preferentially bound to avian influenza virus receptor. However, T192I mutation that might enhance the α 2, 6-linked sialic acid human influenza receptor binding had emerged in HN/1/09 and HN/2/09. D701N mutation of PB2 that increased the virulence in mice was found in HN/1/08. Analysis on drug resistance gene amino acid showed that all 4 viruses were sensitive to amantadine and oseltamivir. CONCLUSION: Highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province from 2006 to 2009 were avian in origin, and the 4 viruses belonged to different genotypes. Some mutations that related to virulence and receptor binding positions had emerged in some of the strains.

[Construction and characterization of reverse genetic system for H9N2 avian influenza].

OBJECTIVE: To provide a technology platform for vaccine development as well as the research on transmission and pathogenesis, the reverse genetic system for H9N2 avian influenza virus was established. METHODS: Eight full-length cDNAs of avian influenza virus A/Guangzhou/333/99 (H9N2) were amplified by RT-PCR and separately cloned into the transcription/expression vector, pCI-polI. The 8 plasmids DNA was cotransfected into 293T cell, the cell supernatant was collected and inoculated into embryonated eggs, the rescued virus from the allantoic fluid was identified by hemagglutinination assay. RESULTS: The avian influenza H9N2 virus was successfully rescued by 8 plasmids co-transfection in 293T cells. The hemagglutinination titer of the rescued virus is up to 2(9)/50 microl and its growth curve remained relatively as to the wild-type virus. CONCLUSION: The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.

[The virus isolation analysis of the H5N1 subtype human avain influenza cases in mainland China from 2005 to 2009].

OBJECTIVE: To analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China. METHODS: The specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs. RESULTS: Most specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens. CONCLUSION: We suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.

[Study on the correlation of human influenza A/H3N2 hemagglutinin gene variation and the epidemic from 1995 to 2005 in China].

To study the correlation of human influenza A/H3N2 hemagglutinin gene variation and the epidemic from 1995 to 2005 in China, 550 HA1 sequences of H3N2 viruses isolated in China were analyzed with phylogenetic tree. The results showed that the evolution of HA1 represented a long trunk with short side branches. The animo acid changes of HA1 mostly located at the antigenic sites or aside of them, but also may occur at the other sites simultaneously. The analysis also showed three possibilities of HA1 variation to cause H3N2 epidemic, the first is multiple site mutations happened simultaneously; the second is mutation sites happened gradually and then accumulated to multiple sites; the third is a single antigenic site mutation occurred simultaneously with the receptor binding site variation.

[Characteristic analysis of NA gene of human influenza viruses (H3N2) isolated from 1996 to 2005 in China].

The NA genes of 395 strains of human H3N2 influenza virus isolated from 1996 to 2005 in China were sequenced, analyzed with bioinformatics tools. The NA nucleotide sequence of phylogenetic tree showed a main evolution branch with multiple short side branches. The strains in the same year may be divided into several branches. There was an obvious lag between vaccine strains recommended by WHO and the Chinese circulating strains in phylogenetic tree of the NA nucleotide. The result also showed no amino acid deletion and insertion in the NA. In NA antigen sites, where including residues 197-199 aa, 431-434 aa and 339-347aa the mutation was higher, in contrast, the residues including 153 aa, 328-336 aa, 367-370aa and 400-403 aa, the mutation was lower. Besides the antigenic determinant sites, there also had the other amino acid mutated highly, such as 18, 23, 30, 93, 143, 208, 216, 221, 249, 265, 267, 307, 385 and 437 aa, among them 143 and 267 mutation were higher than that in antigenic determinant sites, their biological significance are not clear yet. The neuraminidase active-site residues in NA were highly conservative and the same were the disulphide bond and the glycosylation sites in NA. In conclusion, our analysis provides some information for influenza prevention and control and the NA inhibitor medicine application.

[Adamantane resistance among influenza A (H3N2) viruses isolated from the mainland of China].

BACKGROUND: To study the incidence of adamantane resistance among influenza A (H3N2) viruses isolated from the mainland of China since 1989 through our influenza surveillance system, and to provide more information for the clinical usage of adamantane drugs. METHODS: Totally 584 influenza A (H3N2) virus strains were randomly selected from our surveillance network since 1989, the adamantane drug resistance related gene M2 of all 584 strains was sequenced, and the drug sensitivity of viruses was also assayed by using biological methods in cells. RESULTS: No adamantane resistant strains were detected among the strains isolated from 1989 to 1999, but there was a surprisingly increased resistance rate of 56% in 2003 compared with 3.4% in 2002, and in 2005 the resistance rate increased to 77.6%. CONCLUSION: Over 50% of virus among the strains isolated showed adamantane resistance since 2003, and the incidence rate is increasing.