Evidence for human infection with avian influenza A(H9N2) virus via environmental transmission inside live poultry market in Xiamen, China.

H9N2 avian influenza virus (AIV) has become prevalent in the live poultry market (LPM) worldwide, and environmental transmission mode is an important way for AIVs to infect human beings in the LPM. To find evidence of human infection with the influenza A(H9N2) virus via environmental contamination, we evaluated one human isolate and three environmental isolates inside LPMs in Xiamen, China. The phylogeny, transmissibility, and pathogenicity of the four isolates were sorted out systematically. As for the H9N2 virus, which evolved alongside the "Avian-Environment-Human" spreading chain in LPMs from the summer of 2019 to the summer of 2020, its overall efficiency of contact and aerosol transmissibility improved, which might contribute to the increasing probability of human infection. This study indicated that environmental exposure might act as an important source of human infection in LPMs.

Pathogen change of avian influenza virus in the live poultry market before and after vaccination of poultry in southern China.

BACKGROUND: The fifth wave of H7N9 avian influenza virus caused a large number of human infections and a large number of poultry deaths in China. Since September 2017, mainland China has begun to vaccinate poultry with H5 + H7 avian influenza vaccine. We investigated the avian influenza virus infections in different types of live poultry markets and samples before and after genotype H5 + H7 vaccination in Nanchang, and analyzed the changes of the HA subtypes of AIVs. METHODS: From 2016 to 2019, we monitored different live poultry markets and collected specimens, using real-time reverse transcription polymerase chain reaction (RT-PCR) technology to detect the nucleic acid of type A avian influenza virus in the samples. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. The χ2 test was used to compare the differences in the separation rates of different avian influenza subtypes. RESULTS: We analyzed 5,196 samples collected before and after vaccination and found that the infection rate of AIV in wholesale market (21.73%) was lower than that in retail market (24.74%) (P < 0.05). Among all the samples, the positive rate of sewage samples (33.90%) was the highest (P < 0.001). After vaccination, the positive rate of H5 and H7 subtypes decreased, and the positive rate of H9 subtype and untypable HA type increased significantly (P < 0.001). The positive rates of H9 subtype in different types of LPMs and different types of samples increased significantly (P < 0.01), and the positive rates of untypable HA type increased significantly in all environmental samples (P < 0.05). CONCLUSIONS: Since vaccination, the positive rates of H5 and H7 subtypes have decreased, but the positive rates of H9 subtypes have increased to varying degrees in different testing locations and all samples. This results show that the government should establish more complete measures to achieve long-term control of the avian influenza virus.

Different intervention strategies toward live poultry markets against avian influenza A (H7N9) virus: Model-based assessment.

BACKGROUND: Different interventions targeting live poultry markets (LPMs) are applied in China for controlling avian influenza A (H7N9), including LPM closure and "1110" policy (i.e., daily cleaning, weekly disinfection, monthly rest day, zero poultry stock overnight). However, the interventions' effectiveness has not been comprehensively assessed. METHODS: Based on the available data (including reported cases, domestic poultry volume, and climate) collected in Guangdong Province between October 2013 and June 2017, we developed a new compartmental model that enabled us to infer H7N9 transmission dynamics. The model incorporated the intrinsic interplay among humans and poultry as well as the impacts of absolute humidity and LPM intervention, in which intervention strategies were parameterized and estimated by Markov chain Monte Carlo method. RESULTS: There were 258 confirmed human H7N9 cases in Guangdong during the study period. If without interventions, the number would reach 646 (95%CI, 575-718) cases. Temporal, seasonal and permanent closures of LPMs can substantially reduce transmission risk, which might respectively reduce human infections by 67.2% (95%CI, 64.3%-70.1%), 75.6% (95%CI, 73.8%-77.5%), 86.6% (95%CI, 85.7-87.6%) in total four epidemic seasons, and 81.9% (95%CI, 78.7%-85.2%), 91.5% (95%CI, 89.9%-93.1%), 99.0% (95%CI, 98.7%-99.3%) in the last two epidemic seasons. Moreover, implementing the "1110" policy from 2014 to 2017 would reduce the cases by 34.1% (95%CI, 20.1%-48.0%), suggesting its limited role in preventing H7N9 transmission. CONCLUSIONS: Our study quantified the effects of different interventions and execution time toward LPMs for controlling H7N9 transmission. The results highlighted the importance of closing LPMs during epidemic period, and supported permanent closure as a long-term plan.

Interventions in Live Poultry Markets for the Control of Avian Influenza: A Systematic Review and Meta-analysis.

BACKGROUND: This review aimed to provide constructive suggestions for the control and management of avian influenza through quantitative and qualitative evaluation of the impact of different live poultry market (LPM) interventions. METHODS: Both English and Chinese language databases were searched for articles that were published on or before 9 November 2018. After extraction and assessment of the included literature, Stata14.0 was applied to perform a meta-analysis to explore the impacts of LPM interventions. RESULTS: A total of 19 studies were identified. In total, 224 human, 3550 poultry, and 13 773 environment samples were collected before the intervention; 181 people, 4519 poultry, and 9562 environments were sampled after LPM interventions. Avian influenza virus (AIV) detection rates in the LPM environment (odds ratio [OR], 0.393; 95% confidence interval [CI], 0.262-0.589) and the incidence of AIV infection (OR, 0.045; 95% CI, 0.025-0.079) were significantly lower after LPM interventions, while interventions were not significantly effective in reducing AIV detection in poultry samples (OR, 0.803; 95% CI, 0.403-1.597). CONCLUSIONS: LPM interventions can reduce AIV human infections and the detection rate of AIV in market environments.

Live poultry market closure and avian influenza A (H7N9) infection in cities of China, 2013-2017: an ecological study.

BACKGROUND: Previous studies have proven that the closure of live poultry markets (LPMs) was an effective intervention to reduce human risk of avian influenza A (H7N9) infection, but evidence is limited on the impact of scale and duration of LPMs closure on the transmission of H7N9. METHOD: Five cities (i.e., Shanghai, Suzhou, Shenzhen, Guangzhou and Hangzhou) with the largest number of H7N9 cases in mainland China from 2013 to 2017 were selected in this study. Data on laboratory-confirmed H7N9 human cases in those five cities were obtained from the Chinese National Influenza Centre. The detailed information of LPMs closure (i.e., area and duration) was obtained from the Ministry of Agriculture. We used a generalized linear model with a Poisson link to estimate the effect of LPMs closure, reported as relative risk reduction (RRR). We used classification and regression trees (CARTs) model to select and quantify the dominant factor of H7N9 infection. RESULTS: All five cities implemented the LPMs closure, and the risk of H7N9 infection decreased significantly after LPMs closure with RRR ranging from 0.80 to 0.93. Respectively, a long-term LPMs closure for 10-13 weeks elicited a sustained and highly significant risk reduction of H7N9 infection (RRR = 0.98). Short-time LPMs closure with 2 weeks in every epidemic did not reduce the risk of H7N9 infection (p > 0.05). Partially closed LPMs in some suburbs contributed only 35% for reduction rate (RRR = 0.35). Shenzhen implemented partial closure for first 3 epidemics (p > 0.05) and all closure in the latest 2 epidemic waves (RRR = 0.64). CONCLUSION: Our findings suggest that LPMs all closure in whole city can be a highly effective measure comparing with partial closure (i.e. only urban closure, suburb and rural remain open). Extend the duration of closure and consider permanently closing the LPMs will help improve the control effect. The effect of LPMs closure seems greater than that of meteorology on H7N9 transmission.

Genetic and biological characteristics of avian influenza virus subtype H1N8 in environments related to live poultry markets in China.

BACKGROUND: Since 2008, avian influenza surveillance in poultry-related environments has been conducted annually in China. Samples have been collected from environments including live poultry markets, wild bird habitats, slaughterhouses, and poultry farms. Multiple subtypes of avian influenza virus have been identified based on environmental surveillance, and an H1N8 virus was isolated from the drinking water of a live poultry market. METHODS: Virus isolation was performed by inoculating influenza A-positive specimens into embryonated chicken eggs. Next-generation sequencing was used for whole-genome sequencing. A solid-phase binding assay was performed to test the virus receptor binding specificity. Trypsin dependence plaque formation assays and intravenous pathogenicity index tests were used to evaluate virus pathogenicity in vitro and in vivo, respectively. Different cell lines were chosen for comparison of virus replication capacity. RESULTS: According to the phylogenetic trees, the whole gene segments of the virus named A/Environment/Fujian/85144/2014(H1N8) were of Eurasian lineage. The HA, NA, PB1, and M genes showed the highest homology with those of H1N8 or H1N2 subtype viruses isolated from local domestic ducks, while the PB2, PA, NP and NS genes showed high similarity with the genes of H7N9 viruses detected in 2017 and 2018 in the same province. This virus presented an avian receptor binding preference. The plaque formation assay showed that it was a trypsin-dependent virus. The intravenous pathogenicity index (IVPI) in chickens was 0.02. The growth kinetics of the A/Environment/Fujian/85144/2014(H1N8) virus in different cell lines were similar to those of a human-origin virus, A/Brisbane/59/2007(H1N1), but lower than those of the control avian-origin and swine-origin viruses. CONCLUSIONS: The H1N8 virus was identified in avian influenza-related environments in China for the first time and may have served as a gene carrier involved in the evolution of the H7N9 virus in poultry. This work further emphasizes the importance of avian influenza virus surveillance, especially in live poultry markets (LPMs). Active surveillance of avian influenza in LPMs is a major pillar supporting avian influenza control and response.

Westward Spread of Highly Pathogenic Avian Influenza A(H7N9) Virus among Humans, China.

We report infection of humans with highly pathogenic avian influenza A(H7N9) virus in Shaanxi, China, in May 2017. We obtained complete genomes for samples from 5 patients and from live poultry markets or farms in 4 cities. Results indicate that H7N9 is spreading westward from southern and eastern China.

Effect of Live Poultry Market Interventions on Influenza A(H7N9) Virus, Guangdong, China.

Since March 2013, three waves of human infection with avian influenza A(H7N9) virus have been detected in China. To investigate virus transmission within and across epidemic waves, we used surveillance data and whole-genome analysis of viruses sampled in Guangdong during 2013-2015. We observed a geographic shift of human A(H7N9) infections from the second to the third waves. Live poultry market interventions were undertaken in epicenter cities; however, spatial phylogenetic analysis indicated that the third-wave outbreaks in central Guangdong most likely resulted from local virus persistence rather than introduction from elsewhere. Although the number of clinical cases in humans declined by 35% from the second to the third waves, the genetic diversity of third-wave viruses in Guangdong increased. Our results highlight the epidemic risk to a region reporting comparatively few A(H7N9) cases. Moreover, our results suggest that live-poultry market interventions cannot completely halt A(H7N9) virus persistence and dissemination.

Relationship between domestic and wild birds in live poultry market and a novel human H7N9 virus in China.

To trace the source of the avian H7N9 viruses, we collected 99 samples from 4 live poultry markets and the family farms of 3 patients in Hangzhou city of Zhejiang province, China. We found that almost all positive samples came from chickens and ducks in live poultry markets. These results strongly suggest that the live poultry markets are the major source of recent human infections with H7N9 in Hangzhou city, Zhejiang province of China. Therefore, control measures are needed, not only in the domestic bird population, but also in the live poultry markets to reduce human H7N9 infection risk.

Research Note: Factors influencing highly pathogenic avian influenza preventive behavior among live poultry market vendors.

In China, fresh food has always been a top priority and live poultry has been a staple in the diet for thousands of years. As a result, the live poultry market (LPM) remains a popular and important source for purchasing live poultry among the public. However, LPMs also play a crucial role in spreading and retaining highly pathogenic avian influenza (HPAI) due to the high poultry movement and trade volume. Therefore, the preventive behavior of LPM vendors is essential in blocking the transmission of HPAI and reducing occupational exposure. Based on the health belief model, this study utilized structural equation modeling to examine the effect of risk perceptions on preventive behavior among vendors in the live poultry wholesale market (wLPM) and the live poultry retail market (rLPM) in Guangdong Province. The results indicated that perceived severity and perceived benefits positively influenced the wLPM vendors' ability to adopt preventive behavior (i.e., self-efficacy) while perceived barriers negatively affected self-efficacy in both wLPM and rLPM. Moreover, cues to action positively mediated the relationship between perceived severity, perceived benefits, and self-efficacy of wLPM and rLPM vendors. Cues to action also positively mediated the effect of perceived susceptibility among wLPM vendors. To promote preventive behavior among vendors, the market management companies and the government must provide timely and effective HPAI information through various channels and develop differentiated health campaigns according to the market types to raise vendors' awareness about HPAI.

Live poultry markets beyond health risks: Understanding consumer preferences for live poultry in South China.

Live poultry markets (LPMs) are veterinary and public health risks because of potential for zoonotic spillover of pathogens from diseased animals to humans. To control these health risks, veterinary and public health authorities in Asia, including China, have closed or restricted LPMs. In south China, however, LPM closure has been opposed or rendered infeasible by consumers who prefer to purchase live poultry. Previous scholarship has suggested this preference is due to cultural values of freshness. In this study, we present results from detailed interviews with shoppers in south China, including those who prefer live poultry and those who prefer pre-slaughtered poultry. We argue that broader concerns about food safety and quality, rather than freshness alone, drive the demand for LPMs. Live poultry provide sensory information that enable shoppers to evaluate safety and quality in ways that are not possible with pre-slaughtered, refrigerated meat. Based on these findings, we suggest that hygienic interventions into LPMs should recognize that not only freshness, but also trust, must be constructed and maintained in any intervention.

Etiologic characteristics of avian influenza H11 viruses isolated from the live poultry market in southeast coastal region in China.

Since it was first identified in 1956, the H11 subvariant influenza virus has been reported worldwide. However, due to the low pathogenicity of the H11 subvariant and the absence of its widespread transmission among humans, there are only a few reports on the etiology of the H11 subvariant influenza virus. Therefore, in the present study, we isolated a strain of the H11N3 avian influenza virus (AIV) from poultry feces from the live poultry market in the southeast coastal region of China. Considering that the H11 subvariant is known to cause infections in humans and to enrich the knowledge of the H11 subvariant of the avian influenza virus, the genetics, pathogenicity, and transmissibility of the isolate were studied. The phylogenetic analysis indicated that the H11N3 isolate was of Eurasian origin and carried genes closely related to duck H7N2 and H4N6. The receptor binding analysis revealed that the H11N3 isolate only acquired a binding affinity for avian-derived receptors. In the respiratory system of mice, the isolate could directly cause infection without adaptation. In addition, the results from transmission experiments and antibody detection in guinea pigs demonstrated that H11N3 influenza viruses can efficiently transmit through the respiratory tract in mammalian models. Direct infection of the H11N3 influenza virus without adaptation in the mouse models and aerosol transmission between guinea pig models confirms its pandemic potential in mammals, underscoring the importance of monitoring rare influenza virus subtypes in future studies.

Characterisation, whole-genome sequencing and phylogenetic analysis of three H3N2 avian influenza viruses isolated from domestic ducks at live poultry markets of Iran, 2017: First report.

BACKGROUND: Avian influenza type A viruses (AIV) can infect a broad range of hosts including human and birds, making them an important viral pathogen with zoonotic potential. Ducks are a known reservoir for many avian viruses including the AIV. OBJECTIVES: To sequence the entire genome of duck-derived H3N2 and ran comprehensive phylogenetic analysis on them to study their origin. METHODS: In this study, 962 cloacal swabs were collected from domestic ducks at several live poultry markets (LPMs) of Gilan, Mazandaran and Golestan provinces of Iran in the year 2017. RESULTS: Preliminary assays such as haemagglutination inhibition assay (HI), Neuraminidase Inhibition assay(NI) and RT-qPCR suggested that 0.5% of the birds were infected by H3 low pathogenic influenza viruses (LPAI). Three isolates were selected for whole genome sequencing. The cleavage site of the HA genes showed a PEKQTR/GLF motif, an indicator of LPAI. Furthermore, BLAST and phylogenetic analyses of the HA gene showed high homology to the Eurasian lineage of H3N8 AIV (95.5%-97.1% to several European and East Asian isolates). However, the NA genes showed high homology (at most 96.5-96.9%) to those belonging to AIV N2 subtype. Furthermore, internal genes showed high homology (96%-98%) to a variety of duck-origin subtypes and glycoprotein combinations, which were different for each segment. This showed a complex reassortment between different subtypes. DISCUSSION: This report is the first whole genome sequencing and complete characterisation of H3N2 AIV from Iran. CONCLUSION: Such surveillance should continue to study the evolution and possible emergence of viruses with pandemic potential.

Distinct but connected avian influenza virus activities in wetlands and live poultry markets in Bangladesh, 2018-2019.

From April 2018 to October 2019, we continued active surveillance for influenza viruses in Bangladeshi live poultry markets (LPMs) and in Tanguar Haor, a wetland region of Bangladesh where domestic ducks have frequent contact with migratory birds. The predominant virus subtypes circulating in the LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses of the H5N1-R1 genotype, like those found in previous years. Viruses of the H5N1-R2 genotype, which were previously reported as co-circulating with H5N1-R1 genotype viruses in LPM, were not detected. In addition to H9N2 viruses, which were primarily found in chicken and quail, H2N2, H3N8 and H11N3 LPAI viruses were detected in LPMs, exclusively in ducks. Viruses in domestic ducks and/or wild birds in Tanguar Haor were more diverse, with H1N1, H4N6, H7N1, H7N3, H7N4, H7N6, H8N4, H10N3, H10N4 and H11N3 detected. Phylogenetic analyses of these LPAI viruses suggested that some were new to Bangladesh (H2N2, H7N6, H8N4, H10N3 and H10N4), likely introduced by migratory birds of the Central Asian flyway. Our results show a complex dynamic of viral evolution and diversity in Bangladesh based on factors such as host populations and geography. The LPM environment was characterised by maintenance of viruses with demonstrated zoonotic potential and H5N1 genotype turnover. The wetland environment was characterised by greater viral gene pool diversity but a lower overall influenza virus detection rate. The genetic similarity of H11N3 viruses in both environments demonstrates that LPM and wetlands are connected despite their having distinct influenza ecologies.

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.

Live poultry feeding and trading network and the transmission of avian influenza A(H5N6) virus in a large city in China, 2014-2015.

OBJECTIVES: To understand the transmission mechanisms of the avian influenza A(H5N6) virus. METHODS: This study explored the live poultry feeding and trading network (LPFTN) around Changsha city, China. Field epidemiological investigations were performed in Changsha to investigate the LPFTN with the environmental samples systematically collected during 2014-2015 to monitor and analyze the spread of the A(H5N6) virus. Two surveillance systems were also applied to find possible human cases of A(H5N6) infection. RESULT: The information of all the 665 live poultry farming sites, five wholesale markets, and 223 retail markets in Changsha was collected to investigate the LPFTN. Moreover, about 840 environmental samples were systematically collected from the LPFTN during 2014-2015 to monitor the spread of the A(H5N6) virus, with 8.45% (71/840) positive for the N6 subtype. Furthermore, the full genome sequences of 10 A(H5N6) viruses detected from the environmental samples were obtained, which were then characterized and phylogenetically analyzed with the corresponding gene segments of the A(H5N6) virus obtained from GenBank, to determine the source of human infection. CONCLUSION: It was demonstrated that the LPFTN provided a platform for the H5N6 transmission, and formed an infectious pool for the spread of the virus to humans.

More diversified antibiotic resistance genes in chickens and workers of the live poultry markets.

BACKGROUND: Poultry farms and LPMs are a reservoir of antimicrobial resistant bacteria and resistance genes from feces. The LPM is an important interface between humans, farm animals, and environments in a typical urban environment, and it is considered a reservoir for ARGs and viruses. However, the antibiotic resistomes shared between chicken farms and LPMs, and that of LPM workers and people who have no contact with the LPMs remains unknown. METHODS: We characterized the resistome and bacterial microbiome of farm chickens and LPMs and LPM workers and control subjects. The mobile ARGs identified in chickens and the distribution of the mcr-family genes in publicly bacterial genomes and chicken gut metagenomes was analyzed, respectively. In addition, the prevalence of mcr-1 in LPMs following the ban on colistin-positive additives in China was explored. RESULTS: By profiling the microbiomes and resistomes in chicken farms, LPMs, LPM workers, and LPM environments, we found that the bacterial community composition and resistomes were significantly different between the farms and the LPMs, and the LPM samples possessed more diversified ARGs (59 types) than the farms. Some mobile ARGs, such as mcr-1 and tet(X3), identified in chicken farms, LPMs, LPM workers, and LPM environments were also harbored by human clinical pathogens. Moreover, we found that the resistomes were significantly different between the LPM workers and those who have no contact with the LPMs, and more diversified ARGs (188 types) were observed in the LPM workers. It is also worth noting that mcr-10 was identified in both human (5.2%, 96/1,859) and chicken (1.5%, 14/910) gut microbiomes. Although mcr-1 prevalence decreased significantly in the LPMs across the eight provinces in China, from 190/333 (57.1%) samples in September 2016-March 2017 to 208/544 (38.2%) samples in August 2018-May 2019, it is widespread and continuous in the LPMs. CONCLUSION: Live poultry trade has a significant effect on the diversity of ARGs in LPM workers, chickens, and environments in China, driven by human selection with the live poultry trade. Our findings highlight the live poultry trade as ARG disseminators into LPMs, which serve as an interface of LPM environments even LPM workers, and that could urge Government to have better control of LPMs in China. Further studies on the factors that promote antibiotic resistance exchange between LPM environments, human commensals, and pathogens, are warranted.

Molecular characterization and antigenic analysis of reassortant H9N2 subtype avian influenza viruses in Eastern China in 2016.

H9N2 avian influenza viruses (AIVs) can cause respiratory symptoms and decrease the egg production. Additionally, H9N2 AIVs can provide internal genes for reassortment with other subtypes. During the monitoring of live poultry markets in 2016, a total of 32 (32/179, 17.88%) H9N2 AIVs were isolated from poultry in Eastern China, and seven representative strains were selected based on the isolation time, isolation location and sequence homology for further characterization. Phylogenetic analysis of hemagglutinin and neuraminidase showed that these H9N2 AIVs clustered into the Y280 sublineage. And the phylogenetic trees of six internal genes showed that the source of these gene fragments was more abundant, suggesting that extensive reassortment has occurred in these H9N2 viruses. Molecular analysis showed that multiple specific amino acid mutations occurred that increased H9N2 AIVs' infectivity, transmissibility, and affinity to mammals, including Q226L and Q227M in hemagglutinin, E627K in polymerase basic protein 2 (PB2), L13P in polymerase basic protein 1 (PB1), and A70V and S409N in polymerase acidic protein (PA). Pathogenicity tests in mice showed these H9N2 AIVs could replicate in lungs and exhibited slight to moderate virulence. The continuous circulation of these H9N2 viruses suggests the necessity for persistent surveillance of the H9N2 AIVs in poultry.

Genetic analysis and biological characteristics of novel clade 2.3.4.4 reassortment H5N6 avian influenza viruses from poultry in eastern China in 2016.

OBJECTIVES: The continuous evolution of highly pathogenic H5N6 avian influenza viruses (AIVs) causes outbreaks in wildfowl and poultry, and occasional human infections. The aim of this study was to better understand the genetic relationships between these H5N6 AIVs from eastern China and other AIVs. METHODS: In 2016, 1623 cloacal swabs were sampled from poultry in 18 LPMs in eastern China, and subsequently characterized systematically using gene sequencing, phylogenetic studies, and antigenic analysis. In addition, their pathogenicity in mammals was studied in BALB/c mice, which were inoculated with viruses, with survival rate and body weight recorded daily for 14 days. RESULTS: In total, 56 H5N6 AIVs were isolated in eastern China and five representative isolates were selected for further study. In our study, the H5N6 AIVs clustered into clade 2.3.4.4, Group C, and their six internal segments were derived from H6N6 and H9N2 viruses, or both, suggesting extensive reassortant among H5N6 AIVs with other subtypes. These H5N6 viruses could replicate in the lungs without prior adaptation, and exhibited slight-to-moderate virulence in mice. CONCLUSIONS: The continuous circulation of these novel H5N6 viruses suggests the importance of persistent surveillance of H5N6 AIVs in poultry.