The global expansion of avian influenza viruses (AIVs), particularly clade 2.3.4.4b H5 viruses, continues to pose a significant zoonotic threat. Although Chinese vaccination-plus-culling strategy has reduced H5- and H7-exposure disease activity, continued viral evolution and reassortment highlight the need for sustained surveillance at the human-environment interface. Environmental samples from live poultry markets (LPMs), slaughterhouses and poultry farms/backyards, together with respiratory specimens from hospitalized patients with pneumonia of unknown etiology (PUE) were collected in three Chinese cities from December 2021 to December 2023. Specimens were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR) for influenza A virus (IAV), H5, H7, H9 and H10 subtypes. Positive rates among cities and sampling sites were compared using chi-square tests. Monthly positive rates among environmental sample categories were compared using the Kruskal-Wallis test, and paired monthly rates between 2022 and 2023 using the two-sided Wilcoxon signed-rank test. H5, H9 and H10 viruses underwent HA gene sequencing and phylogenetic analysis. Among 8,335 environmental samples, the overall IAV positive rate was 32.2

The outcomes of viral infections typically correlate with viral load in host tissues. In this study, we identified a H3N2 strain A/Environment/Guangxi/44461/2019 (GX19) that induced rapid mortality in mice by 4 days post-infection despite exhibiting low pulmonary replication capacity. Pathological analysis revealed that GX19 at 106 TCID50 (GX19-6) caused more severe lung damage than GX19 at 105 TCID50 (GX19-5), while inducing pulmonary pathology comparable to a H3N8 virus A/Changsha/1000/2022 at 106 TCID50 (CS-6). Both GX19-6 and CS-6 triggered greater cardiac damage than GX19-5. Notably, GX19-6 displayed unique neurovirulence, eliciting significantly more severe brain damage than GX19-5 and CS-6, accompanied by evident cerebral haemorrhage. Gene Set Variation Analysis (GSVA) revealed distinct cardiac gene expression profiles among viral infections. Specifically, GX19-5 up-regulated gene sets associated with arrhythmia, whereas GX19-6 triggered pathways involved in cardiac arrest. Neither of these effects was present in CS-6 infection. In the brain, GX19-6 specifically induced stronger upregulation of cerebral venous thrombosis and acute ischaemic stroke gene sets compared to other groups, consistent with its pronounced neuropathology. Transcriptomic profiling demonstrated significant alterations across all three organs in GX19-6-infected mice, showing suppression of T-cell immunity in the lungs and brain alongside elevated systemic inflammation. In the heart, increased inflammation and apoptosis were accompanied by impaired energy metabolism and reduced cardiac function, potentially contributing to the observed hypoxic responses in the heart, lungs, and brain. Collectively, these findings reveal an inflammation-driven lung-heart-brain axis in influenza virus pathogenicity.

Accurate genome assembly from metagenomic sequencing data remains challenging, particularly in mixed infections involving multiple pathogens, due to data complexity and contaminant sequences. Here, we present GMW (Genomic Microbe-Wise), a novel computational tool that improves pathogen genome assembly accuracy and enhances contaminant removal capabilities by simplifying the post-assembly graph. GMW leverages community detection algorithms, sequence similarity analysis, and coverage patterns to resolve strain mixtures and improve assembly accuracy. Using datasets of influenza A virus subtypes, we demonstrate GMW’s ability to disentangle mixed infections and reconstruct complete viral genomes with high precision. Additionally, GMW outperforms traditional sequence similarity methods in classifying target contigs from contaminants. This tool also provides interactive visualization modules to streamline the inspection of assembly outputs, including simplified representations of complex assembly graphs. By enhancing assembly quality and contamination filtering, GMW emerges as a versatile solution for applications in clinical diagnostics, microbial ecology, and pathogen surveillance.

Background. Avian influenza virus (AIV) H9N2 has a major role in the emergence of influenza pandemic. We assessed the risk of AIV H9N2 to the human population and public health. Method. The hemagglutination inhibition method was used to screen for hemagglutinin antibodies. Microneutralization tests were performed to confirm neutralizing antibodies against the AIV H9N2 subtype. Real-time polymerase chain reaction was conducted to detect the H9 subtype in environmental samples. GraphPad Prism software was used for mapping, and STATA software was used for statistical analysis. Results. The nationwide seroprevalence among these populations was 0.76%. Seroprevalence was compared across regions, genders, and occupational exposure sites. The seroprevalence rates for males and females showed no significant difference. Significant differences were found across regions and occupational exposure environments (P < .05). The south and southwest regions had the highest seroprevalence rates at 1.58% and 1.38%, respectively. The highest seroprevalence was observed in individuals exposed to live poultry market (1.51%). Significant regional differences in H9 nucleic acid positive rates (NAPRs) were found (P < .05), with the southwest and central regions showing the highest rates at 25.99% and 24.35%, respectively. H9 NAPR in live poultry markets (LPMs), farms, and slaughterhouses varied significantly by region (P < .05). Conclusions. Poultry-related environments have become a key factor in AIV H9N2 infection among occupational populations. Exposure to LPM showed the highest seroprevalence among occupational groups. The distribution characteristics of H9N2 across different poultry environments increased the risk of infection in occupationally exposed populations.

The prevalence and transmission of avian influenza viruses (AIVs) in the live poultry market (LPM) is a serious public health concern. This study was to investigate the prevalence of different subtypes of avian influenza viruses in environment of LPM, and to analyze the differences and seasonality of the nucleic acid positive rate (NAPR) of A type, H5, H7, and H9 subtypes in feces, sewage, drinking water, breeding cages, and chopping boards. Feces, breeding cages swabs, drinking water, sewage and chopping boards swabs were collected from live poultry market during 2019–2023 from southern and northern China. Real-time PCR was used to screen for virus subtypes. Viruses were isolated, and deep sequencing was performed to obtain whole-genome sequences. Chi-square test was used for statistical analysis of categorical variable, GraphPad Prism software were used to construct graphs. A total of 64,599 environmental samples were collected from live poultry markets in the southern China and northern China between 2019 and 2023. The average NAPR of the A type was significantly higher in the samples collected from the southern China than in those collected from the northern China (P < 0.05). The NAPR of H5, H7, and H9 subtypes carried by the five types of environmental samples in the southern China were significantly different (P < 0.05), and a higher NAPR was detected in chopping boards (10.84

The H3 subtype avian influenza virus (AIV) has been widely spread in birds and is known as a natural source of mammalian influenza viruses. Based on data from public databases and our surveillance data, we analyzed the ecology, evolution, and spread of H3 AIVs. Sublineages of H3 AIVs have been detected worldwide, infecting various birds, at least 90 species in wild birds and poultry. Important areas for large-scale and local dissemination of H3 AIVs were identified, such as Alaska, Central Asia, and Chinese provinces. The H3 viruses have elevated the HA gene substitution rate after introduction from wild birds to domestic poultry, and even faster in domestic chickens. Our results implied an evolutionary mechanism of H3 AIV cross-species transmission, that viruses from wild birds to domestic poultry have accelerated substitution rate by shorter generation time and host selection. Novel chicken H3 viruses, especially H3N8 G25 viruses that have spilled over to humans, require high attention.

Subtype H10 viruses are known to infect humans in Africa, Oceania, and Asia. In 2021, 2022, and recently in April 2024, a novel H10N3 subtype avian influenza virus was found cause human infection with severe pneumonia. Herein, we comprehensively studied the phylogenetic evolution and biological characteristics of the newly emerged influenza A(H10N3) virus. We found that the human isolated H10N3 virus was generated in early 2019 in domestic poultry. The viruses bound to salic acid α2, 3 receptors, indicating their insufficient ability to infect humans. Although a low pathogenic avian influenza virus, the human isolated H10N3 virus exhibited robust pathogenicity in both BALB/c and C57BL/6 mice, with MLD50 1000 times higher than a homologous environmental isolate. The human isolated H10N3 also showed respiratory droplet transmissibility in ferrets. Considering the continuous circulation in avian populations and repeated transmission to humans, strengthened surveillance of H10 subtype viruses in poultry should be put into effect.

Wild birds are a natural reservoir for zoonotic viruses. To clarify the role of migratory birds in viruses spread in Poyang Lake, we investigated the microbiome of 250 wild bird samples from 19 species in seven orders. The bacterial and viral content abundance and diversity were preliminarily evaluated by Kraken2 and Bracken. After de novo assembly by Megahit and Vamb, viral contigs were identified by CheckV. The reads remapped to viral contigs were quantified using Bowtie2. The bacterial microbiome composition of the samples covers 1526 genera belonging to 175 bacterial orders, while the composition of viruses covers 214 species belonging to 22 viral families. Several taxonomic biomarkers associated with avian carnivory, oral sampling, and raptor migration were identified. Additionally, 17 complete viral genomes belonging to Astroviridae, Caliciviridae, Dicistroviridae, Picornaviridae, and Tombusviridae were characterized, and their phylogenetic relationships were analyzed. This pioneering metagenomic study of migratory birds in Poyang Lake, China illuminates the diverse microbial landscape within these birds. It identifies potential pathogens, and uncovers taxonomic biomarkers relevant to varied bird habitats, feeding habits, ecological classifications, and sample types, underscoring the public health risks associated with wintering migratory birds.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both alpha 2,3 and alpha 2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn. [GRAPHICS]

Pigs are vital genetic mixing vessels for human and avian influenza viruses because their tracheal epitheliums possess both sialic acid α-2,6-Gal and α-2,3-Gal receptors. Cross-species transmission of influenza A viruses from swine to humans occurs occasionally. The first case of human infection with the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) genotype G4 was detected in Jiangsu Province, China, in February 2023, and backtracking epidemiological investigations did not reveal a clear source of the infection. The hemagglutination (HA) and neuraminidase (NA) amino acid variant sites, antiviral drug susceptibility, and antigenic variation of the isolated A/Jiangsu/27271/2023 (JS/27271/23) virus were analyzed, and we evaluated the protective effect of sera collected from occupationally exposed populations in 2024 against the virus. Compared with the vaccine strain, the nucleotide sequence similarities of JS/27271/23 HA and NA were 96.5 % and 95.2 %, respectively. JS/27271/23 was sensitive to polymerase inhibitors (favipiravir and baloxavir), and the antigenicity of its HA protein was 8-fold different from that of the vaccine strain. The percentage of occupationally exposed population with antibody titers of ≥ 40 against A/Hunan/42443/2015 (HN/42443/15) and A/Jiangsu/1/2011 (JS/1/11) were 7.25 % and 2.25 %, respectively, and the geometric mean titers (GMT) were 6.24 and 5.34, respectively. Out of 400 serum samples examined, none had antibody titers of ≥ 40 against JS/27271/23. This suggests that low serum levels of antibodies to EAH1N1 SIVs in occupationally exposed populations may not provide adequate protection because of significant differences in amino acid sites and antigenicity between this virus and the current vaccine strain of EAH1N1 SIVs. There is no evidence of human-to-human transmission of EAH1N1 SIVs. Therefore, surveillance for EAH1N1 SIVs and the development of new vaccine strains are required.

Since 2022, three human cases of a novel H3N8 avian influenza virus infection have been reported in three provinces in China. Specific vaccines are important means of preparing for the potential influenza pandemic. Thus, H3N8 viruses [A/Henan/cnic410/2022 (HN410) and A/Changsha/1000/2022(CS1000)] were isolated from the infected patients as prototype viruses to develop candidate vaccine viruses (CVVs) using the reverse genetics (RG) technology. Five reassortant viruses with different HA and NA combinations were constructed based on the two viruses to get a high-yield and safe CVV. The results showed that all viruses had similar antigenicity but different growth characteristics. Reassortant viruses carrying NA from CS1000 exhibited better growth ability and NA enzyme activity than the ones carrying HN410 NA. Furthermore, the NA gene of CS1000 had one more potential N-glycosylation site at position 46 compared with HN410. The substitution of position 46 showed that adding or removing N-glycosylation sites to different reassortant viruses had different effects on growth ability. A reassortant virus carrying HN410 HA and CS1000 NA with high growth ability was selected as a CVV, which met the requirements for a CVV. These data suggest that different surface gene combinations and the presence or absence of potential N-glycosylation sites on position 46 in the NA gene affect the growth characteristics of H3N8 CVVs.

H4 avian influenza viruses (AIVs) have been widely detected in live poultry markets in China. However, the potential public health impact of H4 AIVs remains largely uncertain. Here, we fully analyzed the distribution and phylogenetic relationship of H4 AIVs in China. We obtained 31 isolates of H4 viruses in China during 2009–2022 through surveillance in poultry-associated environments, such as live poultry markets and poultry farms. Genomic sequence analysis together with publicly available data revealed that frequent reassortment and introduction of H4 AIV from wild birds to poultry may have occurred. We identified 62 genotypes among 127 whole genome sequences of H4 viruses in China, indicating that H4 AIVs had great genetic diversity in China. We also investigated molecular markers and found that drug resistance mutations frequently occurred in the M2 protein and a few mutations related to receptor binding and the host signature in H4 AIVs. Our study demonstrates the cross-species transmission potential of H4 AIVs in China and provides some reference significance for its risk assessment.

BackgroundSince the first human infection with H9N2 virus was reported in 1998, the number of cases of H9N2 infection has exceeded one hundred by 2021. However, there is no systematic description of the biological characteristics of H9N2 viruses isolated from humans.MethodsTherefore, this study analyzed the pathogenicity in mice of all available H9N2 viruses isolated from human cases in China from 2013 to 2021.ResultsAlthough most of the H9N2 viruses analyzed showed low or no pathogenicity in mice, the leucine to glutamine substitution at residue 226 (L226Q) in the hemagglutinin (HA) protein rapidly emerged during the adaptation of H9N2 viruses, and was responsible for severe infections and even fatalities. HA amino acid 226Q conferred a remarkable competitive advantage on H9N2 viruses in mice relative to viruses containing 226L, increasing their virulence, infectivity, and replication.ConclusionThus, our study demonstrates that the adaptive substitution HA L226Q rapidly acquired by H9N2 viruses during the course of infection in mice contributed to their high pathogenicity.

Objective:To improve the understanding of the virome and bacterial microbiome in the wildlife rescue station of Poyang Lake, China.Methods:Ten smear samples were collected in March 2019. Metagenomic sequencing was performed to delineate bacterial and viral diversity. Taxonomic analysis was performed using the Kraken2 and Bracken methods. A maximum-likelihood tree was constructed based on the RNA-dependent RNA polymerase (RdRp) region of picornavirus.Results:We identified 363 bacterial and 6 viral families. A significant difference in microbial and viral abundance was found between samples S01-S09 and S10. In S01-S09, members of Flavobacteriia and Gammaproteobacteria were the most prevalent, while in S10, the most prevalent bacteria class was Actinomycetia. Among S01-S09, members of Myoviridae and Herelleviridae were the most prevalent, while the dominant virus family of S10 was Picornaviridae. The full genome of the pigeon mesivirus-like virus (NC-BM-233) was recovered from S10 and contained an open reading frame of 8,124 nt. It showed the best hit to the pigeon mesivirus 2 polyprotein, with 84.10% amino acid identity. Phylogenetic analysis showed that RdRp clustered into Megrivirus B.Conclusion:This study provides an initial assessment of the bacteria and viruses in the cage-smeared samples, broadens our knowledge of viral and bacterial diversity, and is a way to discover potential pathogens in wild birds.

The continuous evolution of avian influenza viruses (AIVs) of subtype H3 in China and the emergence of human infection with AIV subtype H3N8 highlight their threat to public health. Through surveillance in poultry-associated environments during 2009–2022, we isolated and sequenced 188 H3 AIVs across China. Performing large-scale sequence analysis with publicly available data, we identified 4 sublineages of H3 AIVs established in domestic ducks in China via multiple introductions from wild birds from Eurasia. Using full-genome analysis, we identified 126 distinct genotypes, of which the H3N2 G23 genotype predominated recently. H3N8 G25 viruses, which spilled over from birds to humans, might have been generated by reassortment between H3N2 G23, wild bird H3N8, and poultry H9N2 before February 2021. Mammal-adapted and drug-resistance substitutions occasionally occurred in H3 AIVs. Ongoing surveillance for H3 AIVs and risk assessment are imperative for potential pandemic preparedness.

IntroductionThe continued emergence of human infections of H9N2 avian influenza virus (AIV) poses a serious threat to public health. The prevalent Y280/G9 lineage of H9N2 AIV in Chinese poultry can directly bind to human receptors, increasing the risk of spillover infections to humans. Since 2013, the number of human cases of H9N2 avian influenza has been increasing continuously, and in 2021, China reported the highest number of human cases, at 25.MethodsIn this study, we analyzed the age, geographic, temporal, and sex distributions of humans with H9N2 avian influenza in 2021 using data from the National Influenza Center (Beijing, China). We also conducted evolutionary, gene homology, and molecular characterization analyses of the H9N2 AIVs infecting humans.ResultsOur findings show that children under the age of 12 accounted for 80% of human cases in 2021, and females were more frequently affected than males. More cases occurred in winter than in summer, and most cases were concentrated in southern China. Human-infecting H9N2 viruses showed a high level of genetic homology and belonged to the prevalent G57 genotype. Several additional α2,6-SA-binding sites and sites of mammalian adaptation were also identified in the genomes of human-infecting H9N2 viruses.DiscussionTherefore, continuous monitoring of H9N2 AIV and the implementation of further measures to control the H9N2 virus in poultry are essential to reduce the interspecies transmission of the virus.

H6 avian influenza virus (AIV) is one of the most prevalent AIV subtypes in birds globally. To investigate the current situation and characteristics of H6 AIVs circulating in China, we analysed the epidemiology, genetic evolution and pathogenic features of this subtype. During 2000-2021, H6 subtype AIVs spread widely through Southern China and presented high host diversity. On analysing 171 H6 viruses isolated during 2009-2021, dynamic reassortments were observed among H6 and other co-circulating AIV subtypes, and these generated a total of 16 different genotypes. A few H6N6 strains possessed L226 and S228 mutations of hemagglutinin (H3 numbering), which may enhance the affinity of H6 viruses to human receptors. H6N6 viruses also exhibited divergent pathogenicity and growth profiles in vivo and in vitro. Some of the H6N6 viruses could infect mice without mammalian adaptation, and even caused death in this species. Therefore, our study demonstrated that the H6 AIVs posed a potential threat to human health and highlighted the urgent need for continued surveillance and evaluation of the H6 influenza viruses circulating in the field.

Background The first and only case of human infection with the avian influenza A (H7N4) virus in China emerged in 2018. The H7N4 virus was distinct from previous H7N9 viruses and raised public concerns. Therefore, developing a suitable H7N4 candidate vaccine virus (CVV) remains crucial for potential pandemic preparedness. Methods We constructed a reassortant virus with a (6 + 2) genome composition, then introduced the polymerase basic protein 1 (PB1) from a wild-type virus to develop a (5 + 3) reassortant virus through reverse genetics. We performed whole-genome sequencing to confirm the genome stability, assessed the growth ability in MDCK cells, and analyzed virus antigenicity using hemagglutination inhibition assays. Subsequently, the effect of homologous PB1 on polymerase activity, viral protein yield, and pathogenicity was assessed. Results The (5 + 3) virus harbouring the homologous PB1 gene exhibited significantly improved growth characteristics, higher viral protein yield, and polymerase activity than the (6 + 2) virus. After successive passage in embryonated eggs, glutamic acid (E) substituted glycine(G) at position 218 (H3 numbering) in the hemagglutinin (HA) gene of both (5 + 3) and (6 + 2) viruses. The substitution improved the growth of the (6 + 2) virus but exhibited no significant effect or alteration on the antigenicity of the (5 + 3) virus. Moreover, the (5 + 3) virus exhibited low pathogenicity in chickens and ferrets. Conclusion Homologous PB1 of the H7N4 virus improves the growth ability while sustaining low pathogenicity. Collectively, the gene composition of the (5 + 3) reassortant virus is a suitable H7N4 CVV for potential pandemic preparedness.

Objective:To analyze the antigenicity and genetic characteristics of two strains of Eurasian avian H1N1 swine influenza virus (EAS-H1N1) isolated from 2 human cases found in Gansu province to provide scientific reference for disease prevention and control.Methods:Two cases of human infection with EAS-H1N1 discovered in Gansu province since February 2021 were analyzed for antigenicity and genome comparison, and their genome characteristics were analyzed using software such as Mega7.0.Results:Two human infections with EAS-H1N1 had related environmental exposure history. Two strains were obtained: A/Gansu-Xifeng/1143/2021 and A/Gansu-Xifeng/1194/2021. The two strains of HA, NA genes were all from the Eurasian avian H1N1 swine influenza virus, and are closely related to the human infection EAS-H1N1 virus in Hebei and Tianjin and the EAS-H1N1 virus isolated from the mink in Shandong; the HA receptor binding sites of 2 strains E190D and D225E mutations occurred in all spots, and there were no H274Y and N294S mutations in NA protein.Conclusions:Some important molecular mutations occurred in the genome of people infected with EAS-H1N1 virus in Gansu province, which may lead to increased virulence and potential human-to-human transmission. Strengthening the research and analysis of EAS-H1N1 gene characteristics will help to monitor the mutation and mutation of the virus at the molecular level and scientifically prevent and control the epidemic.

Isolation and propagation of influenza virus from Influenza Like Illness (ILI) clinical sample is essential for the surveillance of circulating virus, such as antigenic and genetic analyses, antiviral sensitivity surveillance, as well as annual influenza vaccine selection. Madin-Darby canine kidney (MDCK) cell is conventionally used for virus isolation in public health laboratories. Throat swap samples of Influenza like Illness (ILI) were collected from two sentinel hospitals and screened seasonal influenza by real-time reverse transcription polymerase chain reaction (RT–PCR). H3N2 positive samples were performed virus isolation in MDCK cells. Samples were stored under different conditions before inoculation, 1-2 days at 2-8°C, 4-5 day or 8-9 days at 2-8°C, and no less than two months at -80°C. The results showed that long term (>2 month) -80°C storage of clinical samples (15.12%) had significantly lower virus isolation rate compare to short term (1-2 days and 4-9 days) under 2-8°C storage (88.37% for 1-2 days and 52.33% for 4-9 days). For those samples stored at 4°C, the shorter of the storage time, the better of sample quality and virus activity could be obtained, resulting in higher isolation rate. This study provides evidence for influenza surveillance and sample quality control.