Summary
Background
The H3N8 avian influenza virus (AIV) has been circulating in wild birds, with occasional interspecies transmission to mammals. The first human infection of H3N8 subtype occurred in Henan Province, China, in April, 2022. We aimed to investigate clinical, epidemiological, and virological data related to a second case identified soon afterwards in Hunan Province, China.
Methods
We analysed clinical, epidemiological, and virological data for a 5-year-old boy diagnosed with H3N8 AIV infection in May, 2022, during influenza-like illness surveillance in Changsha City, Hunan Province, China. H3N8 virus strains from chicken flocks from January, 2021, to April, 2022, were retrospectively investigated in China. The genomes of the viruses were sequenced for phylogenetic analysis of all the eight gene segments. We evaluated the receptor-binding properties of the H3N8 viruses by using a solid-phase binding assay. We used sequence alignment and homology-modelling methods to study the effect of specific mutations on the human receptor-binding properties. We also conducted serological surveillance to detect the H3N8 infections among poultry workers in the two provinces with H3N8 cases.
Findings
The clinical symptoms of the patient were mild, including fever, sore throat, chills, and a runny nose. The patient’s fever subsided on the same day of hospitalisation, and these symptoms disappeared 7 days later, presenting mild influenza symptoms, with no pneumonia. An H3N8 virus was isolated from the patient’s throat swab specimen. The novel H3N8 virus causing human infection was first detected in a chicken farm in Guangdong Province in December, 2021, and subsequently emerged in several provinces. Sequence analyses revealed the novel H3N8 AIVs originated from multiple reassortment events. The haemagglutinin gene could have originated from H3Ny AIVs of duck origin. The neuraminidase gene belongs to North American lineage, and might have originated in Alaska (USA) and been transferred by migratory birds along the east Asian flyway. The six internal genes had originated from G57 genotype H9N2 AIVs that were endemic in chicken flocks. Reassortment events might have occurred in domestic ducks or chickens in the Pearl River Delta area in southern China. The novel H3N8 viruses possess the ability to bind to both avian-type and human-type sialic acid receptors, which pose a threat to human health. No poultry worker in our study was positive for antibodies against the H3N8 virus.
Interpretation
The novel H3N8 virus that caused human infection had originated from chickens, a typical spillover. The virus is a triple reassortment strain with the Eurasian avian H3 gene, North American avian N8 gene, and dynamic internal genes of the H9N2 viruses. The virus already possesses binding ability to human-type receptors, though the risk of the H3N8 virus infection in humans was low, and the cases are rare and sporadic at present. Considering the pandemic potential, comprehensive surveillance of the H3N8 virus in poultry flocks and the environment is imperative, and poultry-to-human transmission should be closely monitored.
Funding
National Natural Science Foundation of China, National Key Research and Development Program of China, Strategic Priority Research Program of the Chinese Academy of Sciences, Hunan Provincial Innovative Construction Special Fund: Emergency response to COVID-19 outbreak, Scientific Research Fund of Hunan Provincial Health Department, and the Hunan Provincial Health Commission Foundation.
Introduction
Since 2000, two viral pandemics, the 2009 influenza A H1N1 pandemic and the recent COVID-19 pandemic, have confronted human society.1Smith GJ Vijaykrishna D Bahl J et al.Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic., 2Zhu N Zhang D Wang W et al.A novel coronavirus from patients with pneumonia in China, 2019. Wild animals are the natural reservoir or gene pool for several emerging and re-emerging viruses, and domestic animals could act as intermediate hosts to transmit these viruses from wild animals to humans. Therefore, the strengthening of virus surveillance in animals and analysing the potential origin of zoonotic transfer is imperative for the control and prevention of emerging and re-emerging infectious diseases.3From “A”IV to “Z”IKV: attacks from emerging and re-emerging pathogens.Research in context
Evidence before this study
The H3 subtype influenza A virus has a wide host range. In addition to circulation among wild birds and poultry, the virus can also infect a variety of mammals. In 1968, a novel recombinant H3N2 influenza virus strain carrying the avian-derived H3 haemagglutinin gene was first reported in Hong Kong and caused the third identified human influenza pandemic. Previous research found that the H3N8 subtype virus could infect poultry, canines, equines, and seals, but no human case infected by this virus strain was reported before this year. In April, 2022, the first human infected with avian influenza A H3N8 virus was reported in Henan Province, China. Soon after, in May 2022, a 5-year-old boy was diagnosed with H3N8 avian influenza virus (AIV) infection in Changsha City, Hunan Province, China. However, the origin and evolutionary genomic characteristics of the human-infecting H3N8 virus were unclear, and the risk of this novel H3N8 virus infection in humans has not been evaluated.
Added value of this study
This study described the clinical symptoms and epidemiological characteristics of the patient infected with H3N8 virus in Hunan Province, China. We retrospectively investigated the prevalence of H3N8-subtype influenza viruses within farm poultry in China and found this novel H3N8 virus originates from chickens through a typical spillover. Phylogenetic analysis revealed that the H3N8 AIVs were evolving as a triple reassortment event with the Eurasian avian H3 gene, the North American avian N8 gene, and the G57 genotype H9N2 internal genes. Receptor binding properties and host genetic marker analysis showed the mammalian adaptation of the human-derived H3N8 viruses. Finally, the serological surveillance to detect the prevalence of virus exposure in poultry workers elucidates a low risk of novel H3N8 virus infection in humans.
Implications of all the available evidence
This study infers the genetic origin and evolution of the human-infecting avian H3N8 influenza virus and illuminates the role of H9N2 virus internal genes for the emerging AIV reassortants. The novel virus was mainly found in meat chicken flocks in China. The dynamic mammal adaptation substitution of human influenza H3N8 virus indicates the in vivo genetic tuning and rapid ongoing host adaptation. The findings emphasise that comprehensive surveillance of the H3N8 virus is imperative in poultry and humans.
Wild waterfowl are the natural reservoir of avian influenza viruses (AIVs), which have caused sporadic human infections since the human-infecting H5N1 AIV was first reported in Hong Kong in 1997.4Yuen KY Chan PKS Peiris M et al.Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. In 2013, a novel H7N9 AIV causing human infections was identified in eastern China and has caused more than 1500 human infections.5Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses., 6Liu WJ Xiao H Dai L et al.Avian influenza A (H7N9) virus: from low pathogenic to highly pathogenic. Since then, other AIVs, including H6N1, H9N2, H10N8, H5N6, H7N4, H10N3, and H5N8, have also been identified as infecting humans.3From “A”IV to “Z”IKV: attacks from emerging and re-emerging pathogens., 7Emerging HxNy influenza A viruses. Of note, H9N2 virus can infect humans directly, and has been shown to donate partial or even whole sets of internal genes to H7N9, H5N6, H7N4, H10N8, and H10N3 reassortants, which are responsible for human infection.7Emerging HxNy influenza A viruses., 8Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus., 9Current situation of H9N2 subtype avian influenza in China. AIVs have thus evolved more complicated genotypes, posing a substantial threat to public health.Among influenza A virus subtypes, the H3Ny virus subtype has the broadest host spectrum, including wild birds, poultry, humans, swine, canines, equines, cats, and seals.10Bean WJ Schell M Katz J et al.Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts., 11Karlsson EA Ip HS Hall JS et al.Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal. Seroarcheological evidence indicates that the 1889–93 pandemic might have been caused by a H3N8 virus.12Worobey M Han GZ Rambaut A Genesis and pathogenesis of the 1918 pandemic H1N1 influenza A virus. In 1968, a novel recombinant H3N2 influenza virus strain carrying the avian-derived H3 haemagglutinin gene was first reported in Hong Kong and caused the third identified human influenza pandemic.13Cockburn WC Delon PJ Ferreira W Origin and progress of the 1968-69 Hong Kong influenza epidemic. This evidence suggest that H3Ny influenza virus can easily cross species barriers and poses a threat to human health.On April 26, 2022, a novel avian influenza A H3N8 virus causing infection in a 4-year-old boy was identified in Henan Province, China, which is the first time a human has been infected by this virus strain. In May, 2022, we detected a positive specimen of H3N8 AIV in a 5-year-old boy in Changsha City, Hunan Province, China. In this study, we aimed to investigate the clinical, epidemiological, and virological features of the case during the emergency responses to the human H3N8 infection. We also serologically surveyed potential H3N8 virus infection in workers working with live poultry. As the H3Ny subtype influenza virus might have the potential to cause another pandemic,14Yang J Yang L Zhu W Wang D Shu Y Epidemiological and genetic characteristics of the H3 subtype avian influenza viruses in China. we propose the possible origin of this novel H3N8 virus and further illustrate the gene reassortment events and genetic diversity by phylogenetic and coalescent analyses.
Methods
Patient identification and sample collection
On May 10, 2022, a 5-year-old boy with influenza-like illness attended The First Hospital of Changsha in Changsha City, Hunan Province, China. A throat swab specimen of the patient was collected for influenza virus testing as part of routine surveillance. Clinical and epidemiological data of the patient was collected by the doctors at admission, including clinical signs and symptoms of influenza, demographic characteristics, medical history, influenza vaccinations, contact with poultry or pigs or exposure to live poultry markets in the past 10 days, chest CT, laboratory test results, clinical complications, clinical efficacy after treatment, and outcome. The boy’s parents gave informed consent.
The human serum sampling and data collection were determined by Henan and Hunan provincial centres for disease control and prevention to be part of the sentinel surveillance system for influenza, and therefore exempt from institutional review board assessment.
Laboratory testing
On May 10, 2022, a throat swab specimen from the boy was collected by the First Hospital of Changsha. On May 12, 2022, the sample was tested by the Changsha Municipal Center for Disease Control and Prevention. Firstly, the QIAamp Viral RNA Mini Kit (Qiagen) was used to extract the RNA from a throat swab specimen. Seasonal influenza viruses (ie, H1, H3 subtypes, and B lineages) and AIVs (ie, H5N1, H7N9, H9N2, H10N8, and H10N3) and also SARS-CoV-2 were tested by specific real-time RT-PCR. Then, real-time RT-PCR testing was done by use of sets of specific primers and probes, designed by the Chinese National Influenza Center, for the detection of H1–16 and N1–9 subtypes to verify the viral subtype. Madin–Darby canine kidney cells were selected for the isolation of the influenza A virus. Five environmental samples were collected from the patient’s house, and 46 samples, including environmental surfaces, faeces, and poultry drinking water were collected from the market the patient visited. The specimens were tested for influenza A virus with RT- PCR.
Avian H3N8 virus isolation and identification
Specimens of tracheal swabs or lung tissues from diseased poultry were collected and diagnosed by the Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs, China. From January, 2021, to April, 2022, 378 chicken farms in 13 provinces, autonomous regions, or municipality with high-density poultry populations (ie, Anhui, Guangdong, Fujian, Hebei, Henan, Jiangsu, Jiangxi, Jilin, Liaoning, Ningxia, Shandong, Sichuan, and Tianjin) sent samples for diagnosis. In these cases, the sampled chicken flocks exhibited overt respiratory illness or reduced egg production. Virus isolation and the identification of haemagglutinin and neuraminidase subtype were performed as previously described.8Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus. To exclude other viral infections, the H3N8 positive samples were tested for Newcastle disease virus, infectious bronchitis virus, and circulating influenza viruses (eg, H5, H7, and H9 subtypes) by RT-PCR. H3N8-containing allantoic fluid was harvested and stored at –80°C in China Agricultural University until use.
Genomic sequencing of H3N8 virus
The H3N8 virus derived from the Hunan patient and 15 H3N8 viruses derived from chicken flocks were used for full genome sequencing. Genomic sequencing was done as previously described.8Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus. All nucleotide sequences of segments from the H3N8 influenza virus isolates detected in this study have been deposited in the Global Initiative on Sharing Avian Influenza Data database. The accession numbers for the H3N8 sequences reported in this paper are: EPI_ISL_12703722, 12951098, 12950567, 12950247, 12949914, 12949277, 12947104, 12946882, 12946278, 12945954, 12945655, 12945224, 12944913, 12944402, 12943865, and 12935439.
Evolutionary analyses and homology modelling
The genome sequences of the H3N8 viruses were compared with all the publicly available sequences, as of June, 2022. Phylogenetic trees of each genomic segment were inferred based on the maximum likelihood method. Next, more detailed analyses using the Bayesian Markov chain Monte Carlo method were performed to jointly estimate phylogenies, divergence times, and other evolutionary parameters. AlphaFold2 was used for our homology modelling of the ectodomain protein sequences of H3 haemagglutinin.15Tunyasuvunakool K Adler J Wu Z et al.Highly accurate protein structure prediction for the human proteome. Detailed information for methods and models are provided in the appendix (pp 1, 17).
Genotypic analysis
To elaborate genetic diversity of the novel H3N8 viruses, genotypes were determined on the basis of the combination of cluster assignments of each of the six internal genes. A distinct cluster was identified considering the maximum likelihood bootstrap support of more than 75%. The genotypes of the isolates were determined by the combination of cluster assignments of each of the internal gene segments.
Receptor-binding assays
α2–6 glycans (6ʹ SLN: Neu5Acα2–6Galβ1–4GlcNAcβ-sp3-PAA-biotin) and α2–3 glycans (3’ SLN: Neu5Acα2–3Galβ1–4Glcβ-sp4-PAA-biotin) were purchased from Glyco (Auckland, New Zealand). Receptor-binding specificity was determined by a solid-phase direct binding assay as previously described.16Itoh Y Shinya K Kiso M et al.In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.
Serological survey of the H3N8 virus infection in humans
Serum samples obtained from the patient were assessed with the haemagglutination-inhibition assay for antibody titre against the H3N8 virus A/Changsha/1000/2022. To determine the risk of H3N8 AIV infection, we conducted serological surveillance for antibodies to H3N8 virus among poultry workers in Henan and Hunan provinces, where the recent human infections of H3N8 had occurred. In May, 2022, 81 serum samples were collected from poultry workers in Henan (n=30) and Hunan (n=51) provinces. Human H3N8 virus A/Changsha/1000/2022 and human H3N2 virus A/Cambodia/e0826360/2020 were used as antigens. Haemagglutination-inhibition tests were done in accordance with WHO guidelines.17WHO
WHO manual on animal influenza diagnosis and surveillance. 1% turkey erythrocytes were used in haemagglutination-inhibition assays. On the basis of the WHO guideline, titres of 40 or more haemagglutination-inhibition antibodies was considered positive.
Neuraminidase inhibition assay
To assess the sensitivity of the human H3N8 virus A/Changsha/1000/2022 to the neuraminidase inhibitors oseltamivir and zanamivir, neuraminidase inhibition assays were performed as described previously.16Itoh Y Shinya K Kiso M et al.In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.
Role of the funding source
The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.
Results
On May 9, 2022, the patient developed symptoms of a fever, sore throat, chills, and runny nose. Their temperature reached a peak at 40°C in the early morning of May 10, 2022. The patient had no symptoms of cough, diarrhoea, or vomiting. The patient went to the fever clinic of The First Hospital of Changsha on May 10 and was diagnosed with acute tonsillitis. A blood test on May 10 showed a reduced lymphocyte ratio (14·1%) and eosinophil ratio (0·3%), but high neutrophil ratio (77·5%; Table 1, Table 2). Chest CT showed no signs of pneumonia (appendix p 2). A throat swab specimen was collected on May 10. The patient’s temperature returned to normal when treated with amoxicillin–clavulanic acid, Kaihoujian spray, and Xiaoerchiqiao Qingre granules, which are traditional Chinese medicines for sore throat and influenza treatment in children. The patient’s condition was stable afterwards and no symptoms such as fever and cough were present since the end of the day on May 10. A blood test on May 21, 2022, showed the recovery of most indexes of the patient (appendix p 12).
Table 1Demographic, epidemiological, and clinical characteristics of the boy with avian influenza A (H3N8) virus infection
Table 2Clinical blood cell and biochemistry tests on admission
The blood sample was collected for biochemistry testing on May 10, 2022.
On May 12, a RT-PCR test of the throat swab specimen was positive for H3N8 influenza A virus. Furthermore, tests for the haemagglutinin genes of H3N2, H5N1, H5N6, H7N9, H9N2, H10N8, and SARS-CoV-2 were all negative. Subsequently, the Changsha Center for Disease Control and Prevention obtained the whole-genome sequence through next-generation sequencing, named A/Changsha/1000/2022(H3N8). The sequence alignment by Basic Local Alignment Search Tool proved that the newly obtained H3N8 strain sequence was an avian-origin H3N8 virus. 3 days after inoculation of the specimen within Madin–Darby canine kidney cells, cytopathic effects could be observed, and the haemagglutinin titre of the culture supernatant was 1:128. The serial patient serum antibody titres were tested by haemagglutination-inhibition and a significant increasing of antibody titre against this H3N8 strain was observed (appendix p 13).Epidemiological investigation showed the patient visited a fresh market near their place of residence 6 days before illness onset. In this market, the live poultry species traded were chickens, ducks, and pigeons, but the patient did not handle the poultry. The patient did not leave Hunan Province or have any contact ever with the person with H3N8 infection from Henan Province. Five environmental samples from the patient’s house were negative. 33 (72%) of 46 of the environmental samples from the market were positive for AIVs, among which 14 samples were H3N8 positive (appendix p 13).To evaluate the risk of the novel H3N8 virus infections in humans, serological surveillance was conducted to detect the prevalence of virus exposure in poultry workers of Hunan and Henan provinces, where the recent human infections of H3N8 occurred. 81 poultry workers (51 in Hunan Province and 30 in Henan Province) were included in this investigation. Haemagglutination-inhibition tests showed that 26 (32%) of the 81 poultry workers were seropositive for human H3N2 influenza virus A/Cambodia/e0826360/2020. No poultry worker was positive for antibodies against the H3N8 influenza virus A/Changsha/1000/2022 (appendix p 13). These findings indicate that the risk of the H3N8 virus infection in humans was low, and the cases were rare and sporadic at the time of testing.To explore the origin of this novel H3N8 virus, we retrospectively investigated the infection of farm poultry with H3N8-subtype influenza viruses in 13 provinces in China with high-density poultry populations from January, 2021, to April, 2022. In December, 2021, an H3N8 subtype AIV, A/chicken/Guangdong/F1201/2021, was isolated from the chickens that showed typical respiratory disease signs in a farm in Guangdong Province. Subsequently, from January, to April, 2022, 14 strains of H3N8 were further isolated from diseased chickens from the commercial chicken meat farms in five provinces (ie, Guangdong, Fujian, Anhui, Jiangsu, and Henan Province; appendix p 3). Previous surveillance of AIVs in the mainland of China showed that H3Ny viruses are dynamically circulating in waterfowl, usually without resulting in any apparent illness, and no H3N8 virus was detected from chicken flocks.14Yang J Yang L Zhu W Wang D Shu Y Epidemiological and genetic characteristics of the H3 subtype avian influenza viruses in China. However, in the present study, all 15 H3N8 viruses were isolated from diseased-meat chickens, indicating that the novel H3N8 viruses were adapted to chickens and shown to be pathogenic to chickens. Genome sequence alignment and pairwise comparison revealed that the haemagglutinin and neuraminidase genes of the human H3N8 strain A/Henan/4-10/2022 and A/Changsha/1000/2022 showed the highest identity (sequence identity >98·7%; appendix p 14) with the chicken isolates, indicating that the novel H3N8 viruses causing human infection originated from chickens.The phylogeny of globally available H3 gene sequences showed four main independent lineages: Eurasian avian, North American avian, seasonal human, and equine lineages. All the chicken-derived H3N8 strains and the two human isolates fell within the Eurasian avian lineage and formed an independent phylogenetic subclade with high bootstrap support (100%; figure 1, appendix p 4). Evolutionary analyses showed that this independent branch had likely originated from H3Ny viruses circulating in domestic ducks in southern China. The estimated time to the most recent common ancestor of the novel H3N8 strains was roughly April, 2019 (mean April 22, 2019, 95% highest posterior density, March 6, 2018–May 12, 2020; Figure 1, Figure 2).
Figure 1Phylogenetic tree of haemagglutinin gene of H3 subtype influenza virus
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(A) Phylogenetic tree of the haemagglutinin gene of the H3 subtype. Yellow indicates seasonal human lineage, grey indicates North American avian lineage, and purple indicates equine lineage. (B) Phylogenetic tree of haemagglutinin gene of the Eurasian avian H3 subtype. Blue node bars represent 95% credible intervals of the lineage divergence times. Human H3N8 virus is red and is indicated with a solid circle. Chicken H3N8 viruses isolated in this study are blue and viral sequences named in black were downloaded from the Global Initiative on Sharing Avian Influenza Data and the Influenza Virus Resource at the National Center for Biotechnology Information.
Figure 2Spatial and temporal model of the origins of the novel avian influenza A H3N8 virus
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(A) Origins and evolution of the gene segments of the novel H3N8 virus. The eight gene segments (ie, horizontal bars) are, PB2, PB1, PA, haemagglutinin, NP, neuraminidase, MP, and NS, from top to bottom. (B) Dynamic reassortments of the novel H3N8 virus. Host and lineage origins of each gene segment and most closely related sequences of the novel H3N8 virus are shown. Solid circles represent the estimated times of the most recent common ancestors.
The phylogeny of the N8 gene showed two main independent lineages, the Eurasian avian and North American avian lineages. All the chicken-derived H3N8 strains and the two human isolates fell within the North American avian lineage. Similar to the H3 phylogeny, the N8 gene of these H3N8 viruses also formed an independent phylogenetic subclade (bootstrap value, 100%). These H3N8 viruses were distinctly different from the human H10N8 viruses18Chen H Yuan H Gao R et al.Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. found in China and the human H5N8 virus found in Russia (figure 3; appendix p 5). The N8 gene of these H3N8 viruses could be derived from the H3N8 or H6N8 viruses, which were found in migratory birds in Russia or Japan in 2020. The estimated time to the most recent common ancestor of the neuraminidase gene was roughly August 2020 (mean August 11, 2020, 95% highest posterior density, Jan 2, 2020–Feb 26, 2021; Figure 2, Figure 3).
Figure 3Phylogenetic tree of neuraminidase gene of N8 subtype influenza virus
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(A) Phylogenetic tree of the neuraminidase gene of the N8 subtype. The sequences inside the green box were selected for more detailed analyses using the Bayesian Markov chain Monte Carlo method shown in panel B. (B) Phylogenetic tree of the neuraminidase gene of the N8 subtype derived from the North American avian lineage. Blue node bars represent 95% credible intervals of lineage divergence times. Human H3N8 virus is red and is indicated with a solid circle. H3N8 viruses isolated in this study are blue and viral sequences named in black were downloaded from the databases.
The six internal genes of the novel H3N8 virus had originated from G57 H9N2 viruses (appendix pp 6–11). Although these viruses belong to the same clade, they could be grouped into approximately three to four clusters, with a major cluster comprising the majority of the strains and the minor clusters containing only one or two sequences (appendix pp 6–11). On the basis of this genomic diversity, we categorised the H3N8 viruses into seven different genotypes (figure 2, appendix pp 6–11). Genotype 0 (G0) was the most dominant genotype, and the virus strains with this genotype were isolated from chickens in Guangdong, Anhui, Jiangsu, and Henan Province. The human isolate A/Henan/4-10/2022 also belongs to this genotype. The G1–G5 genotype viruses were detected at a lower frequency and were only isolated from chickens in Guangdong and Fujian Province. The human isolate A/Changsha/1000/2022 belongs to G6, the haemagglutinin, neuraminidase, PA, and NS genes were located in the same clade with chicken-derived H3N8 viruses, while the PB2, PB1, NP, and M genes were located in the same clade with H9N2 viruses isolated in Hunan Province (appendix pp 6–11), implying that the local H9N2 AIVs in Hunan Province could contribute to the reassortment.Methodological limitations including model misspecification can have a substantial effect on phylogenetic estimation. To address these methodological concerns, we applied comprehensive methods and different models to infer the evolution of the novel H3N8 virus. The congruence between the results obtained using different methods and models ensured the robustness of our analysis to method selection. On the basis of the epidemiological and phylogenetic analyses, we extrapolated the potential evolutionary pathway of the H3N8 viruses and routes of reassortment events. The HxN8 viruses of the North American avian lineage were circulating in migratory waterfowls in Alaska, USA and migrated to Russia or Japan in 2020, followed by transmission to southern China. The HxN8 virus then underwent complex reassortment events with the H3Ny and G57 H9N2 viruses in chickens or ducks, resulting in multiple genotypes of H3N8 being formed and circulated in chicken flocks in the Pearl River Delta area in southern China before December, 2021, and they were transferred to other provinces, causing human infection (figure 2). Similar to the human-infecting H7N9 AIV that emerged in 2013,19Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus. the H3N8 AIV also constitutes multiple reassortment events—that is, independent origins of haemagglutinin and neuraminidase, and continuous dynamic reassortments of H9N2-originated internal genes.Direct binding assays with SAα2–3Gal and SAα2–6Gal sialylglycopolymers were done with the A/Changsha/1000/2022 and five representative chicken-derived H3N8 virus strains. We found that all the H3N8 viruses could bind to both SAα2–6Gal and SAα2–3Gal receptors, although their affinity for SAα2–6Gal was lower than that for SAα2–3Gal (figure 4). Key residues in the receptor-binding site revealed that the human isolate A/Changsha/1000/2022 and all the chicken-derived H3N8 isolates still maintain the avian signature (Q226 and G228) in the 220-loop, but have residues W222, S227, and N193 in the receptor-binding site (appendix p 15). The residue N193 could form potential hydrogen bonds with the α2–6 glycan and the residues W222 and S227 might change the conformational flexibility of the 220-loop,20Xu R McBride R Nycholat CM Paulson JC Wilson IA Structural characterization of the hemagglutinin receptor specificity from the 2009 H1N1 influenza pandemic. hence conferring the chicken H3N8 isolates with dual receptor-binding properties (figure 4). Although the human H3N8 A/Henan/4-10/2022 haemagglutinin has a degenerative codon in the position 228, which could be residue G or S (figure 4B, 4C), previous study with the A/Hong Kong/68 (H3N2) strain has revealed that the residue S228 can form additional hydrogen bonds with the sialic acid moiety of the glycan receptors, and then greatly enhance both avian and human receptor-binding capacities.21Matrosovich M Tuzikov A Bovin N et al.Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals.
Figure 4Receptor binding and homology-modelling structural analysis of haemagglutinins derived from influenza A H3N8 virus
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(A) Binding affinity of influenza viruses to α2–6-linked (red) and α2–3-linked (blue) sialic acid polymers. (B) Structural model of A/Changsha/1000/2022 (H3N8) haemagglutinin and its comparison with avian A/duck/Ukraine/1/1963 (H3N8) haemagglutinin (PDB: 1MQM), A/Hong Kong/1/1968 (H3N2) haemagglutinin (PDB: 6TZB), and A/harbor seal/New Hampshire/179629/2011 (H3N8) haemagglutinin (PDB: 4WA2) bound to avian or human receptor analogues. The structural model of the G228S mutant of the A/Changsha/1000/2022 (H3N8) haemagglutinin bound to human receptor analogue is also shown. The three secondary structural elements of the binding site (the 130-loop, 190-helix, and 220-loop) are labelled in ribbon representation, together with selected residues in stick representation. (C) Sequence alignment of the key residues in the receptor binding site region of the haemagglutinins from representative H3 strains.
The haemagglutinin cleavage peptides of the H3N8 viruses were PEKQTR/GL, implying that the strain is a low pathogenic AIV among poultry. Neuraminidase gene had Ile312Val mutations, which might be resistant to oseltamivir; however, the phenotypic test result for resistance to neuraminidase showed that the H3N8 virus A/Changsha/1000/2022 was sensitive to oseltamivir and zanamivir (appendix p 16). The Ser31Asn mutation found in the M2 protein, showed that the strains were resistant to amantadine. All these H3N8 viruses showed genetic markers for mammalian adaptation in PB2 (Ile292Val and Lys318Arg), PB1 (Leu13Pro and Leu473Val), PA (Lys26Glu, Val160Asp, and Lys356Arg), NP (Lys398Gln), M1(Val15Ile, Asn30Asp, and Thr215Ala), and NS1 (Pro42Ser and Glu227Lys).22H9 Influenza viruses: an emerging challenge. Of note, the Glu627Lys mutation in PB2 was only found in the A/Henan/4-10/2022 virus, and the Glu627Val mutation in PB2 and the Ser20Asn mutation in M2 was only found in the A/Changsha/1000/2022 virus (appendix p15). The dynamic haemagglutinin-Gly228Ser, PB2-Glu627Lys/Val substitution of human influenza H3N8 virus indicates the in-vivo genetic tuning and rapid host adaptation.
Discussion
In 2022, a novel avian influenza A H3N8 virus causing infection in two boys was detected by the sentinel surveillance system for influenza-like illnesses in China, which was the first time that humans had been infected by this virus strain. Here, we propose that the H3N8 AIVs were evolving as a triple reassortment event with the Eurasian avian H3 gene, the North American avian N8 gene, and G57 genotype H9N2 internal genes. This novel virus had emerged before December, 2021, and it has been endemic in chicken flocks in southern China since December, 2021.
Epidemiological data showed that the two patients had a history of exposure to live poultry. H3Ny subtype viruses have become enzootic in domestic ducks in farms and live poultry markets in southern China.14Yang J Yang L Zhu W Wang D Shu Y Epidemiological and genetic characteristics of the H3 subtype avian influenza viruses in China. The combination of industrial and mixed-animal backyard farming practices, and the live poultry markets, construct an ideal environment for virus reassortment and interspecies transmission.23Bao CJ Cui LB Zhou MH Hong L Gao GF Wang H Live-animal markets and influenza A (H7N9) virus infection. This complicated ecosystem could have facilitated the emergence of novel influenza viruses in the region, such as H5N1, H7N9, H10N8, H5N6, H10N3, and the novel H3N8 virus.24Dominant subtype switch in avian influenza viruses during 2016-2019 in China. Furthermore, the influenza infection reported in this study was found soon after the discovery of human H3N8 infection in Henan Province, which is not adjacent to Hunan Province (appendix p 3). This fact indicates a potential high risk for the interspecies transmission of this newly human-infecting AIV.Among the HxN8 AIVs, in addition to H3N8 viruses, H10N8 and H5N8 viruses were also found to be able to infect humans.7Emerging HxNy influenza A viruses. The N8 gene of human infecting H5N8 virus belongs to Eurasian lineage, whereas the N8 genes of the human-infecting H10N8 and H3N8 viruses belong to North American lineage. Generally, the viruses of Eurasian lineage and North American lineage circulate predominantly in the Eurasian region and in the North American region, respectively. In 2005, for the first time, H5N1 influenza virus infected wild waterfowls at a population level and was disseminated by long-distance migration.25Liu J Xiao H Lei F et al.Highly pathogenic H5N1 influenza virus infection in migratory birds. Afterwards, the H5Ny influenza viruses spread from the Eurasian continent to the American region, and have been continuously circulated bilaterally between Eurasian and North American regions.26Lewis NS Banyard AC Whittard E et al.Emergence and spread of novel H5N8, H5N5 and H5N1 clade 2.3.4.4 highly pathogenic avian influenza in 2020. Molecular epidemiological analysis revealed that the gene reassortments between the Eurasian and North American lineage are common in H3Ny viruses circulating in domestic poultry in southern China.14Yang J Yang L Zhu W Wang D Shu Y Epidemiological and genetic characteristics of the H3 subtype avian influenza viruses in China. In the case of H3N8, the N8 genes derived from the North American lineage might have been transferred to southern China by wild birds through migration along the east Asian flyway. These facts indicate that the migratory birds could promote the globalisation of the transmission of influenza viruses. Therefore, control and prevention of influenza viruses is not an issue for a single country or region, but rather it has become a global challenge, highlighting the consensus; one world, one health, for confronting emerging infectious diseases.27Public health priorities for China–Africa cooperation.Since 2010, the G57 genotype H9N2 virus has been the most predominant AIV across China. The G57 H9N2 virus not only infects humans directly, but also provides partial or whole sets of internal genes for the emerging AIVs reassortants. Multiple AIVs, including H7N9, H5N6, H10N8, and H10N3 have been recognised to infect humans with the internal genes from the G57 H9N2 influenza virus.9Current situation of H9N2 subtype avian influenza in China. Here, the H3N8 viruses also acquired the six internal genes of the G57 H9N2 viruses; however, they fell within 3–4 separate clusters, implying high genetic diversity. Seemingly, the endemic and widespread prevailing of the G57 H9N2 virus in poultry could increase the opportunity of multiple reassortments with the H3N8 viruses circulating in the same population. Thus, a combination of multiple viral and epidemiological factors could be driving the reassortment events. Therefore, control and prevention of H9N2 viruses in domestic poultry28Poultry carrying H9N2 act as incubators for novel human avian influenza viruses. is a promising measure to reduce the occurrence of novel AIVs.The haemagglutinin gene of the 1968 Hong Kong H3N2 pandemic strain originated from the wild bird virus.13Cockburn WC Delon PJ Ferreira W Origin and progress of the 1968-69 Hong Kong influenza epidemic. Subsequently, following Gln226Leu and Gly228Ser substitutions, the haemagglutinin protein has driven a shift of the receptor-binding property of the virus from avian-receptor preference to human-receptor preference, which enables rapid transmission in human populations.29Ha Y Stevens DJ Skehel JJ Wiley DC X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus. Here, we found that the novel H3N8 influenza viruses obtained dual receptor-binding properties. Sequence analysis of key residues in the receptor-binding site of haemagglutinin revealed that the chicken H3N8 strains and the human H3N8 A/Changsha/1000/2022 did not acquire the Gln226Leu and Gly228Ser substitutions. However, the observation of a degenerative codon in position 228 of haemagglutinin in H3N8 A/Henan/4-10/2022 protein sequences showed that the novel virus could be quickly adapted to human-receptor binding by dynamic substitution of key residues of viral proteins, which also included the simultaneous PB2-Glu627Lys/Val substitution. This dynamic viral adaptation during infection was termed genetic tuning of AIVs in new hosts, and we proposed this concept with the observed phenomenon of dynamic PB2-Glu627Lys substitution in influenza H7N9 virus in 2013.30Zhou J Wang D Gao R et al.Biological features of novel avian influenza A (H7N9) virus., 31Dynamic PB2-E627K substitution of influenza H7N9 virus indicates the in vivo genetic tuning and rapid host adaptation. Continuous human infection with the novel H3N8 virus might further drive the virus to acquire a human-receptor-binding preference, which is a prerequisite for pandemic potential. The limited serological investigation in poultry workers in Henan and Hunan Province indicate that the risk of the novel H3N8 virus infection in humans was low and the cases were rare and sporadic at the time of investigation. Ongoing serological studies are needed to assess the risk of human infections with the H3N8 viruses.
The shortcomings of this study are that there was insufficient data from long-term monitoring of the H3N8 subtype in the local live poultry markets, which reduces traceability of this case, and the collection of subsequent surveillance samples lagged behind due to the late AIV infection confirmation in the case with mild symptoms.
In conclusion, the avian H3N8 influenza virus that infected humans is a novel virus generated by dynamic genetic reassortments, which acquires the H3 gene from Eurasian lineage viruses, the N8 gene from North American lineage viruses, and the internal genes from G57 genotype H9N2 viruses. The novel viruses were found in meat chicken flocks in southern China. Comprehensive surveillance of the H3N8 AIVs in domestic poultry, especially in meat chickens with respiratory disease signs, is imperative and control of the virus endemic in poultry could decrease the risk of emerging human infections.
JinL, GFG, LG, XO, HSu, and FG designed the study. HSu, QT, QH, JiyL, HL, JY, RP, WY, DY, and YW did the experiments. RY, HSu, FG, QT, HSo, QH, JiyL, YL, MX, XZ, ZZ, BY, SC, YZ, JQi, CH, YG, JQiu, MR, BL, JP, YSh, and YSu collected and analysed the data. GFG, JinL, ZH, KL, GZ, HSu, FG, HSo, and YSh interpreted the data. HSu, WJL, FG, HSo, XO, LG, YSh, JinL, and GFG wrote the paper.
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