Spatiotemporal Associations and Molecular Evolution of Highly Pathogenic Avian Influenza A H7N9 Virus in China from 2017 to 2021.

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China.

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control.

The adaptability of H9N2 avian influenza A virus to humans: A comparative docking simulation study.

Influenza A virus, the H9N2 subtype, is an avian influenza virus that has long been circulating in the worldwide poultry industry and is occasionally found to be transmissible to humans. Evidence from genomic analysis suggests that H9N2 provides the genes for the H5N1 and H7N9 subtypes, which have been found to infect mammals and pose a threat to human health. However, due to the lack of a structural model of the interaction between H9N2 and host cells, the mechanism of the extensive adaptability and strong transformation capacity of H9N2 is not fully understood. In this paper, we collected 40 representative H9N2 virus samples reported recently, mainly in China and neighboring countries, and investigated the interactions between H9N2 hemagglutinin and the mammalian receptor, the polysaccharide α-2,6-linked lactoseries tetrasaccharide c, at the atomic level using docking simulation tools. We categorized the mutations of studied H9N2 hemagglutinin according to their effects on ligand-binding interactions and the phylogenetic analysis. The calculations indicated that all the studied H9N2 viruses can establish a tight binding with LSTc although the mutations caused a variety of perturbations to the local conformation of the binding pocket. Our calculations suggested that a marginal equilibrium is established between the conservative ligand-receptor interaction and the conformational dynamics of the binding pocket, and it might be this equilibrium that allows the virus to accommodate mutations to adapt to a variety of environments. Our results provided a way to understand the adaptive mechanisms of H9N2 viruses, which may help predict its propensity to spread in mammals.

Differential Modulation of Innate Immune Responses in Human Primary Cells by Influenza A Viruses Carrying Human or Avian Nonstructural Protein 1.

The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-ß) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.

A field-deployable insulated isothermal RT-PCR assay for identification of influenza A (H7N9) shows good performance in the laboratory.

BACKGROUND: Avian influenza A (H7N9) remains circulating in China. For countries at risk of introduction of H7N9, such as Vietnam, early detection of H7N9 virus is essential for the early containment of the virus. Insulated isothermal reverse transcriptase PCR (iiRT-PCR) is a portable PCR system that can be deployed under field conditions to identify pathogens at the sampling site. Applying PCR at the sampling site will greatly reduce the time to obtain a diagnostic result which allows the veterinary authority to take immediate action to contain disease spreading. OBJECTIVE: To determine analytical and diagnostic sensitivity and specificity of the portable iiRT-PCR for H7N9 virus detection. METHODS: A panel of 59 virus isolates, including H7N9, avian influenza viruses of subtype H1 to H13, swine and human influenza viruses, Newcastle disease virus, and infectious bursal disease virus, were tested by H7 and N9 iiRT-PCR reagents, using probes and primers specific to H7 or N9, in comparison with laboratory-based real-time RT-PCR assays to determine analytical sensitivity and specificity. Fifty oropharyngeal samples from experimentally infected chicken and ducks with H7N9 and 50 non-infected control swabs were tested by the H7 iiRT-PCR to determine diagnostic sensitivity and specificity. RESULTS: The H7 and N9 iiRT-PCR reagents yielded comparable levels of analytical sensitivity and specificity with real-time RT-PCR for the detection of H7N9 virus. Diagnostic sensitivity and specificity of H7 iiRT-PCR were 98% and 100%, respectively. CONCLUSION: The observed high sensitivity and specificity of iiRT-PCR for H7N9 detection show its potential for early detection of H7N9 in risk-based surveillance.

Delayed peak of human infections and ongoing reassortment of H7N9 avian influenza virus in the newly affected western Chinese provinces during Wave Five.

OBJECTIVES: Eight additional provinces in western China reported human infections for the first time during the fifth wave of human H7N9 infections. The aim of this study was to analyze the epidemiological and virological characteristics of this outbreak. METHODS: The epidemiological data of H7N9 cases from the newly affected western Chinese provinces were collected and analyzed. Full-length genome sequences of H7N9 virus were downloaded from the GenBank and GISAID databases, and phylogenetic, genotyping, and genetic analyses were conducted. RESULTS: The peak of human infections in the newly affected western Chinese provinces was delayed by 4 months compared to the eastern Chinese provinces, and both low pathogenic (LP) and highly pathogenic (HP) H7N9-infected cases were found. The LP- and HP-H7N9 virus belonged to 10 different genotypes (including four new genotypes), of which G11 and G3 were the dominant genotypes, respectively. Almost all of these viruses originated from eastern and southern China and were most probably imported from neighboring provinces. Genetic characteristics of the circulating viruses were similar to those of the viruses from previously affected provinces during Wave Five. CONCLUSIONS: A delayed peak of human infections was observed in the newly affected western Chinese provinces, and reassortment has been ongoing since the introduction of H7N9 viruses. This study highlights the importance of continued surveillance of the circulation and evolution of H7N9 virus in western China.

Evolutionary dynamics of the H7N9 avian influenza virus based on large-scale sequence analysis.

Since 2013, epidemics caused by novel H7N9 avian influenza A viruses (AIVs) have become a considerable public health issue. This study investigated the evolution of these viruses at the population level. Compared to H7 and N9 before 2013, there were 18 and 24 substitutions in the majority of novel H7N9 AIVs, respectively. Nine of these in HA and six in NA were rare before 2013, and four of these in HA and two in NA displayed host tropism. S136(128)N and A143(135)V are located on the receptor binding sites of the HA1 subunit and might be important factors in determining the host species of novel H7N9 AIV. On an overall scale, the evolution of H7 and N9, both in terms of time distribution and host species, is under negative selection. However, both in HA and NA, several sites were under positive selection. In both the overall epidemics and the human-derived H7N9 AIVs, eight positive selection sites were identified in HA1, with some located within the known antigen epitopes or the receptor binding site(RBS) domain. This may induce variations in H7N9 AIV with positive selection. It is necessary to strengthen the surveillance of novel H7N9 AIVs, both in human and bird population to determine whether a new virus has emerged through selection pressure and to prevent future epidemics from occurring.

A hospital cluster combined with a family cluster of avian influenza H7N9 infection in Anhui Province, China.

OBJECTIVES: To identify human-to-human transmission of H7N9 avian influenza virus, we investigated a hospital cluster combined with family cluster in this study. METHODS: We obtained and analyzed clinical, epidemiological and virological data from the three patients. RT-PCR, viral culture and sequencing were conducted for determination of causative pathogen. RESULTS: The index case presented developed pneumonia with fever after exposure to chicken in a poultry farm. Case A presented pneumonia with high fever on day 3 after she shared a hospital room with the index case. Case B, the father of the index case, presented pneumonia with high fever on day 15 after he took care of the index case. H7N9 virus circulated in the local farm to which the index case was exposed. Full genomic sequence of virus showed 99.8-100% identity shared between the index case and case A or case B. Compared to the earliest virus of Anhui, a total of 29 amino acid variation sites were observed in the 8 segments. CONCLUSIONS: A hospital cluster combined with family cluster of H7N9 avian influenza infection was identified. Air transmission resulted in the hospital cluster possibly. A poultry farm was the initially infectious source of the cluster.

Characterization of substitutions in the neuraminidase of A(H7N9) influenza viruses selected following serial passage in the presence of different neuraminidase inhibitors.

Avian A(H7N9) infections in humans have been reported in China since 2013 and are of public health concern due to their severity and pandemic potential. Oseltamivir and peramivir are neuraminidase inhibitors (NAIs) routinely used for the treatment of A(H7N9) infections, but variants with reduced sensitivity to these drugs can emerge in patients during treatment. Zanamivir and laninamivir are NAIs that are used less frequently. Herein, we performed in vitro serial passaging experiments with recombinant viruses, containing the neuraminidase (NA) from influenza A/Anhui/1/13 (H7N9) virus, in the presence of each NAI, to determine whether variants with reduced sensitivity would emerge. NA substitutions were characterized for their effect on the NA enzymatic activity and surface expression of the A/Anhui/1/13 (Anhui/1) NA, as well as NAs originating from contemporary A(H7N9) viruses of the Yangtze River Delta and Pearl River Delta lineages. In vitro passage in the presence of oseltamivir, peramivir and laninamivir selected for substitutions associated with reduced sensitivity (E119D, R292K and R152K), whereas passage in the presence of zanamivir did not select for any viruses with reduced sensitivity. All the NA substitutions significantly reduced activity, but not the expression of the Anhui/1 NA. In contemporary N9 NAs, all substitutions tested significantly reduced NA enzyme function in the Yangtze River lineage background, but not in the Pearl River Delta lineage background. Overall, these findings suggest that zanamivir may be less likely than the other NAIs to select for resistance in A(H7N9) viruses and that the impact of substitutions that reduce NAI susceptibility or enzyme function may be less in A(H7N9) viruses from the Pearl River lineage.

Influenza A(H7N9) virus emerged and resulted in human infections in Chongqing, southwestern China since 2017.

OBJECTIVES: Influenza A(H7N9) virus has emerged and resulted in human infections in Chongqing, southwestern China since 2017. This study aimed to describe the epidemiological characteristics of the first epidemic in this region. METHODS: The epidemiological data of patients were collected. Live poultry markets (LPMs), commercial poultry farms (CPFs) and backyard poultry farms (BPFs) were monitored, and poultry sources were registered. Samples derived from the patients, their close contacts, and the environments were tested for influenza A(H7N9) virus by real-time reverse transcriptase polymerase chain reaction. Genetic sequencing and phylogenetic analysis were also conducted. RESULTS: Since the confirmation of the first patient infected with influenza A(H7N9) virus on March 5, 2017, nine patients had been identified within four months in Chongqing. Their mean age was 45 years, 77.8% were male, 66.7% were urban residents and 55.6% were of poultry related occupation. All patients became infected after exposure to live chickens. The median time interval from initial detection of influenza A(H7N9) virus in Chongqing to the patients' onset was 75 days. Since initial detection in February 2017, influenza A(H7N9) virus was detected in 21 (53.8%) counties within four months. The proportion of positive samples was 2.94% (337/11,451) from February 2017 to May 2018, and was higher (χ2=75.78, P<0.001) in LPMs (3.66%, 329/8979) than that in CPFs (0.41%, 5/1229) and BPFs (0.24%, 3/1243). The proportion of positive samples (34.4%, 22/64) at the premises to which the patients were exposed was significantly higher than that (5.7%, 257/4474) in premises with no patients. Phylogenetic analysis indicated that the viruses isolated in Chongqing belonged to the Yangtze River Delta lineage and resembled those circulated in Jiangsu and Anhui provinces between late 2016 and early 2017. CONCLUSION: Influenza A(H7N9) virus was newly introduced into Chongqing most likely between late 2016 and early 2017, which swept across half of Chongqing territory and resulted in human infections within months. The most impacted premises and population were LPMs and poultry related workers respectively in the epidemic.

Pathogenicity of two novel human-origin H7N9 highly pathogenic avian influenza viruses in chickens and ducks.

Human infection by low-pathogenic avian influenza viruses of the H7N9 subtype was first reported in March 2013 in China. Subsequently, these viruses caused five outbreaks through September 2017. In the fifth outbreak, H7N9 virus possessing a multiple basic amino acid insertion in the cleavage site of hemagglutinin emerged and caused 4% of all human infections in that period. To date, H7N9 highly pathogenic avian influenza viruses (HPAIVs) have been isolated from poultry, mostly chickens, as well as the environment. To evaluate the relative infectivity of these viruses in poultry, chickens and ducks were subjected to experimental infection with two H7N9 HPAIVs isolated from humans, namely A/Guangdong/17SF003/2016 and A/Taiwan/1/2017. When chickens were inoculated with the HPAIVs at a dose of 106 50% egg infectious dose (EID50), all chickens died within 2-5 days after inoculation, and the viruses replicated in most of the internal organs examined. The 50% lethal doses of A/Guangdong/17SF003/2016 and A/Taiwan/1/2017 in chickens were calculated as 103.3 and 104.7 EID50, respectively. Conversely, none of the ducks inoculated with either virus displayed any clinical signs, and less-efficient virus replication and less shedding were observed in ducks compared to chickens. These findings indicate that chickens, but not ducks, are highly permissive hosts for emerging H7N9 HPAIVs.

Identifying key bird species and geographical hotspots of avian influenza A (H7N9) virus in China.

BACKGROUND: In China since the first human infection of avian influenza A (H7N9) virus was identified in 2013, it has caused serious public health concerns due to its wide spread and high mortality rate. Evidence shows that bird migration plays an essential role in global spread of avian influenza viruses. Accordingly, in this paper, we aim to identify key bird species and geographical hotspots that are relevant to the transmission of avian influenza A (H7N9) virus in China. METHODS: We first conducted phylogenetic analysis on 626 viral sequences of avian influenza A (H7N9) virus isolated in chicken, which were collected from the Global Initiative on Sharing All Influenza Data (GISAID), to reveal geographical spread and molecular evolution of the virus in China. Then, we adopted the cross correlation function (CCF) to explore the relationship between the identified influenza A (H7N9) cases and the spatiotemporal distribution of migratory birds. Here, the spatiotemporal distribution of bird species was generated based on bird observation data collected from China Bird Reports, which consists of 157 272 observation records about 1145 bird species. Finally, we employed a kernel density estimator to identify geographical hotspots of bird habitat/stopover that are relevant to the influenza A (H7N9) infections. RESULTS: Phylogenetic analysis reveals the evolutionary and geographical patterns of influenza A (H7N9) infections, where cases in the same or nearby municipality/provinces are clustered together with small evolutionary differences. Moreover, three epidemic waves in chicken along the East Asian-Australasian flyway in China are distinguished from the phylogenetic tree. The CCF analysis identifies possible migratory bird species that are relevant to the influenza A(H7N9) infections in Shanghai, Jiangsu, Zhejiang, Fujian, Jiangxi, and Guangdong in China, where the six municipality/provinces account for 91.2% of the total number of isolated H7N9 cases in chicken in GISAID. Based on the spatial distribution of identified bird species, geographical hotspots are further estimated and illustrated within these typical municipality/provinces. CONCLUSIONS: In this paper, we have identified key bird species and geographical hotspots that are relevant to the spread of influenza A (H7N9) virus. The results and findings could provide sentinel signal and evidence for active surveillance, as well as strategic control of influenza A (H7N9) transmission in China.

A family cluster of two fatal cases infected with influenza A (H7N9) virus in Kunming China, 2017.

Two imported family cases (mother and daughter) of fatal H7N9 infection in Kunming, China were reported in 2017. Epidemiological investigation showed that the two family members had both been exposed to sick chickens in a poultry market. The onset of illness and death of the mother was 7 days later than her daughter, raising concerns about human-to-human transmission of H7N9 in the locality. Sequence alignment and phylogenetic analysis of the virus strains isolated from the two patients revealed high sequence similarity (≥ 99%) and homology to each other. The two virus strains shared a PEIPKGR/G cleavage motif and the same key amino acid mutations across 8 viral genes except for a R292K mutation in the neuraminidase (NA) gene isolated from the mother who had been treated with oseltamivir in the clinic. Moreover, the isolated H7N9 virus possesses avian and human dual-receptor specificity and is able to efficiently proliferate in human cell lines in vitro. Further epidemiological study demonstrated that five family members who had close contacted with the patients were free of illness and negative for the H7N9 genomic test. Collectively, the H7N9 virus described here is still limited to transmit efficiently from human-to-human.

Molecular Evolution, Diversity, and Adaptation of Influenza A(H7N9) Viruses in China.

The substantial increase in prevalence and emergence of antigenically divergent or highly pathogenic influenza A(H7N9) viruses during 2016-17 raises concerns about the epizootic potential of these viruses. We investigated the evolution and adaptation of H7N9 viruses by analyzing available data and newly generated virus sequences isolated in Guangdong Province, China, during 2015-2017. Phylogenetic analyses showed that circulating H7N9 viruses belong to distinct lineages with differing spatial distributions. Hemagglutination inhibition assays performed on serum samples from patients infected with these viruses identified 3 antigenic clusters for 16 strains of different virus lineages. We used ancestral sequence reconstruction to identify parallel amino acid changes on multiple separate lineages. We inferred that mutations in hemagglutinin occur primarily at sites involved in receptor recognition or antigenicity. Our results indicate that highly pathogenic strains likely emerged from viruses circulating in eastern Guangdong Province during March 2016 and are associated with a high rate of adaptive molecular evolution.

Genetic and biological characterization of H9N2 avian influenza viruses isolated in China from 2011 to 2014.

The genotypes of the H9N2 avian influenza viruses have changed since 2013 when almost all H9N2 viruses circulating in chickens in China were genotype 57 (G57) with the fittest lineage of each gene. To characterize the H9N2 variant viruses from 2011 to 2014, 28 H9N2 influenza viruses were isolated from live poultry markets in China from 2011-2014 and were analyzed by genetic and biological characterization. Our findings showed that 16 residues that changed antigenicity, two potential N-linked glycosylation sites, and one amino acid in the receptor binding site of the HA protein changed significantly from 2011-2014. Moreover, the HA and NA genes in the phylogenetic tree were mainly clustered into two independent branches, A and B, based on the year of isolation. H9N2 virus internal genes were related to those from the human-infected avian influenza viruses H5N1, H7N9, and H10N8. In particular, the NS gene in the phylogenetic tree revealed genetic divergence of the virus gene into three branches labeled A, B, and C, which were related to the H9N2, H10N8, and H7N9 viruses, respectively. Additionally, the isolates also showed varying levels of infection and airborne transmission. These results indicated that the H9N2 virus had undergone an adaptive evolution and variation from 2011-2014.

Structural and Molecular Characterization of the Hemagglutinin from the Fifth-Epidemic-Wave A(H7N9) Influenza Viruses.

The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that, compared to the 2013 virus HAs, the fifth-wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9) HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth-wave H7 HAs analyzed in this study.IMPORTANCE The A(H7N9) viruses circulating in China are of great public health concern. Here, we report a molecular and structural study of the major surface proteins from several recent A(H7N9) influenza viruses. Our results improve the understanding of these evolving viruses and provide important information on their receptor preference that is central to ongoing pandemic risk assessment.

Cross-reactive mouse monoclonal antibodies raised against the hemagglutinin of A/Shanghai/1/2013 (H7N9) protect against novel H7 virus isolates in the mouse model.

Influenza viruses remain a major global public health risk. In addition to seasonal influenza viruses, epizootic influenza A H7 subtype viruses of both the Asian and North American lineage are of concern due to their pandemic potential. In China, the simultaneous occurrence of H7N9 zoonotic episodes and seasonal influenza virus epidemics could potentially lead to novel reassortant viruses with the ability to efficiently spread among humans. Recently, the H7N9 virus has evolved into two new lineages, the Pearl River Delta and the Yangtze River Delta clade. This development has also resulted in viruses with a polybasic cleavage site in the hemagglutinin that are highly pathogenic in avian species and have caused human infections. In addition, an outbreak of a highly pathogenic H7N8 strain was reported in the US state of Indiana in 2016. Furthermore, an H7N2 feline virus strain caused an outbreak in cats in an animal shelter in New York City in 2016, resulting in one human zoonotic event. In this study, mouse monoclonal antibodies previously raised against the hemagglutinin of the A/Shanghai/1/2013 (H7N9) virus were tested for their (cross-) reactivity to these novel H7 viruses. Moreover, the functionality of these antibodies was assessed in vitro in hemagglutination inhibition and microneutralization assays. The therapeutic and prophylactic efficacy of the broadly reactive antibodies against novel H7 viruses was determined in vivo in mouse passive transfer-viral challenge experiments. Our results provide data about the conservation of critical H7 epitopes and could inform the selection of pre-pandemic H7 vaccine strains.

Evolutionary dynamics of avian influenza A H7N9 virus across five waves in mainland China, 2013-2017.

Since its emergence in March 2013, novel avian influenza A H7N9 virus has triggered five epidemics of human infections in China. This raises concerns about the pandemic threat of this quickly evolving H7N9 subtype for humans. In this study, we evaluated all available genomes for H7N9 and H9N2 influenza A viruses. Our assessment discovered that H7N9 of the 1st wave had the lowest nucleotide diversity, which then experienced substantial and rapid population expansion from a small founder population. From the 2nd wave, their nucleotide diversity increased quickly, indicating that H7N9 viruses had acquired larger populations and mutations after their initial emergence in 2013. After the phylogeographic divergence in the 2nd wave, although the HA and NA genes from different regions differed, compared to previous epidemics, the evolving H7N9 viruses in the 5th wave lost most of their previous clades. The highly pathogenic avian influenza (HPAI) H7N9 viruses in the 5th wave clustered together, and clustered close to the low pathogenic avian influenza (LPAI) virus isolated from the Pearl River Delta in the 3rd and 4th waves. This result supports the origin of HPAI H7N9 viruses was in the Pearl River Delta. In the 5th wave, although both HPAI and LPAI H7N9 viruses were isolated from the Pearl River Delta, their HA and NA genes were phylogenetically distinct.

Computational analysis of the receptor binding specificity of novel influenza A/H7N9 viruses.

BACKGROUND: Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches. RESULTS: The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference. CONCLUSION: The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses.

Avian influenza viruses (AIVs) H9N2 are in the course of reassorting into novel AIVs.

In 2013, two episodes of influenza emerged in China and caused worldwide concern. A new H7N9 avian influenza virus (AIV) first appeared in China on February 19, 2013. By August 31, 2013, the virus had spread to ten provinces and two metropolitan cities. Of 134 patients with H7N9 influenza, 45 died. From then on, epidemics emerged sporadically in China and resulted in several victims. On November 30, 2013, a 73-year-old woman presented with an influenza-like illness. She developed multiple organ failure and died 9 d after the onset of disease. A novel reassortant AIV, H10N8, was isolated from a tracheal aspirate specimen that was obtained from the patient 7 d after onset. This case was the first human case of influenza A subtype H10N8. On 4 February, 2014, another death due to H10N8 avian influenza was reported in Jiangxi Province, China.