Double mutations in the H9N2 avian influenza virus PB2 gene act cooperatively to increase viral host adaptation and replication for human infections.

Avian H9N2 influenza viruses in East Asia are genetically diversified and multiple genotypes (A-W) have been established in poultry. Genotype S strains are currently the most prevalent strains, have caused many human infections and pose a public health threat. In this study, human adaptation mutations in the PB2 polymerase in genotype S strains were identified by database screening. Several PB2 double mutations were identified that acted cooperatively to produce higher genotype S virus polymerase activity and replication in human cells than in avian cells and to increase viral growth and virulence in mice. These mutations were chronologically and phylogenetically clustered in a new group within genotype S viruses. Most of the relevant human virus isolates carry the PB2-A588V mutation together with another PB2 mutation (i.e. K526R, E627V or E627K), indicating a host adaptation advantage for these double mutations. The prevalence of PB2 double mutations in human H9N2 virus isolates has also been found in genetically related human H7N9 and H10N8 viruses. These results suggested that PB2 double mutations in viruses in the field acted cooperatively to increase human adaptation of the currently prevalent H9N2 genotype S strains. This may have contributed to the recent surge of H9N2 infections and may be applicable to the human adaptation of several other avian influenza viruses. Our study provides a better understanding of the human adaptation pathways of genetically related H9N2, H7N9 and H10N8 viruses in nature.

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.

Bayesian phylodynamics of avian influenza A virus H9N2 in Asia with time-dependent predictors of migration.

Model-based phylodynamic approaches recently employed generalized linear models (GLMs) to uncover potential predictors of viral spread. Very recently some of these models have allowed both the predictors and their coefficients to be time-dependent. However, these studies mainly focused on predictors that are assumed to be constant through time. Here we inferred the phylodynamics of avian influenza A virus H9N2 isolated in 12 Asian countries and regions under both discrete trait analysis (DTA) and structured coalescent (MASCOT) approaches. Using MASCOT we applied a new time-dependent GLM to uncover the underlying factors behind H9N2 spread. We curated a rich set of time-series predictors including annual international live poultry trade and national poultry production figures. This time-dependent phylodynamic prediction model was compared to commonly employed time-independent alternatives. Additionally the time-dependent MASCOT model allowed for the estimation of viral effective sub-population sizes and their changes through time, and these effective population dynamics within each country were predicted by a GLM. International annual poultry trade is a strongly supported predictor of virus migration rates. There was also strong support for geographic proximity as a predictor of migration rate in all GLMs investigated. In time-dependent MASCOT models, national poultry production was also identified as a predictor of virus genetic diversity through time and this signal was obvious in mainland China. Our application of a recently introduced time-dependent GLM predictors integrated rich time-series data in Bayesian phylodynamic prediction. We demonstrated the contribution of poultry trade and geographic proximity (potentially unheralded wild bird movements) to avian influenza spread in Asia. To gain a better understanding of the drivers of H9N2 spread, we suggest increased surveillance of the H9N2 virus in countries that are currently under-sampled as well as in wild bird populations in the most affected countries.

Historical origins and zoonotic potential of avian influenza virus H9N2 in Tunisia revealed by Bayesian analysis and molecular characterization.

During 2009-2012, several outbreaks of avian influenza virus H9N2 were reported in Tunisian poultry. The circulating strains carried in their hemagglutinins the human-like marker 226L, which is known to be important for avian-to-human viral transmission. To investigate the origins and zoonotic potential of the Tunisian H9N2 viruses, five new isolates were identified during 2012-2016 and their whole genomes were sequenced. Bayesian-based phylogeny showed that the HA, NA, M and NP segments belong to the G1-like lineage. The PB1, PB2, PA and NS segments appeared to have undergone multiple intersubtype reassortments and to be only distantly related to all of the Eurasian lineages (G1-like, Y280-like and Korean-like). The spatiotemporal dynamic of virus spread revealed that the H9N2 virus was transferred to Tunisia from the UAE through Asian and European pathways. As indicated by Bayesian analysis of host traits, ducks and terrestrial birds played an important role in virus transmission to Tunisia. The subtype phylodynamics showed that the history of the PB1 and PB2 segments was marked by intersubtype reassortments with H4N6, H10N4 and H2N2 subtypes. Most of these transitions between locations, hosts and subtypes were statistically supported (BF > 3) and not influenced by sampling bias. Evidence of genetic evolution was observed in the predicted amino acid sequences of the viral proteins of recent Tunisian H9N2 viruses, which were characterized by the acquisition of new mutations involved in virus adaptation to avian and mammalian hosts and amantadine resistance. This study is the first comprehensive analysis of the evolutionary history of Tunisian H9N2 viruses and highlights the zoonotic risk associated with their circulation in poultry, indicating the need for continuous surveillance of their molecular evolution.

Laboratory-Confirmed Avian Influenza A(H9N2) Virus Infection, India, 2019.

A 17-month-old boy in India with severe acute respiratory infection was laboratory confirmed to have avian influenza A(H9N2) virus infection. Complete genome analysis of the strain indicated a mixed lineage of G1 and H7N3. The strain also was found to be susceptible to adamantanes and neuraminidase inhibitors.

The evolution and molecular characteristics of H9N2 avian influenza viruses in Jiangxi of China.

To understand the evolution and molecular characteristics of Jiangxi H9N2 viruses, we isolated 17 viruses in 2011 and analyzed their characteristics. Phylogenetic analyses revealed that their hemagglutinin genes originate from JS/1/00-like sublineage, neuraminidase genes originate from BJ/94-like sublineage, PB1, PA, NP, and NS genes all come from SH/F/98-like sublineage, PB2 genes originate from ST/163/04-like sublineage, while M genes come from G1-like sublineage. Genotype analysis showed that our isolates were classified as genotype 57. Molecular analyses indicated that our strains contained specific sites characteristic of low-pathogenic viruses. The current study once again highlights the necessity for continued surveillance of novel H9N2 viruses.

Amantadine resistance markers among low pathogenic avian influenza H9N2 viruses isolated from poultry in India, during 2009-2017.

Antiviral susceptibility screening of avian influenza (AI) H9N2 viruses is crucial considering their role at the animal-human interface and potential to cause human infections. The Matrix 2 (M2) inhibitors (amantadine and rimantadine) have been used for prophylaxis and treatment of influenza A virus infections, however, resistance to these drugs has been widely reported. Information about amantadine susceptibility of H9N2 viruses from India is scanty. Matrix genes of 48H9N2 viruses isolated from India during 2009-2017 were sequenced and M2 trans-membrane region sequences were screened for mutations which are known to confer resistance to amantadine namely, L26F, V27A, A30 T/V, S31N and G34E. All the viruses isolated during the year 2009 were sensitive to amantadine. However, resistance started to appear since the year 2010 and all the viruses isolated from the year 2015 onwards showed presence of molecular markers conferring resistance to amantadine. Majority of the resistant viruses exhibited S31 N mutation. Four isolates showed presence of V27A + S31 N dual mutations. Comparison of the M2 sequences from other Asian countries showed different patterns of amantadine resistance wherein phylogenetic analysis of the M genes of the strains from Pakistan formed a separate cluster. In conclusion, the present study reports prevalence and gradual increase of amantadine resistance among AI H9N2 viruses in India, emphasizing the importance of the antiviral surveillance.

Molecular evolutionary and antigenic characteristics of newly isolated H9N2 avian influenza viruses in Guangdong province, China.

Four new H9N2 avian influenza viruses (AIVs) were isolated from domestic birds in Guangdong between December 2015 and April 2016. Nucleotide sequence comparisons indicated that most of the internal genes of these four strains were highly similar to those of human H7N9 viruses. Amino acid substitutions and deletions found in the HA and NA proteins indicated that all four of these new isolates may have an enhanced ability to infect humans and other mammals. A cross-hemagglutinin-inhibition assay, conducted with two vaccine strains that are broadly used in China, suggested that antisera against vaccine candidates could not provide complete inhibition of the new isolates.

The genesis and source of the H7N9 influenza viruses causing human infections in China.

A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility.

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses.

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 103.0EID50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Evidence of infection with avian, human, and swine influenza viruses in pigs in Cairo, Egypt.

The majority of the Egyptian swine population was culled in the aftermath of the 2009 H1N1 pandemic, but small-scale growing remains. We sampled pigs from piggeries and an abattoir in Cairo. We found virological evidence of infection with avian H9N2 and H5N1 viruses as well as human pandemic H1N1 influenza virus. Serological evidence suggested previous exposure to avian H5N1 and H9N2, human pandemic H1N1, and swine avian-like and human-like viruses. This raises concern about potential reassortment of influenza viruses in pigs and highlights the need for better control and prevention of influenza virus infection in pigs.

Genetic characterization of H9N2 avian influenza viruses isolated from poultry in Poland during 2013/2014.

The study presents molecular characterization of H9N2 avian influenza (AI) isolates from field outbreaks in turkeys that occurred in Poland in 2013-2014. Sequences of all gene segments of one isolate from 2013 (A/turkey/Poland/14/2013(H9N2)) and two isolates from 2014 (A/turkey/Poland/08/2014(H9N2), A/turkey/Poland/09/2014(H9N2)) were obtained and analyzed in search of the phylogenetic relationship and molecular markers of zoonotic potential or increased pathogenicity. All gene segments were shown to originate from the wild bird reservoir and the close relationship of the analyzed isolates proved the link between the outbreaks in 2013 and 2014. However, remarkable molecular differences between isolates from 2013 to 2014 were identified, including mutation in the HA cleavage site (CS) leading to conversion from the PAASNR*GLF to the PAASKR*GLF motif and truncation of the PB1-F2 protein. Additionally, T97I substitution in the PA protein in A/turkey/Poland/08/2014 was detected which can be responsible for enhanced activity of viral polymerase in mammalian cells. However, experimental infection of mice with both isolates from 2014 showed their low pathogenicity, and no statistically significant differences in virus replication were observed between the viruses. Nevertheless, these findings indicate the dynamic evolution of H9N2 in the field emphasizing the need for monitoring of the situation in terms of H9N2 AI in Europe.

Influenza A(H9N2) Virus, Burkina Faso.

We identified influenza A(H9N2) virus G1 lineage in poultry in Burkina Faso. Urgent actions are needed to raise awareness about the risk associated with spread of this zoonotic virus subtype in the area and to construct a strategy for effective prevention and control of influenza caused by this virus.

Influenza A(H9N2) Virus, Myanmar, 2014-2015.

Routine surveillance of influenza A virus was conducted in Myanmar during 2014-2015. Influenza A(H9N2) virus was isolated in Shan State, upper Myanmar. Whole-genome sequencing showed that H9N2 virus from Myanmar was closely related to H9N2 virus of clade 4.2.5 from China.

Assessing evidence for avian-to-human transmission of influenza A/H9N2 virus in rural farming communities in northern Vietnam.

Rural farming communities in northern Vietnam do not routinely practice vaccination for influenza A viruses (IAV) for either humans or poultry, which enables us to study transmission intensity via seroepidemiology. Using samples from a longitudinal cohort of farming households, we determined the number of symptomatic and asymptomatic human infections for seasonal IAV and avian A/H9 over 2 years. As expected, we detected virologically confirmed acute cases of seasonal IAV in humans, as well as large numbers of subclinical seroconversions to A/H1pdm [55/265 (21 %)], A/H3 [95/265 (36 %)] and A/H9 [24/265 (9 %)]. Five of the A/H9 human seroconverters likely represented true infections rather than heterosubtypic immunity, because the individuals seroconverted solely to A/H9. Among co-located poultry, we found significantly higher seroprevalance for A/H5 compared to A/H9 in both chickens and ducks [for northern study sites overall, 337/1105 (30.5 %) seropositive for A/H5 and 123/1105 (11.1 %) seropositive for A/H9].

Avian influenza H9N2 virus isolated from air samples in LPMs in Jiangxi, China.

BACKGROUND: Recently, avian influenza virus has caused repeated worldwide outbreaks in humans. Live Poultry Markets (LPMs) play an important role in the circulation and reassortment of novel Avian Influenza Virus (AIVs). Aerosol transmission is one of the most important pathways for influenza virus to spread among poultry, from poultry to mammals, and among mammals. METHODS: In this study, air samples were collected from LPMs in Nanchang city between April 2014 and March 2015 to investigate possible aerosol transmission of AIVs. Air samples were detected for Flu A by Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR). If samples were positive for Flu A, they were inoculated into 9- to 10-day-old specific-pathogen-free embryonated eggs. If the result was positive, the whole genome of the virus was sequenced by MiSeq. Phylogenetic trees of all 8 segments were constructed using MEGA 6.05 software. RESULTS: To investigate the possible aerosol transmission of AIVs, 807 air samples were collected from LPMs in Nanchang city between April 2014 and March 2015. Based on RRT-PCR results, 275 samples (34.1%) were Flu A positive, and one virus was successfully isolated with embryonated eggs. The virus shared high nucleotide homology with H9N2 AIVs from South China. CONCLUSIONS: Our study provides further evidence that the air in LPMs can be contaminated by influenza viruses and their nucleic acids, and this should be considered when choosing and evaluating disinfection strategies in LPMs, such as regular air disinfection. Aerosolized viruses such as the H9N2 virus detected in this study can increase the risk of human infection when people are exposed in LPMs.

Genetic characterization of H9N2 avian influenza virus in plateau pikas in the Qinghai Lake region of China.

Qinghai Lake is a major migratory-bird breeding site that has experienced several highly pathogenic avian influenza virus (AIV) epizootics. Plateau pikas (Ochotona curzoniae) have previously been implicated in the ecology of avian influenza virus in this region. We first isolated an H9N2 AIV (A/Pika/Menyuan/01/2008) from plateau pikas between November 2008 and October 2009. Sequence analysis showed that the A/Pika/Menyuan/01/2008 AIV was closely related to the H9N2 AIV strain (A/Turkey/Wisconsin/ 1/1966). Our findings suggested that plateau pikas may contribute to AIV epidemiology in the Qinghai Lake region.

A brief summary of the epidemiology and genetic relatedness of avian influenza H9N2 virus in birds and mammals in the Middle East and North Africa.

H9N2 is the most widespread avian influenza virus subtype in poultry worldwide. It infects a broad spectrum of host species including birds and mammals. Infections in poultry and humans vary from silent to fatal. Importantly, all AIV, which are fatal in humans (e.g. H5N1, H7N9) acquired their 'internal' gene segments from H9N2 viruses. Although H9N2 is endemic in the Middle East (ME) and North Africa since the late 1990s, little is known about its epidemiology and genetics on a regional level. In this review, we summarised the epidemiological situation of H9N2 in poultry and mammals in Iran, Iraq, Kuwait, Qatar, United Arab Emirates, Oman, Bahrain, Yemen, Saudi Arabia, Jordan, Palestine, Israel, Syria, Lebanon, Turkey, Egypt, Sudan, Libya, Tunisia, Algeria and Morocco. The virus has been isolated from humans in Egypt and serosurveys indicated widespread infection particularly among poultry workers and pigs in some countries. Some isolates replicated well in experimentally inoculated dogs, mice, hamsters and ferrets. Insufficient protection of immunised poultry was frequently reported most likely due to concurrent viral or bacterial infections and antigenic drift of the field viruses from outdated vaccine strains. Genetic analysis indicated several distinct phylogroups including a panzootic genotype in the Asian and African parts of the ME, which may be useful for the development of vaccines. The extensive circulation of H9N2 for about 20 years in this region where the H5N1 virus is also endemic in some countries, poses a serious public health threat. Regional surveillance and control strategy are highly recommended.

Diversity and evolution of avian influenza viruses in live poultry markets, free-range poultry and wild wetland birds in China.

The wide circulation of novel avian influenza viruses (AIVs) highlights the risk of pandemic influenza emergence in China. To investigate the prevalence and genetic diversity of AIVs in different ecological contexts, we surveyed AIVs in live poultry markets (LPMs), free-range poultry and the wetland habitats of wild birds in Zhejiang and Hubei provinces. Notably, LPMs contained the highest frequency of AIV infection, and the greatest number of subtypes (n = 9) and subtype co-infections (n = 14), as well as frequent reassortment, suggesting that they play an active role in fuelling AIV transmission. AIV-positive samples were also identified in wild birds in both provinces and free-range poultry in one sampling site close to a wetland region in Hubei. H9N2, H7N9 and H5N1 were the most commonly sampled subtypes in the LPMs from Zhejiang, whilst H5N6 and H9N2 were the dominant subtypes in the LPMs from Hubei. Phylogenetic analyses of the whole-genome sequences of 43 AIVs revealed that three reassortant H5 subtypes were circulating in LMPs in both geographical regions. Notably, the viruses sampled from the wetland regions and free-range poultry contained complex reassortants, for which the origins of some segments were unclear. Overall, our study highlights the extent of AIV genetic diversity in two highly populated parts of central and south-eastern China, particularly in LPMs, and emphasizes the need for continual surveillance.

Serological evidence of H9N2 avian influenza virus exposure among poultry workers from Fars province of Iran.

BACKGROUND: Since the 1990s, influenza A viruses of the H9N2 subtype have been causing infections in the poultry population around the globe. This influenza subtype is widely circulating in poultry and human cases of AI H9N2 have been sporadically reported in countries where this virus is endemic in domestic birds. The wide circulation of H9N2 viruses throughout Europe and Asia along with their ability to cause direct infection in mammals and humans, raises public health concerns. H9N2 AI was reported for the first time in Iran in 1998 and at present it is endemic in poultry. This study was carried out to evaluate the exposure to H9N2 AI viruses among poultry workers from the Fars province. METHODS: 100 poultry workers and 100 healthy individuals with no professional exposure to poultry took part in this study. Serum samples were tested for antibodies against two distinct H9N2 avian influenza viruses, which showed different phylogenetic clustering and important molecular differences, such as at the amino acid (aa) position 226 (Q/L) (H3 numbering), using haemagglutination inhibition (HI) and microneutralization (MN) assays. RESULTS: Results showed that 17 % of the poultry workers were positive for the A/chicken/Iran/10VIR/854-5/2008 virus in MN test and 12 % in HI test using the titer ≥40 as positive cut-off value. Only 2 % of the poultry workers were positive for the A/chicken/Iran/12VIR/9630/1998 virus. Seroprevalence of non exposed individuals for both H9N2 strains was below 3 % by both tests. Statistical analyses models showed that exposure to poultry significantly increases the risk of infection with H9N2 virus. CONCLUSIONS: The results have demonstrated that exposure to avian H9N2 viruses had occurred among poultry workers in the Fars province of Iran. Continuous surveillance programmes should be implemented to monitor the presence of avian influenza infections in humans and to evaluate their potential threat to poultry workers and public health.