Influenza Virus In China Research Articles

Evaluation of the Viricidal Efficacy of Four Chemical Disinfectants against Emerged H7N9 Avian Influenza Virus in China

Background: Establishment of scientific disinfection measures in domestic poultry farms and live-poultry markets (LPMs) are critical to prevent the spread of H7N9 subtype avian influenza that outbreaks in poultry and infection in humans. The current study was aimed to evaluate the efficacy of four common disinfectants to inactivate H7N9 subtype avian influenza virus (AIV). Methods: According to the disinfection technical specification, the average inactivation logarithmic values of four disinfectants against the epidemic H7N9 subtype AIV, including deciquam solution, glutaral and benzalkonium bromide solution, sodium dichloroisocyanurate powder, and peroxyacetic acid solution, were systematically evaluated. Result: Our data showed that incubation of 0.005% (w/v) deciquam solution for 30 min contact time, or 0.05% (v/v) glutaral and benzalkonium bromide solution for 10 min, or 0.24 g/L sodium dichloroisocyanurate powder for 30 min, or 0.05% (v/v) peroxyacetic acid solution for 10 min, can completely inactivate H7N9 subtype AIV. These results suggested that glutaral and benzalkonium bromide solution as well as peroxyacetic acid solution at recommended concentrations can be effectively used to cut off the spread of H7N9 subtype AIV in poultry farms and LPMs, however, deciquam solution and sodium dichloroisocyanurate powder need to increase their working concentration.

Phylogenetic tracing and biological characterization of a novel clade 2.3.2.1 reassortant of H5N6 subtype avian influenza virus in China.

In recent years in China, clade 2.3.4.4 H5N6 plus clade 2.3.2.1 H5N1 subtype highly pathogenic avian influenza (HPAI) viruses have gradually become endemic in poultry, and their co-circulation could inevitably facilitate the gene reassortment between each other. During our routine surveillance in live poultry markets (LPMs) in eastern China in 2017-2018, a novel reassortant H5N6 strain with the HA gene derived from clade 2.3.2.1 was isolated from the cloacal swabs of apparently healthy ducks. Phylogenetic tracing analysis indicated that another two clade 2.3.2.1 H5N1 strains with divergent lineages of PB1 gene and one clade 2.3.4.4 H5N6 isolate of the dominant genotype sharing spatio-temporal proximity were intimately involved in the generation of this rarely reported clade 2.3.2.1 H5N6 reassortant. Distinct with the other three HPAI H5 viruses showing moderate virulence in mice, the H5N1 strain of the homologous internal gene constellation against the clade 2.3.2.1 H5N6 reassortant was highly pathogenic, which might probably attribute to the H3 subtype-derived PB1 gene. However, as compared to the clade 2.3.4.4 H5N6 ancestor, the clade 2.3.2.1 H5N6 reassortant displayed a broader tissue distribution and higher viral titres in mice, which could likely facilitate the viral maintenance and spread in nature. Therefore, our results highlight that continuous epidemiological survey of H5 subtype HPAI viruses in LPMs needs to be strengthened to prevent the potential poultry or even public health threat of the novel reassortants from endemic viruses.

Epidemiology and Genotypic Diversity of Eurasian Avian-Like H1N1 Swine Influenza Viruses in China.

Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) outside European countries was first detected in Hong Kong Special Administrative Region (Hong Kong, SAR) of China in 2001. Afterwards, EA H1N1 SIVs have become predominant in pig population in this country. However, the epidemiology and genotypic diversity of EA H1N1 SIVs in China are still unknown. Here, we collected the EA H1N1 SIVs sequences from China between 2001 and 2018 and analyzed the epidemic and phylogenic features, and key molecular markers of these EA H1N1 SIVs. Our results showed that EA H1N1 SIVs distributed in nineteen provinces/municipalities of China. After a long-time evolution and transmission, EA H1N1 SIVs were continuously reassorted with other co-circulated influenza viruses, including 2009 pandemic H1N1 (A(H1N1)pdm09), and triple reassortment H1N2 (TR H1N2) influenza viruses, generated 11 genotypes. Genotype 3 and 5, both of which were the reassortments among EA H1N1, A(H1N1)pdm09 and TR H1N2 viruses with different origins of M genes, have become predominant in pig population. Furthermore, key molecular signatures were identified in EA H1N1 SIVs. Our study has drawn a genotypic diversity image of EA H1N1 viruses, and could help to evaluate thepotential risk of EA H1N1 for pandemic preparedness and response.

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China.

The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry, wild bird ecology and human health. Circulation of H5 viruses between poultry and wild birds is a significant public health threat in China. Thus, viral migration networks in this region need to be urgently studied. Here, we conducted molecular genetic analyses of the hemagglutinin genes of H5 highly pathogenic avian influenza viruses in multiple hosts from 2000 to 2018 in China. Our aim was to clarify the roles of different hosts in the evolution of H5 viruses. We used a flexible Bayesian statistical framework to simulate viral space diffusion and continuous-time Markov chains to infer the dynamic evolutionary process of spatiotemporal dissemination. Bayesian phylogeographic analysis of H5 viruses showed for the first time that H5 viruses in poultry and wild birds were present in Guangdong Province. Furthermore, Guangdong, Jiangsu, Shanghai and Hunan acted as the epicenters for the spread of various H5 subtypes viruses in poultry, and Henan, Shanghai, Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds. Thus, H5 viruses exhibited distinct evolutionary dynamics in poultry and wild birds. Our findings extend our understanding of the transmission and spread of highly pathogenic H5 avian influenza viruses in China.

Dynamic and integrative approaches to understanding pathogen spillover.

Dynamic and integrative approaches to understanding pathogen spillover.

Prevalence and genetic evolution of EA-H1N1 swine influenza virus in China

H1N1 swine influenza virus (SIV) has posed a potential threat to public health. Genetic evolution and amino acid substitutions of whole genome sequence of influenza virus have been shown to be associated with the virulence and pathogenicity of the virus. We made a statistical analysis of the prevalence of swine influenza virus in China and found that Eurasian avian-like H1N1 (EA-H1N1) SIVs were the main prevalent SIVs in China. We further analyzed the genotypes of EA-H1N1 SIVs, and the results showed that these viruses were divided into 11 genotypes, and there is an increasing number of reassorted EA-H1N1 SIVs in recent years, especially for those viruses whose viral genes PB2, PB1, PA and NP derived from pandemic H1N1/2009. Besides, we isolated a novel EA-H1N1 SIV (A/swine/Hubei/221/2006 (H1N1)). We find that this isolated virus is highly homologous to a previous human isolated EA-H1N1 virus A/Hunan/42443/2015 (H1N1). Taken together, this study analyzes the prevalence and genetic evolution of EA-H1N1 SIVs in China, which provides a theoretical basis for the prevention and control of swine flu.

Compatibility between haemagglutinin and neuraminidase drives the recent emergence of novel clade 2.3.4.4 H5Nx avian influenza viruses in China.

Genetic reassortments between highly pathogenic avian influenza (HPAI) H5 subtype viruses with different neuraminidase (NA) subtypes have increased in prevalence since 2010 in wild birds and poultry from China. The HA gene slightly evolved from clade 2.3.4 to clade 2.3.4.4, raising the question of whether novel clade 2.3.4.4 HA broke the balance with N1 but is matched well with NAx to drive viral epidemics. To clarify the role of compatibility between HA and NA on the prevalence of H5Nx subtypes, we constructed 10 recombinant viruses in which the clade 2.3.4 or clade 2.3.4.4 HA genes were matched with different NA (N1, N2 and N8) genes and evaluated viral characteristics and pathogenicity. Combinations between clade 2.3.4 HA and N1 or between clade 2.3.4.4 HA and NAx, but not between clade 2.3.4.4 HA and N1, or between clade 2.3.4 HA and NAx, promoted viral growth, NA activity, thermostability, low-pH stability and pathogenicity in chicken and mice. These findings suggest that both clade 2.3.4 HA/N1 and clade 2.3.4.4 HA/NAx displayed a better match, which could promote the increased prevalence of clade 2.3.4 H5N1 AIV (prior to 2010) and clade 2.3.4.4 H5Nx AIV (since 2010) in China, respectively.

Genetic analysis and biological characteristics of different internal gene origin H5N6 reassortment avian influenza virus in China in 2016

Clade 2.3.4.4 of H5N6 subtype Avian Influenza Viruses (AIVs) has become dominant clade in South-East Asia. So far, a total of 16 cases of human infection, including 6 deaths, have been confirmed since 2014. In this study, we systematically investigated the genetic evolution and biological characteristics of these viruses. We first carried out phylogenetic and statistical analysis of all H5N6 viruses that were downloaded from Influenza Research Database, GISAID and isolates from our lab. We found that H5N6 AIVs continued to reassort with other AIVs subtypes since 2014. Among these H5N6 reassortments, four main gene types were identified: A (internal genes of H5N1-origin), B (PB2 of H6-origin, and others of H5N1-origin), C (internal genes of H9-origin) and D (PB2 of H6-origin and PB1of H3-origin, and others of H5N1). In addition, after several years of evolution, gene type D is currently the dominant gene type. To systematically compare the genetic and evolutionary characteristics and pathogenicity of these viruses, four H5N6 AIVs of different gene types were selected for further analysis. S4, XZ6, GD1602 and YZ587 virus represented gene type A, B, C and D, respectively. Their NA genes were all originated from H6 and their whole genome showed a high similarity with human isolates. All these isolates could both bind with SA-α2,3 Gal and SA-α2,6 Gal receptors. Pathogenicity test showed that these viruses were highly pathogenic in chickens, while YZ587 showed the lowest virulence. Moreover, XZ6 and S4 viruses were highly pathogenic in ducks and moderately pathogenic in mice, while GD1602 and YZ587 viruses were no-pathogenic in these animals. Interestingly, GD1602 and YZ587-like viruses were responsible for 4 and 2 human infection cases in 2016, respectively. Therefore, our study showed that the YZ587 virus which has mixed internal genes, showed lower virulence in avian species and mammals compared to other genotype viruses. Overall, our findings suggest that the H5N6 avian influenza virus is undergoing constantly evolving and reassortment. Thus, our study highlights the necessary of continued surveillance of the H5N6 AIVs in birds and paying close attention to the spread of these novel reassortment viruses.

A59 Expansion of genetic diversity and interspecies transmission dynamics of swine influenza viruses in China

A59 Expansion of genetic diversity and interspecies transmission dynamics of swine influenza viruses in China

Isolation and molecular characterization of an H5N1 swine influenza virus in China in 2015

In 2015, an H5N1 influenza virus was isolated from a pig in Zhejiang Province, Eastern China. This strain was characterized by whole-genome sequencing with subsequent phylogenetic analysis. Phylogenetic analysis showed that all segments from this strain belonged to clade 2.3.2 and that it had received its genes from poultry influenza viruses in China. A Glu627Lys mutation associated with pathogenicity was observed in the PB2 protein. This strain was moderately pathogenic in mice and was able to replicate without prior adaptation. These results suggest that active surveillance of swine influenza should be used as an early warning system for influenza outbreaks in mammals.

Fc functional antibodies in humans with severe H7N9 and seasonal influenza.

Both seasonal and novel avian influenza viruses can result in severe infections requiring hospitalization. Anti-influenza antibodies (Abs) with Fc-mediated effector functions, such as Ab-dependent cellular cytotoxicity (ADCC), are of growing interest in control of influenza but have not previously been studied during severe human infections. As such, the objective of this study was to examine Fc-mediated Ab functions in humans hospitalized with influenza infection. Serum Ab response was studied in subjects hospitalized with either pandemic H7N9 avian influenza virus in China (n = 18) or circulating seasonal influenza viruses in Melbourne, Australia (n = 16). Recombinant soluble Fc receptor dimer ELISAs, natural killer (NK) cell activation assays, and Ab-dependent killing assays with influenza-infected target cells were used to assess the Fc functionality of anti-influenza hemagglutinin (HA) Abs during severe human influenza infection. We found that the peak generation of Fc functional HA Abs preceded that of neutralizing Abs for both severe H7N9 and seasonal influenza infections. Subjects who succumbed to complications of H7N9 infection demonstrated reduced HA-specific Fc receptor-binding Abs (in magnitude and breadth) immediately prior to death compared with those who survived. Subjects who recovered from H7N9 and severe seasonal influenza infections demonstrated increased Fc receptor-binding Abs not only against the homologous infecting strain but against HAs from different influenza A subtypes. Collectively, survivors of severe influenza infection rapidly generate a functional Ab response capable of mediating ADCC against divergent influenza viruses. Broadly binding HA Abs with Fc-mediated functions may be a useful component of protective immunity to severe influenza infection. The National Health and Medical Research Council ([NHMRC] grants 1023294, 1041832, and 1071916), the Australian Department of Health, and the joint University of Melbourne/Fudan University International Research and Research Training Fund provided funding for this study.

Both haemagglutinin-specific antibody and T cell responses induced by a chimpanzee adenoviral vaccine confer protection against influenza H7N9 viral challenge

Since 2013, the outbreak or sporadic infection of a new reassortant H7N9 influenza virus in China has resulted in hundreds of deaths and thousands of illnesses. An H7N9 vaccine is urgently needed, as a licensed human vaccine against H7N9 influenza is currently not available. Here, we developed a recombinant adenovirus-based vaccine, AdC68-H7HA, by cloning the H7N9 haemagglutinin (HA) gene into the chimpanzee adenoviral vector AdC68. The efficacy of AdC68-H7HA was evaluated in mice as well as guinea pigs. For comparison, an H7N9 DNA vaccine based on HA was also generated and tested in mice and guinea pigs. The results demonstrated that both AdC68-H7HA and the DNA vaccine prime-adenovirus boost regimen induced potent immune responses in animals and completely protected mice from lethal H7N9 influenza viral challenge. A post-immunization serum transfer experiment showed that antibody responses could completely protect against lethal challenge, while a T cell depletion experiment indicated that HA-specific CD8+ T cells responses also contributed to protection. Therefore, both HA-specific humoral immunity and cellular immunity play important roles in the protection. These data suggest that the chimpanzee adenovirus expressing HA is a promising vaccine candidate for H7N9 virus or other influenza viral subtypes.

Genetic Characterization of Continually Evolving Highly Pathogenic H5N6 Influenza Viruses in China, 2012-2016.

H5N6 is a highly pathogenic avian influenza (HPAI) and a zoonotic disease that causes recurring endemics in East Asia. At least 155 H5N6 outbreaks, including 15 human infections, have been reported in China. These repeated outbreaks have increased concern that the H5N6 virus may cross over to humans and cause a pandemic. In February, 2016, peafowls in a breeding farm exhibited a highly contagious disease. Post-mortem examinations, including RT-PCR, and virus isolation, confirmed that the highly pathogenic H5N6 influenza virus was the causative agent, and the strain was named A/Pavo Cristatus/Jiangxi/JA1/2016. In animal experiments, it exhibited high pathogenicity in chickens and an estimated median lethal dose in mice of ~104.3 TCID50. A phylogenetic analysis showed that JA1/2016 was clustered in H5 clade 2.3.4.4. FG594-like H5N6 virus from Guangdong Province was the probable predecessor of JA1/2016, and the estimated divergence time was June 2014. Furthermore, we found that H5N6 influenza viruses can be classified into the two following groups: Group 1 and Group 2. Group 2 influenza viruses have not been detected since the end of 2014, whereas Group 1 influenza viruses have continually evolved and reassorted with the “gene pool” circulating in south China, resulting in the rise of novel subtypes of this influenza virus. An increase in the number of its identified hosts, the expanding range of its distribution, and the continual evolution of H5N6 AIVs enhance the risk that an H5N6 virus may spread to other continents and cause a pandemic.

Quantifying predictors for the spatial diffusion of avian influenza virus in China

BackgroundAvian influenza virus (AIV) causes both severe outbreaks and endemic disease among poultry and has caused sporadic human infections in Asia, furthermore the routes of transmission in avian species between geographic regions can be numerous and complex. Using nucleotide sequences from the internal protein coding segments of AIV, we performed a Bayesian phylogeographic study to uncover regional routes of transmission and factors predictive of the rate of viral diffusion within China.ResultsWe found that the Central area and Pan-Pearl River Delta were the two main sources of AIV diffusion, while the East Coast areas especially the Yangtze River delta, were the major targets of viral invasion. Next we investigated the extent to which economic, agricultural, environmental and climatic regional data was predictive of viral diffusion by fitting phylogeographic discrete trait models using generalised linear models.ConclusionsOur results highlighted that the economic-agricultural predictors, especially the poultry population density and the number of farm product markets, are the key determinants of spatial diffusion of AIV in China; high human density and freight transportation are also important predictors of high rates of viral transmission; Climate features (e.g. temperature) were correlated to the viral invasion in the destination to some degree; while little or no impacts were found from natural environment factors (such as surface water coverage). This study uncovers the risk factors and enhances our understanding of the spatial dynamics of AIV in bird populations.

Human infections with novel reassortant H5N6 avian influenza viruses in China

Emerging Microbes & Infections (2017) 6, e50; doi:10.1038/emi.2017.38; published online 7 June 2017

Endemic Variation of H9N2 Avian Influenza Virus in China.

Forty-two H9N2 subtype AIV strains were isolated from vaccinated commercial chickens in China from 2012 to 2015. Their HA genes had nucleotide sequence homology from 86.7% to 99.7%, and similarity to the classic vaccine strain was 88.6%-92.6%. A comparison was carried out with published HA genes (410 H9 strains) and whole genomes (306 strains) isolated in China during 2012-2015. Interestingly, 99.1% (448/452) of Chinese H9N2 AIV belonged to lineage h9.4.2, and 98.5% (445/452) of the viruses belonged to h9.4.2.5. Meanwhile, 99.6% (443/445) of lineage 9.4.2.5 viruses had PSRSSR↓GLF instead of PARSSR↓GLF motifs in the HA cleavage sites; 98.2% (444/452) of HA genes showed human receptor binding associated mutation Q226L. A total of 96.8% (337/348) of the viruses had three amino-acid deletions at 63-65 in the NA stalk, associated with enhanced virulence in chickens and mice; 97.1% (338/348) of M2 proteins had the S31N mutation associated with adamantane resistance in humans. Two H9 viruses isolated in this study were highly homologous to the human-origin H9N2 virus reported in 2013. The isolates were divided into four different genotypes (U, S, V, and W). Genotype S was the major one, accounting for 94.8% (330/348). Genotypes V and W were new reassortment genotypes, with genotype W recombined with the PB2 gene originating from the new wild waterfowl-like lineage. According to the cross-HI antibody titer data, HA gene evolution, and isolation history, the isolates were divided into A, B, and C antigenic groups successively. All the antigenic group viruses were found to circulate throughout China. This study emphasizes the importance of updated vaccine and strengthened veterinary biosecurity on poultry farms and trade markets.

Detection and typing of human-infecting influenza viruses in China by using a multiplex DNA biochip assay

Rapid identification of the infections of specific subtypes of influenza viruses is critical for patient treatment and pandemic control. Here we report the application of multiplex reverse transcription polymerase chain reaction (RT-PCR) coupled with membrane-based DNA biochip to the detection and discrimination of the type (A and B) and subtype (human H1N1, human H3N2, avian H5N1 and avian H7N9) of influenza viruses in circulation in China. A multiplex one-step RT-PCR assay was designed to simultaneously amplify the HA and NA genes of the four subtypes of influenza A viruses and NS genes to discriminate type A and B viruses. PCR products were analyzed by a membrane-based biochip. The analytical sensitivity of the assay was determined at a range of 2–100 copies/reactions for each of the gene transcripts. Eighty one clinical samples, containing 66 positive samples with evident seasonal influenza virus infections, were tested, which gives the clinical sensitivity and specificity of 95.5% and 100% respectively. For the avian influenza samples, 3 out of 4 H5N1 samples and 2 out of 2 H7N9 avian samples were correctly identified. We argue this method could allow a rapid, reliable and inexpensive detection and differentiation of human-infecting influenza viruses.

Monitoring potential geographical distribution of four wild bird species in China

The outbreak of highly pathogenic avian influenza (HPAI) of the H5N1 subtypes in wild birds and poultry have caught worldwide attention. To explore the association between wild bird migration and avian influenza virus transmission, we monitored potential geographical distribution of four wild bird species that might carry the avian influenza viruses in China. They are bar-headed geese (Anser indicus), ruddy shelducks (Tadorna ferruginea), whooper swans (Cygnus cygnus) and black-headed gulls (Larus ridibundus). They served as major reservoir of the avian influenza viruses. We used bird watching records with the precise latitude/longitude coordinates from January 2002 to August 2014, and environmental variables with a pixel resolution of 5 km × 5 km from 2002 to 2014. The study utilized maximum entropy (MaxEnt) model based on ecological niche model approaches, and got the following results: (1) MaxEnt model have good discriminatory ability with the area under the curve (AUC) of the receiver operating curve (ROC) of 0.86–0.97; (2) the four wild bird species were estimated to concentrate in the North China Plain, the middle and lower region of the Yangtze River, Qinghai Lake, Tianshan Mountain and Tarim Basin, part of Tibet Plateau, and Hengduan Mountains; (3) radiation and temperature were found to provide the most significant information. Our findings will help to understand the spread of avian influenza viruses by wild bird migration in China, which benefits for effective monitoring strategies and prevention measures.

Comparison of pathogenicities of H7 avian influenza viruses via intranasal and conjunctival inoculation in cynomolgus macaques

The outbreak of H7N9 low pathogenic avian influenza viruses in China has attracted attention to H7 influenza virus infection in humans. Since we have shown that the pathogenicity of H1N1 and H5N1 influenza viruses in macaques was almost the same as that in humans, we compared the pathogenicities of H7 avian influenza viruses in cynomolgus macaques via intranasal and conjunctival inoculation, which mimics natural infection in humans. H7N9 virus, as well as H7N7 highly pathogenic avian influenza virus, showed more efficient replication and higher pathogenicity in macaques than did H7N1 and H7N3 highly pathogenic avian influenza viruses. These results are different from pathogenicity in chickens as reported previously. Therefore, our results obtained in macaques help to estimate the pathogenicity of H7 avian influenza viruses in humans.

H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep–wake regulatory neurons in mice

An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009-2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep-wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep-wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.