Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls.We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010.Out of 1036 cloacal swabs collected,three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards.All the isolates were genetically and phylogenetically characterized.The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics(QTRGL for H3N2 viruses,PEKASR for H4N6 virus) of lowly pathogenic avian influenza(LPAI) virus.The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses.The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses.In addition,co-infection of H3N2 and H4N6 in the same farm was observed.And interestingly,we isolated two subtypes viruses(H3N2 and H4N6) and their progeny virus(H3N2) with evidence of genome reassortment from the same farm,in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain.The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice,which was consistent with the results of genetic analysis.     

Orisin and future distribution of the new A （H 1N1 ） influenza virus emerging in North America in 2009

The origin of the new A （H1N1） influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.     

Mutations in Hemagglutinin of a Novel Avian-Origin H7N9 Virus That Are Critical for Receptor Binding Specificity

A novel avian-origin H7N9 influenza virus was discovered in March in China and has caused a total of131 people infected including 39 deaths in China as of June 9, 2013. Adaptation of avian viruses to efficiently infect humans requires the viral hemagglutinin（HA） binding switches from avian to human type receptors with help of some mutations in HA. As such it is critical for pandemic assessment to discover these mutations as hallmarks of adaptation. To continue our previous study of this novel H7N9 virus, we identified two sets of mutations in HA. The first set of mutations are present in the current circulating strains of 2013 H7N9 in China, and the second set are potential mutations that were found when compared to the HAs of previous human H7 subtype. These two sets of mutations exhibited unique features. The first group of mutations, on average, enhanced the HA binding to human type receptors whereas reduced that to avian types. Further the reduction of avian binding was almost three times of the increase of the human binding. The second group increased the binding to both human and avian types.But the increase in human types was almost three times of that in the avian types. Though different in their way of changing the binding preference, these two sets of mutations both contained more mutations to decrease the avian binding and increase the human binding than those that did the opposite. Our research highlighted the pandemic potential of this novel virus by showing the important mutations that could potentially help it to adapt to human hosts. Our findings offered new insights into the current state of evolution of this virus, which might be helpful for the continued surveillance of the emergence of H7N9 strains having the ability of human-to-human transmission.     

Epidemiological and risk analysis of the H7N9 subtype influenza outbreak in China at its early stage

Dozens of human cases infected with H7N9 subtype avian influenza virus (AIV) have been confirmed in China since March, 2013. Distribution data of sexes, ages, professions and regions of the cases were analyzed in this report. The results showed that the elderly cases, especially the male elderly, were significantly more than expected, which is different from human cases of H5N1 avian influenza and human cases of the pandemic H1N1 influenza. The outbreak was rated as a Grade Ⅲ (severe) outbreak, and it would evolve into a Grade IV (very severe) outbreak soon, using a method reported previously. The H7N9 AIV will probably circulate in humans, birds and pigs for years. Moreover, with the driving force of natural selection, the virus will probably evolve into highly pathogenic AIV in birds, and into a deadly pandemic influenza virus in humans. Therefore, the H7N9 outbreak has been assumed severe, and it is likely to become very or extremely severe in the future, highlighting the emergent need of forceful scientific measures to eliminate any infected animal flocks. We also described two possible mild scenarios of the future evolution of the outbreak.     

Molecular patterns of avian influenza A viruses

Avian influenza A viruses could get across the species barrier and be fatal to humans. Highly pathogenic avian influenza H5N1 virus was an example. The mechanism of interspecies transmission is not clear as yet. In this research, the protein sequences of 237 influenza A viruses with different subtypes were transformed into pseudo-signals. The energy features were extracted by the method of wavelet packet decomposition and used for virus classification by the method of hierarchical clustering. The clustering results showed that five patterns existed in avian influenza A viruses, which associated with the phenotype of interspecies transmission, and that avian viruses with patterns C and E could across species barrier and those with patterns A, B and D might not have the abilities. The results could be used to construct an early warning system to predict the transmissibility of avian influenza A viruses to humans.     

Sequence and epitope analysis of surface proteins of avian influenza H5N1 viruses from Asian patients

Avian influenza virus strain H5N1 is a highly patho- genic type A influenza virus that has caused several outbreaks of severe poultry plagues in the past. H5N1 preferentially binds to receptors with α2-3 linked sialic acids on the surface of avian epithe…     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.     

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin （HA） cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.     

Special features of the 2009 pandemic swine-origin influenza A H1N1 hemagglutinin and neuraminidase

Since the 2009 pandemic H1N1 swine-origin influenza A virus (09 S-OIV) has reminded the world about the global threat of the ever changing influenza virus,many questions regarding the detailed re-assortment of influenza viruses yet remain unanswered.Influenza A virus is the causative agent of the pandemic flu and contains 2 major antigenic glycoproteins on its surface:(i) hemagglutinin (HA);and (ii) neuraminidase (NA).The structures of the 09 S-OIV HA and NA proteins (09H1 and 09N1) have recently been resolved in our laboratory and provide some clues as to why the 09 S-OIV re-assortment virus is highly infectious with severe consequences in humans.For example,the 09H1 is highly similar to the HA of the 1918 influenza A pandemic virus in overall structure and especially in regards to its 5 defined antibody binding epitopes.For 09N1,its most distinctive feature is the lack of a 150-loop active site cavity,which was previously predicted to be present in all N1 NAs,and we hypothesize that the 150-loop may play a important role in the substrate specificity (α2,3 or α2,6 linked sialic acid receptors) and enzymatic mechanism of influenza NA.Combination of the HA and NA with special characteristics for the 09 S-OIV might contribute to its high increased transmissibility in humans.     

Urban structure and the risk of influenza A （H1N1） outbreaks in municipal districts

Changsha was one of the most affected areas during the 2009 A （H 1N 1） influenza pandemic in China. Here, we analyze the spatial-temporal dynamics of the 2009 pan- demic across Changsha municipal districts, evaluate the relationship between case incidence and the local urban spatial structure and predict high-risk areas of influenza A （H1NI）. We obtained epidemiological data on all cases of influenza A （H1NI） reported across municipal districts in Changsha dur- ing period May 2009-December 2010 and data on population density and basic geographic characteristics for 239 primary schools, 97 middle schools, 347 universities, 96 mails and markets, 674 business districts and 121 hospitals. Spatial- temporal K functions, proximity models and logistic regres- sion were used to analyze the spatial distribution pattern of influenza A （H1N1） incidence and the association between influenza A （HINI） cases and spatial risk factors and predict the infection risks. We found that the 2009 influenza A （H 1N 1 ） was driven by a transmission wave from the center of the study area to surrounding areas and reported cases increased significantly after September 2009. We also found that the distribution of influenza A （H 1N1） cases was associ- ated with population density and the presence of nearest public places, especially universities （OR = 10.166）. The final pre- dictive risk map based on the multivariate logistic analysis showed high-risk areas concentrated in the center areas of the study area associated with high population density. Our find- ings support the identification of spatial risk factors and high- risk areas to guide the prioritization of preventive and miti- gation efforts against future influenza pandemics.     

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.     

Avian flu: isolation of drug-resistant H5N1 virus

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug.     

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.     

江浙沪地区H7N9禽流感事件中的脆弱性与康复力影响因素

2013年春季在我国发生的H7N9禽流感事件,是一次公共卫生领域的恶性突发事件,在全国产生了严重的影响。研究和评估该事件的脆弱性与康复力,对于我国突发公共卫生体系的建设具有较大的借鉴意义。本文选择江浙沪地区感染H7N9禽流感的病例为研究对象,从承灾体脆弱性以及响应主体康复力的角度分析、审视我国突发性公共卫生事件的管理体系,以期探求改进我国公共卫生疫情处置的最佳途径和方法。     

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.     

Avian flu: multiple introductions of H5N1 in Nigeria

As the avian influenza virus H5N1 swept from Asia across Russia to Europe, Nigeria was the first country in Africa to report the emergence of this highly pathogenic virus. Here we analyse H5N1 sequences in poultry from two different farms in Lagos state and find that three H5N1 lineages were independently introduced through routes that coincide with the flight paths of migratory birds, although independent trade imports cannot be excluded.     

Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2-4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003-04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.