Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

The influenza M2 ectodomain (M2e) is well conserved across human influenza A subtypes, but there are few residue changes among avian and swine origin influenza A viruses. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses using the yeast expression system. Intramuscular immunization of mice with AS04-adjuvanted M2e5x protein vaccines was effective in inducing M2e-specific antibodies reactive to M2e peptide and native M2 proteins on the infected cells with human, swine, or avian influenza virus, mucosal and systemic memory cellular immune responses, and cross-protection against H3N2 virus. Importantly, M2e5x immune sera were found to confer protection against different subtypes of H1N1 and H5N1 influenza A viruses in naïve mice. Also, M2e5x-immune complexes of virus-infected cells stimulated macrophages to secrete cytokines via Fc receptors, indicating a possible mechanism of protection. The present study provides evidence that M2e5x proteins produced in yeast cells could be developed as a potential universal influenza vaccine.     

Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins

Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets

Background

Influenza A viruses generate an extreme genetic diversity through point mutation and gene segment exchange, resulting in many new strains that emerge from the animal reservoirs, among which was the recent highly pathogenic H5N1 virus. This genetic diversity also endows these viruses with a dynamic adaptability to their habitats, one result being the rapid selection of genomic variants that resist the immune responses of infected hosts. With the possibility of an influenza A pandemic, a critical need is a vaccine that will recognize and protect against any influenza A pathogen. One feasible approach is a vaccine containing conserved immunogenic protein sequences that represent the genotypic diversity of all current and future avian and human influenza viruses as an alternative to current vaccines that address only the known circulating virus strains.

Methodology/Principal Findings

Methodologies for large-scale analysis of the evolutionary variability of the influenza A virus proteins recorded in public databases were developed and used to elucidate the amino acid sequence diversity and conservation of 36,343 sequences of the 11 viral proteins of the recorded virus isolates of the past 30 years. Technologies were also applied to identify the conserved amino acid sequences from isolates of the past decade, and to evaluate the predicted human lymphocyte antigen (HLA) supertype-restricted class I and II T-cell epitopes of the conserved sequences. Fifty-five (55) sequences of 9 or more amino acids of the polymerases (PB2, PB1, and PA), nucleoprotein (NP), and matrix 1 (M1) proteins were completely conserved in at least 80%, many in 95 to 100%, of the avian and human influenza A virus isolates despite the marked evolutionary variability of the viruses. Almost all (50) of these conserved sequences contained putative supertype HLA class I or class II epitopes as predicted by 4 peptide-HLA binding algorithms. Additionally, data of the Immune Epitope Database (IEDB) include 29 experimentally identified HLA class I and II T-cell epitopes present in 14 of the conserved sequences.

Conclusions/Significance

This study of all reported influenza A virus protein sequences, avian and human, has identified 55 highly conserved sequences, most of which are predicted to have immune relevance as T-cell epitopes. This is a necessary first step in the design and analysis of a polyepitope, pan-influenza A vaccine. In addition to the application described herein, these technologies can be applied to other pathogens and to other therapeutic modalities designed to attack DNA, RNA, or protein sequences critical to pathogen function.     

Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus

Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.     

Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.

We determined the origin and evolutionary pathways of the PB1 genes of influenza A viruses responsible for the 1957 and 1968 human pandemics and obtained information on the variable or conserved region of the PB1 protein. The evolutionary tree constructed from nucleotide sequences suggested the following: (i) the PB1 gene of the 1957 human pandemic strain, A/Singapore/1/57 (H2N2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, A/NT/60/68 (H3N2), the PB1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human H3N2 virus inherited the PB1 gene from an A/NT/60/68-like virus. Nucleotide sequence analysis also showed that the avian PB1 gene was introduced into pigs. Hence, transmission of the PB1 gene from avian to mammalian species is a relatively frequent event. Comparative analysis of deduced amino acid sequences disclosed highly conserved regions in PB1 proteins, which may be key structures required for PB1 activities.     

Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004

The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.     

Molecular genetic characterization of H5N1 influenza virus strains isolated from poultry in the Kurgan region in 2005

In the second half of 2005, a large-scale outbreak of influenza in poultry and wild birds was caused by a highly pathogenic H5N1 influenza virus in Russia. The level of pathogenicity is a polygenic trait, and most individual genes contribute to the influenza A virus pathogenicity in birds, animals, and humans. The full-length nucleotide sequences were determined for H5N1 strains isolated in the Kurgan region (Western Siberia). The structure of viral proteins was analyzed using the deduced amino acid sequences. The receptor-binding site of hemagglutinin (HA) in strains A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. The structure of the basic amino acid cluster located within the HA cleavage site was identical in all isolates: QGERRRKKR. According to the neuraminidase structure, all H5N1 isolates from the Kurgan region were assigned to the Z genotype. Amino acid residues typical for the avian influenza virus were revealed in 30 out of 32 positions of M1, M2, NP, PA, and PB2, determining the host range specificity. One of the strains contained Lys at position 627 of PB2. Isolates from the Kurgan region were shown to have a remantadine-sensitive genotype. Both strains contained Glu at position 92 of NS1, indicating that the virus is interferon-resistant. Phylogenetic analysis related the Kurgan isolates to subclade 2 of clade 2 of highly pathogenic H5N1 influenza viruses.     

Sequence comparison between the extracellular domain of M2 protein human and avian influenza A virus provides new information for bivalent influenza vaccine design

To prevent the human and economic losses caused by human and avian influenza viruses, it is necessary to prepare safe bivalent influenza vaccines. Recent studies found that human influenza vaccines based on the extracellular domain of influenza M2 protein (M2e) induced broad-spectrum protective immunity in various antigen constructs. A prerequisite for using the M2e protein as a bivalent influenza vaccine component was to find out the sequence differences between human and non-human (avian or swine) influenza M2e proteins. Here, we completed such a comparison using 716 influenza M2e sequences available in Genbank. The results found one region on M2e protein consistent with host restriction specificities: PIRNEWGCRCN, PTRNGWECKCS and PIRNGWECRCN (aa10-20; the human, avian and swine specific M2e sequence, respectively). Interestingly, the comparison result was then validated by immunoblotting and enzyme-linked immunosorbent assay. The monoclonal antibody against the EVETPIRN sequence (aa6-13) of human M2e protein could weakly recognize avian M2e proteins bearing the EVETPTRN sequence (aa6-13) but failed to recognize avian M2e proteins bearing the EVETLTRN sequence (aa6-13). The data in this study provided useful information in the race to develop bivalent influenza vaccines against avian and human influenza A virus infection in human beings.     

2017–2019年我国南方地区高致病性H5N6亚型禽流感病毒血凝素蛋白分子特征分析

【背景】自2014年以来,H5N6禽流感病毒在我国家禽和活禽市场持续进化,成为人类和动物健康的重大威胁。【目的】对2017–2019年中国南方地区93株高致病性H5N6禽流感病毒的HA基因进行分子进化分析。【方法】接种9–11日龄鸡胚分离核酸检测阳性的H5N6标本,运用下一代测序平台对病毒分离物进行全基因组测序,从NCBI和GISAID数据库下载参考序列,利用BLAST、MEGA6.1及Clustal X等软件进行序列分析。【结果】2017–2019年,从189份江苏省H5亚型禽类/环境标本和1名H5N6患者咽拭子标本中共分离到43株病毒,完成了33株H5N6病毒的全基因组测序。下载网上同时期中国其他地区流行的H5N6毒株序列,对总计93株H5N6病毒的HA基因进行分子进化分析。93株H5N6病毒中有78株属于Clade 2.3.4.4h,9株病毒属于Clade 2.3.4.4e,4株H5N6病毒属于Clade 2.3.4.4b,1株属于Clade 2.3.4.4f,1株属于Clade 2.3.4.4g。所有93株病毒HA蛋白的裂解位点含有多个碱性氨基酸,表明它们都属于高致病性禽流感病...     

Persistent host markers in pandemic and H5N1 influenza viruses

Avian influenza viruses have adapted to human hosts, causing pandemics in humans. The key host-specific amino acid mutations required for an avian influenza virus to function in humans are unknown. Through multiple-sequence alignment and statistical testing of each aligned amino acid, we identified markers that discriminate human influenza viruses from avian influenza viruses. We applied strict thresholds to select only markers which are highly preserved in human influenza virus isolates over time. We found that a subset of these persistent host markers exist in all human pandemic influenza virus sequences from 1918, 1957, and 1968, while others are acquired as the virus becomes a seasonal influenza virus. We also show that human H5N1 influenza viruses are significantly more likely to contain the amino acid predominant in human strains for a few persistent host markers than avian H5N1 influenza viruses. This sporadic enrichment of amino acids present in human-hosted viruses may indicate that some H5N1 viruses have made modest adaptations to their new hosts in the recent past. The markers reported here should be useful in monitoring potential pandemic influenza viruses.     

Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.     

H9N2亚型禽流感病毒NS1基因的进化分析

利用RT-PCR方法,扩增了1998～2005年间分离的9株H9N2亚型禽流感病毒的NS1基因,对其进行了序列测定和进化分析.序列分析表明,9株AIV NS1基因完整的阅读框均为654bp,编码217个氨基酸,其核苷酸和推导的氨基酸同源性分别为95.4%～99.8%和93.6%～100%;9株病毒的NS1蛋白的C端均有13个氨基酸的缺失;进化分析表明,9株AIV属于A群,且形成一个独立分支,在该分支中,只有Ck/HN/A3/98株属于Ck/HK/Y280/97-like亚类,且与Ck/BJ/8/98的进化关系最近,其余8株属于Ck/SH/F/98-like亚类,说明Ck/SH/F/98-like亚类的H9N2亚型AIV在中国大陆的鸡群中广泛存在.NS1基因的进化及其编码产物的特性分析,为AIV的毒力变异、致病机制、药物靶位点的设计及鉴别诊断的研究奠定了基础.     

A Single Amino Acid in the M1 Protein Responsible for the Different Pathogenic Potentials of H5N1 Highly Pathogenic Avian Influenza Virus Strains

Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.     

Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong

The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of "internal" virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.     

Evolution of the nucleoprotein gene of influenza A virus

Nucleotide sequences of 24 nucleoprotein (NP) genes isolated from a wide range of hosts, geographic regions, and influenza A virus serotypes and 18 published NP gene sequences were analyzed to determine evolutionary relationships. The phylogeny of NP genes was determined by a maximum-parsimony analysis of nucleotide sequences. Phylogenetic analysis showed that NP genes have evolved into five host-specific lineages, including (i) Equine/Prague/56 (EQPR56), (ii) recent equine strains, (iii) classic swine (H1N1 swine, e.g., A/Swine/Iowa/15/30) and human strains, (iv) gull H13 viruses, and (v) avian strains (including North American, Australian, and Old World subgroups). These NP lineages match the five RNA hybridization groups identified by W. J. Bean (Virology 133:438-442, 1984). Maximum nucleotide differences among the NPs was 18.5%, but maximum amino acid differences reached only 10.8%, reflecting the conservative nature of the NP protein. Evolutionary rates varied among lineages; the human lineage showed the highest rate (2.54 nucleotide changes per year), followed by the Old World avian lineage (2.17 changes per year) and the recent equine lineage (1.22 changes per year). The per-nucleotide rates of human and avian NP gene evolution (1.62 x 10(-3) to 1.39 x 10(-3) changes per year) are lower than that reported for human NS genes (2.0 x 10(-3) changes per year; D. A. Buonagurio, S. Nakada, J. D. Parvin, M. Krystal, P. Palese, and W. M. Fitch, Science 232:980-982, 1986). Of the five NP lineages, the human lineage showed the greatest evolution at the amino acid level; over a period of 50 years, human NPs have accumulated 39 amino acid changes. In contrast, the avian lineage showed remarkable conservatism; over the same period, avian NP proteins changed by 0 to 10 amino acids. The specificity of the H13 NP in gulls and its distinct evolutionary separation from the classic avian lineage suggests that H13 NPs may have a large degree of adaptation to gulls. The presence of avian and human NPs in some swine isolates demonstrates the susceptibility of swine to different virus strains and supports the hypothesis that swine may serve as intermediates for the introduction of avian influenza virus genes into the human virus gene pool. EQPR56 is relatively distantly related to all other NP lineages, which suggests that this NP is rooted closest to the ancestor of all contemporary NPs. On the basis of estimation of evolutionary rates from nucleotide branch distances, current NP lineages are at least 100 years old, and the EQPR56 NP is much older.(ABSTRACT TRUNCATED AT 400 WORDS)     

一株人感染高致病性禽流感(H5N1)病毒基因组分子特征分析

【背景】H5N1禽流感病毒可以感染人类导致重症呼吸道感染,致死率高。【目的】研究我中心确认的一例人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015的可能起源及基因组分子特征。【方法】对病人痰液样本中的H5N1病毒进行全基因组测序,使用CLC Genomics Workbench 9.0对序列进行拼接,使用BLAST和MEGA 5.22软件进行同源性比对和各片段分子特征分析。【结果】该株禽流感病毒属于H5亚型的2.3.2.1c家系,其8个片段均与江浙地区禽类中分离的病毒高度同源,未发现有明显的重配。分子特征显示,该病毒血凝素(Hemagglutinin,HA)蛋白裂解位点为PQRERRRR/G,受体结合位点呈现禽类受体特点,但出现D94N、S133A和T188I氨基酸置换增强了病毒对人类受体的亲和性。神经氨酸酶(Neuraminidase,NA)蛋白颈部在49-68位缺失20个氨基酸,非结构蛋白1 (Non-structure protein,NS1)存在P42S置换和80-84位氨基酸的缺失。其他蛋白中也存在多个增强病毒致病力和对人类细胞亲和力的氨基酸突变。对耐药位点分析发现存在对奥司他韦的耐药突变H_274Y,病毒对金刚烷胺仍旧敏感。【结论】人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015属于2.3.2.1c家系,禽类来源,关键位点较保守,但仍出现了多个氨基酸的进化与变异使其更利于感染人类。H5N1禽流感病毒进化活跃,持续动态监测不能放松。     

Uncovering the Potential Pan Proteomes Encoded by Genomic Strand RNAs of Influenza A Viruses

Influenza A virus genomes are composed of eight negative sense RNAs. In total, 16 proteins encoded by eight positive sense RNAs were identified. One putative protein coding sequence (PCS) encoded by genomic strand RNA of segment 8 has been previously proposed. In this study, 95,608, 123,965 and 35,699 genomic strand RNA sequences from influenza A viruses from avian, human and mammalian hosts, respectively, were used to identify PCSs encoded by the genomic strand RNAs. In total, 326,069 PCSs with lengths equal to or longer than 80 amino acids were identified and clustered into 270 PCS groups. Twenty of the 270 PCS groups which have greater than 10% proportion in influenza A viruses from avian, human or mammalian hosts were selected for detailed study. Maps of the 20 PCSGs in the influenza A virus genomes were constructed. The proportions of the 20 PCSGs in influenza A viruses from different hosts and serotypes were analyzed. One secretory and five membrane proteins predicted from the PCS groups encoded by genomic strand RNAs of segments 1, 2, 4, 6, 7 and 8 were identified. These results suggest the possibility of the ambisense nature of the influenza A virus genomic RNAs and a potential coding sequence reservoir encoding potential pan proteomes of influenza A viruses.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

The variable codons of H5N1 avian influenza A virus haemagglutinin genes

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsynonymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in viruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected positions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.