一株鹅源H5N2亚型高致病性禽流感病毒的分离鉴定及生物学特性分析

分离到一株鹅源 H5N2亚型高致病性禽流感病毒,SPF鸡静脉接种致病指数为2.99,但鸭子对该病毒不敏感．病毒感染小鼠后不致病,但能够在肺内有效复制,表明其具有感染哺乳动物的潜在风险．血凝素(hemagglutinin, HA)蛋白裂解位点上插入有多个连续的碱性氨基酸(-RRRKKR-),从分子上证实这是一株高致病性禽流感病毒．核酸序列比较分析表明,分离的流感病毒HA基因与A/chicken/Hubei/489/2004 (H5N1)同源率达到99.4%,神经氨酸酶(neuraminidase, NA)基因与A/chicken/Jilin/53/01(H9N2)同源率达到99.8%;氨基酸水平上,HA与2004年分离到的A/chicken/Hubei/489/2004(H5N1)、A/swan/Guangxi/307/2004(H5N1)、A/wildduck/Guangdong/314/　2004(H5N1)和A/chicken/Henan/210/2004(H5N1)同源率均为99.3%,NA 与A/chicken/Jilin/53/01(H9N2)同源率为99.6%．进化树分析结果表明,该流感病毒分离株可能是由H5N1和H9N2两个亚型病毒重排而来．     

Phylogenetic Analysis of the Neuraminidase Gene Reveals that the H5N1 Strains Prevalent in Chickens During 2006 Bird Flu Outbreaks in Two Regions of Maharashtra, India Are Genetically Different

In February 2006, two outbreaks of highly pathogenic avian influenza A virus subtype H5N1 occurred in chickens in two neighboring districts (first in Nandurbar and second in Jaigaon) of Maharashtra, India, in a span of 12 days. In the present study, the neuraminidase (NA) gene of the two Indian H5N1 isolates was taken into consideration to find if the two strains are genetically similar. Phylogenetic analysis of the NA gene showed that the H5N1 strains isolated from the two outbreaks were not originated from the same source. The first Indian isolate (Nandubar/7972/06) was clustered closest to an isolate from chicken in Vietnam in 2004, whereas the second Indian isolate (Jalgaon/8824/06) showed resemblance to strains isolated from swan in Italy and Iran in 2006. Moreover, amino acid sequence analysis showed varying hot spots for substitutions between these two Indian isolates, and three substitutions were found at functional domain sites. Secondary structure changes due to these substitutions were also reported. This study reveals that the H5N1 strains isolated from chickens during 2006 bird flu outbreaks in two neighboring districts of Maharashtra, India are genetically different.     

海南省2016-2018年A(H1N1)pdm09亚型流感病毒基因特性分析

2009年A(H1N1)pdm09亚型流感病毒在墨西哥暴发,之后在全世界流行。为了解海南省2016-2018年A(H1N1)pdm09亚型流感病毒流行态势,分析血凝素(HA)与神经氨酸酶(NA)基因遗传进化特征与变异情况,本研究从中国流感监测信息系统获取海南省2016-2018年流感病毒病原学监测数据,选取5家流感监测网络实验室分离鉴定的37株A(H1N1)pdm09亚型流感毒株进行HA与NA基因测序,利用MEGA 10.1.8构建HA与NA基因种系进化树,并分析其氨基酸变异情况。结果显示,2016-2018年共出现3次A(H1N1)pdm09亚型流感病毒活动高峰。2017年10月份以后的分离株(4/8)与2018年大部分分离株(21/22)独立于疫苗株A/Michigan/45/2015聚为一个小支,发生20余处HA与NA氨基酸位点变异。与疫苗株A/California/7/2009(2010-2016)相比,2016-2018年流感病毒分离株在HA基因抗原决定簇上发生7处氨基酸变异并有一个潜在糖基化位点,未发现HA基因受体结合位点变异与NA基因耐药性变异。本研究提示,2016-2018年,A(H1N1)pdm09亚型流感病毒逐步发生规律性进化,氨基酸变异频率有增加趋势,今后应持续加强流感病毒病原学监测,密切追踪A(H1N1)pdm09亚型流感病毒基因变异情况,为科学防控提供理论依据。     

华东地区家鸭中N1亚型禽流感病毒NA基因的遗传进化分析

为了解华东地区家鸭内禽流感病毒的遗传进化情况,对2002~2006年分离自华东地区家鸭的3种主要N1亚型的禽流感病毒:2株H1N1、10株H3N1和14株H5N1,共26株病毒的NA基因进行了遗传进化分析。结果表明,华东地区家鸭中的N1亚型的禽流感病毒正处于不断进化状态中。14株H5N1禽流感病毒均在NA的茎部缺失20个氨基酸(49~68位),而其他N1亚型的禽流感病毒的NA都未见发生此缺失。H3N1病毒可能与H1N1病毒发生了NA基因的重排,但是目前还没有直接证据表明华东地区家鸭中H5N1禽流感参与了基因重排。     

Genome sequencing and phylogenetic analysis of three avian influenza H9N2 subtypes in Guangxi

Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.     

神经氨酸酶茎部氨基酸缺失对H5N1亚型禽流感病毒生物学特性的影响

近年来H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)茎部15~20个氨基酸的自发缺失时有报道,突变对于AIV生物学特性的影响还没有得到系统研究。应用反向遗传操作技术,拯救获得5株具有不同NA茎部长度的H5N1/PR8重组AIV。重组病毒的内部基因和血凝素(HA)基因来源相同,NA基因来源不同,并在NA茎部进行20个氨基酸的删除或添加突变。通过研究其生物学特性发现,5株重组病毒在SPF鸡胚中繁殖良好,其EID50、MDT和平均病毒滴度相似;NA茎部长短影响病毒的解凝能力,长茎病毒红细胞解脱能力比短茎病毒强;NA茎部15或20个氨基酸删除突变提高了重组病毒在MDCK细胞上的繁殖能力,短茎病毒释放出的病毒粒子数量是长茎病毒的10~100倍,释放时间提前6~10h,短茎病毒在MDCK细胞上形成的空斑也明显比长茎病毒的空斑大。实验结果揭示了AIV NA茎部氨基酸缺失突变的生物学意义,NA茎部15或20个氨基酸删除突变增强了AIV的细胞适应性,可能与现阶段H5N1亚型AIV宿主范围进一步扩大有关。利用反向遗传技术成功拯救了5株H5N1/PR8重组流感病毒,为流感病毒基因功能研究和重组疫苗研究建立了技术平台。通过对AIV NA茎部氨基酸的删除突变提高了病毒在MDCK细胞上的繁殖产量,为流感病毒细胞苗的生产提供了新的思路。     

Epidemiological Surveillance of Low Pathogenic Avian Influenza Virus (LPAIV) from Poultry in Guangxi Province,Southern China

Low pathogenic avian influenza virus (LPAIV) usually causes mild disease or asymptomatic infection in poultry. However, some LPAIV strains can be transmitted to humans and cause severe infection. Genetic rearrangement and recombination of even low pathogenic influenza may generate a novel virus with increased virulence, posing a substantial risk to public health. Southern China is regarded as the world “influenza epicenter”, due to a rash of outbreaks of influenza in recent years. In this study, we conducted an epidemiological survey of LPAIV at different live bird markets (LBMs) in Guangxi province, Southern China. From January 2009 to December 2011, we collected 3,121 cotton swab samples of larynx, trachea and cloaca from the poultry at LBMs in Guangxi. Virus isolation, hemagglutination inhibition (HI) assay, and RT-PCR were used to detect and subtype LPAIV in the collected samples. Of the 3,121 samples, 336 samples (10.8%) were LPAIV positive, including 54 (1.7%) in chicken and 282 (9.1%) in duck. The identified LPAIV were H3N1, H3N2, H6N1, H6N2, H6N5, H6N6, H6N8, and H9N2, which are combinations of seven HA subtypes (H1, H3, H4, H6, H9, H10 and H11) and five NA subtypes (N1, N2, N5, N6 and N8). The H3 and H9 subtypes are predominant in the identified LPAIVs. Among the 336 cases, 29 types of mixed infection of different HA subtypes were identified in 87 of the cases (25.9%). The mixed infections may provide opportunities for genetic recombination. Our results suggest that the LPAIV epidemiology in poultry in the Guangxi province in southern China is complicated and highlights the need for further epidemiological and genetic studies of LPAIV in this area.     

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

Influenza neuraminidase (NA) proteins expressed in TK⁻ cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.     

Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009

The 2009 pandemic H1N1 (pH1N1), of apparent swine origin, may have evolved in pigs unnoticed because of insufficient surveillance. Consequently, the need for surveillance of influenza viruses circulating in pigs has received added attention. In this study we characterized H1N1 viruses isolated from Canadian pigs in 2009. Isolates from May 2009 were comprised of hemagglutinin and neuraminidase (NA) genes of classical SIV origin in combination with the North American triple-reassortant internal gene (TRIG) cassette, here termed contemporary SIV (conSIV) H1N1. These conSIV H1N1 viruses were contiguous with the North American αH1 cluster, which was distinct from the pH1N1 isolates that were antigenically more related to the γH1 cluster. After the initial isolation of pH1N1 from an Alberta pig farm in early May 2009, pH1N1 was found several times in Canadian pigs. These pH1N1 isolates were genetically and antigenically homogeneous. In addition, H1N1 viruses bearing seasonal human H1 and N1 genes together with the TRIG cassette and an NA encoding an oseltamivir-resistance marker were isolated from pigs. The NS gene of one of these seasonal human-like SIV (shSIV) H1N1 isolates was homologous to pH1N1 NS, implicating reassortment between the two strains. Antigenic cross-reactivity was observed between pH1N1 and conSIV but not with shSIV H1N1. In summary, although there was cocirculation of pH1N1 with conSIV and shSIV H1N1 in Canadian pigs after May 2009, there was no evidence supporting the presence of pH1N1 in pigs prior to May 2009. The possibility for further reassortants being generated exists and should be closely monitored.     

Genome sequence analysis of H5N1 influenza a virus isolated from a vietnamese in 2007

Highly pathogenic H5N1 avian influenza A virus (AIV) crossed the species barrier and caused a number of deaths in humans in Vietnam and 14 other countries. Since the last report of human H5N1 infection in November 2005, the first documented H5N1 human infection was reported in June 2007 in Vietnam and was followed by 7 more cases, including 5 fatalities. In this study, we isolated and analyzed the full length of the H5N1 genome from a sample from the first patient in 2007. Phylogenetic analysis of eight genomic segments of the H5N1 virus strain (A/Vietnam/HN/2007, VNH07) revealed that this strain appears to be of genotype V and contains the HA gene, which is classified into clade 2.3.4. The deduced amino acid sequence of the HA protein has a typical affinity sequence for α2,3 linkage (SAα2,3-Gal) receptors and typical multibasic cleavage sequences. Compared with other H5N1 isolates, VNH07 showed that the possible reassortments for the NA and NP segments occurred between A/goose/Guangxi/3017/2005-like iso?lates (2.3.2) and A/human/Zhejiang/16/2006-like isolates (2.3.4).     

Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity

The 1957 and 1968 human pandemic influenza A virus strains as well as duck viruses possess sialidase activity under low-pH conditions, but human H3N2 strains isolated after 1968 do not possess such activity. We investigated the transition of avian (duck)-like low-pH stability of sialidase activities with the evolution of N2 neuraminidase (NA) genes in human influenza A virus strains. We found that the NA genes of H3N2 viruses isolated from 1971 to 1982 had evolved from the side branches of NA genes of H2N2 epidemic strains isolated in 1968 that were characterized by the low-pH-unstable sialidase activities, though the NA genes of the 1968 pandemic strains preserved the low-pH-stable sialidase. These findings suggest that the prototype of the H3N2 epidemic influenza strains isolated after 1968 probably acquired the NA gene from the H2N2 low-pH-unstable sialidase strain by second genetic reassortment in humans.     

Pseudovirus-based neuraminidase inhibition assays reveal potential H5N1 drug-resistant mutations

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. F or a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identifi ed to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.     

深圳市首例人感染H5N1病毒病例的实验室诊断及分子特点分析

对深圳首例疑似人禽流感病人的标本,进行了RT-PCR、Real-time PCR检测及病毒分离培养、血清中和试验、抗原比检测及发病早期不同病程多份标本的病毒载量分析;对分离物进行了HA基因、NA基因及M基因的核酸检测.结果表明:患者气管吸出物的H5N1亚型和A型流感病毒的特异核酸均呈阳性,并通过细胞培养分离到禽流感病毒A/Guangdong/2/06(H5N1)株.气管吸取物病毒载量随着病程延长逐渐减少,而血清中和抗体水平逐渐上升达到1∶160之后又缓缓下降.A/Guangdong/2/06株8个片段的核苷酸序列显示,其与2005～2006年中国南部的禽流感分离株高度同源,与越南、泰国、印度尼西亚等分离到的禽流感分离株存在明显的差异.     

苏州活禽市场禽流感病毒(H7N9)季节变化及HA、NA、PB2基因系统进化分析

自2013年3月中国首次发现新型禽流感病毒H7N9以来,其于2013-2014年期间发生流行,2015年也有散发性感染。该病毒的流行不仅危及家禽养殖业,还对公共卫生安全造成严重威胁。为调查活禽市场中H7N9的进化史和季节性变化,本研究于2013年7-12月在H7N9主要流行地区之一江苏省苏州市活禽市场采集2 655份鸡、鸭咽拭子样本,对样本中流感病毒核酸进行检测。结果显示,冬季样本中H7N9阳性率显著高于夏季样本,同时发现样本中存在H5、H7和H9亚型毒株之间的混合感染。进一步对H7N9阳性样本的HA、NA和PB2基因序列进行分析,结果表明阳性样本中HA、NA和PB2基因序列与新型H7N9病毒的相应基因序列同源,其在家禽体内传代时也在继续进化。特别是一些样品中PB2基因序列与H5N1病毒PB2基因序列的同源性较高。结果提示,苏州存在一种新型H7N9病毒基因重排的可能性,建议在活禽市场对所有禽流感病毒亚型进行持续监控,从而有助于流感病毒的及时防控。     

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...     

The Short Stalk Length of Highly Pathogenic Avian Influenza H5N1 Virus Neuraminidase Limits Transmission of Pandemic H1N1 Virus in Ferrets

H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.     

The Surface Glycoproteins of H5 Influenza Viruses Isolated from Humans, Chickens, and Wild Aquatic Birds Have Distinguishable Properties

In 1997, 18 confirmed cases of human influenza arising from multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5 viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds. All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in the stalk of the NA and the presence of a carbohydrate at the globular head of the HA. We found that a deletion in the NA decreased its ability to release the virus from cells, whereas carbohydrate at the HA head decreased the affinity of the virus for cell receptors. Comparison of amino acid sequences from GenBank of the HAs and NAs from different avian species revealed that additional glycosylation of the HA and a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses. This finding indicates that changes in both HA and NA may be required for the adaptation of influenza viruses from wild aquatic birds to domestic chickens and raises the possibility that chickens may be a possible intermediate host in zoonotic transmission.     

Molecular analysis of the first avian influenza H5N1 isolates from fowl in Romania

Since the events of avian influenza (AI) caused by H5N1 subtype from Hong Kong (1997), the people worldwide have been confronted with new waves of epizootic influenza. In 2005 in Romania an unprecedent H5N1 epizootic occurred in domestic and wild birds. Therefore an immediate investigation by molecular approach of this highly pathogenic H5N1 strain was necessary. The virus isolation and the RNA extraction were performed in the Institute of Diagnosis and Animal Health while PCR and sequencing were carried out in Cantacuzino Institute. Herein we report the first evidence of H5N1 presence in Romanian fowls. The phylogenetic analysis of haemagglutinin and neuraminidase gene indicated a close relationship of Romanian strains to those from Siberia and China. The virological and molecular analysis of the first strains of avian virus from Romania confirmed the presence of H5N1 subtype, belonging to the genetic line Z. These results indicate that the avian virus from this genetic line is directly derived from the highly pathogenic viruses isolated in China and Russia in 2005.     

Molecular and genetic analysis of influenza A viruses isolated in Russia, based on the neuraminidase and M2 protein gene sequence

The results of molecular analysis of 15 influenza A(H3N2) and 17-A(H1N1) epidemic strains isolated in the Russian Federation in 1995-2007 are described. The analysis on the M2 and neuraminidase influenza A virus genes was performed. The M2 sequences analysis among the remantadin resistant viruses demonstrated the S31N substitution in all strains. Besides S31N substitution, additional mutations were detected in both proteins. Mutations associated with S31N substitution were detected in each virus subtype, which may be considered as new markers for the identification of remantadin-resistant strains. The sequencing of the NA segments from all viruses showed no amino acid substitutions known to cause resistance to neuraminidase inhibitors, which indicates susceptibility to NA inhibitors among the strains.