一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

21世纪的首次大流行：2009甲型（H1N1）流感

2009年4月初,在墨西哥和美国出现一种新型甲型（H1N1）流感病毒。该病毒通过人-人传播迅速在全球范围蔓延。该病毒拥有来自人流感病毒、禽流感病毒和猪流感病毒的基因片段,其HA基因与引发1918年大流行的流感病毒株的HA基因同源性很高。该病毒倾向于感染儿童、青少年、孕妇,以及具有心肺疾病的人。据观察,它在人群中的传播能力高于季节性流感。部分感染患者具有在季节性流感中罕见的呕吐和腹泻症状。先前的流感病毒大流行和2009年爆发的甲型H1N1流感病毒大流行表明,由于流感病毒变异速度快、容易发生基因重排,新产生的变异毒株很可能造成新的大流行,威胁人类健康。由于禽流感病毒和人流感病毒都能感染猪,猪被认为是通过基因重排生成新的大流行病毒的＂混合容器＂。     

猪源性甲型H1N1流感病毒研究概况

2009年3月在美国和墨西哥流感样患者的呼吸道标本中鉴定出新的猪源性甲型H1N1流感病毒。该病毒可人-人传播,已蔓延到112个国家和地区。为了遏制不断重组或重配的流感病毒,各国学者对甲型H1N1流感病毒的分子生物学特征、复制周期及实验室诊断做了细致的研究,以研发相应的药物或疫苗,这些成就为世界各国防控今年新鉴定的猪源性甲型H1N1流感病毒感染发挥了重要作用。现就猪源性甲型H1N1流感病毒的鉴定、基因组结构特征做一综述。     

新世纪流感大流行的思考

2009年从墨西哥开始暴发了一场席卷全世界的流感疫情．此次大流行的毒株,甲型H1N1病毒,包含了猪源、禽源和人源流感病毒的基因片段．研究该毒株的基因重配、进化历程及其生物学特性,将对防控此次流行具有重要意义．目前,该毒株的遗传进化关系已明确,通过遗传性状分析可获知该毒株可能的生物学性状,但流感大流行动向、毒株遗传变化、毒力及致病性变化仍在密切监控中．流感病毒生态系统具有复杂性,其基因组易突变、易重配、易在自然宿主保存,使得流感大流行存在一定的必然性．正视流感大流行的威胁,积极提高流感病毒在生态系统中的监控,加强流行病学调查,发展疫苗与药物,建立有效公共卫生保障体系,才能降低流感大流行的破坏性．     

2004年山东分离的一株H5N1禽流感病毒被检测为X基因型

A/Duck/Shandong/093/2004被报道是一株重配的H5N1禽流感病毒.为此,对它进行了基因型研究.对其8个独立的基因片段的贝叶斯分析和对全基因组的邻接法分析都表明它属于基因型X,尽管基因型X在中国南部在2002年已被基因型Z所取代.     

Emerging influenza virus: A global threat

Since 1918, influenza virus has been one of the major causes of morbidity and mortality, especially among young children. Though the commonly circulating strain of the virus is not virulent enough to cause mortality, the ability of the virus genome to mutate at a very high rate may lead to the emergence of a highly virulent strain that may become the cause of the next pandemic. Apart from the influenza virus strain circulating in humans (H1N1 and H3N2), the avian influenza H5N1 H7 and H9 virus strains have also been reported to have caused human infections, H5N1 H7 and H9 have shown their ability to cross the species barrier from birds to humans and further replicate in humans. This review addresses the biological and epidemiological aspects of influenza virus and efforts to have a control on the virus globally.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

甲型H5N1流感病毒M2与HA双基因载体疫苗在小鼠体内的免疫学评价

高致病性H5N1亚型禽流感病毒 (AIV) 严重威胁到人类健康,因此研制高效、安全的禽流感疫苗具有重要意义。以我国分离的首株人H5N1亚型禽流感病毒 (A/Anhui/1/2005) 作为研究对象,PCR扩增基质蛋白2 (M2) 和血凝素 (HA) 基因全长开放阅读框片段,构建共表达H5N1亚型AIV膜蛋白基因 M2和HA的重组质粒pStar-M2/HA。此外,还通过同源重组以293细胞包装出表达M2基因的重组腺病毒Ad-M2以及表达HA基因的重组腺病毒Ad-HA。用间接免疫荧光 (IFA) 方法检测到了各载体上插入基因的表达。按初免-加强程序分别用重组质粒pStar-M2/HA和重组腺病毒Ad-HA+Ad-M2免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集血清用于检测体液免疫应答,末次免疫后14 d采集脾淋巴细胞用于检测细胞免疫应答。血凝抑制 (HI) 实验检测到免疫后小鼠血清中的HI活性。ELISA实验检测到免疫后小鼠血清中抗H5N1亚型流感病毒表面蛋白的IgG抗体。ELISPOT实验检测到免疫后小鼠针对M2蛋白和HA蛋白的特异性细胞免疫应答。流感病毒M2与HA双基因共免疫的研究,为研究开发新型重组流感疫苗奠定了基础。     

甲型H1N1流感病毒HA基因RT-LAMP检测方法的建立与初步应用

目的:建立反转录环介导等温扩增技术(RT-LAMP)检测甲型H1N1流感病毒HA基因的方法,并用于检测临床样本.方法:通过在线软件Primer Explorer V4设计H1N1 HA基因的RT-LAMP引物,建立RT-LAMP检测方法,并评价其灵敏度和特异度.结果:与传统RT-PCR方法相比,RT-LAMP检测方法具备更高的灵敏度,达到10个拷贝,并且具有良好的特异性;在76份呼吸道感染儿童的咽拭子标本中检测到2份阳性,与RT-PCR方法检测结果相同.结论:建立的H1N1 RT-LAMP检测方法灵敏特异,简单快速,具备H1N1现场检测应用前景.     

H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其应用

为建立H1亚型猪流感病毒抗体检测方法,扩增了H1N1亚型猪流感病毒流行株的血凝素基因HA1部分,构建原核表达载体pET30a-HA1,并转化大肠杆菌BL21表达重组蛋白。对重组蛋白包涵体进行变性、复性和Ni-NTA亲和层析纯化。以纯化后的蛋白作包被抗原,建立间接ELISA检测方法。利用该检测方法检测了2008?2009年采集的猪血清785份,阳性率为15.54％,不同省份的阳性率存在差异 (8％~47％)。以IDEXX相关试剂盒检测结果作为参照,该方法的诊断特异性达到91％,诊断敏感性达到95％。     

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.     

1株含H9N2内部基因的H5N1亚型基因重配禽流感病毒的全基因测序及遗传进化分析

在对华东地区家养水禽中流感病毒的带毒状况的流行病学监测过程中,从表观健康家鸭体内分离到一株H5N1亚型禽流感病毒A/duck/Shandong/009/2008(简称Dk/SD/009/08)。为了解该毒株的基因组构成,对该分离株进行全基因测序。测序结果显示:该毒株HA裂解位点处的氨基酸序列为PLRERRRK-R/GL,符合高致病性禽流感病毒的分子特征,且参照H5N1国际统一命名准则,Dk/SD/009/08的HA基因属于2.3.4进化支。BLAST结果显示,HA、NA、NP及NS基因均与H5N1亚型病毒的核苷酸一致性最高,而RNA聚合酶基因(PB2、PB1、PA)及M基因则与H9N2亚型病毒的亲缘关系最近,故推测该分离株可能是一株天然重组病毒;遗传进化分析进一步表明,流行于华南地区鹌鹑中的G1-like H9N2亚型病毒可能为该分离株提供部分的内部基因。     

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96–99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.     

利用原子力显微镜对A型流感病毒的形态学研究

利用透射电子显微镜(TEM)和原子力显微镜(AFM)观察流感病毒(H1N1),探讨AFM在病毒形态研究中的应用,为病毒形态学研究提供一种新型、简便、快捷的工具.TEM采用磷钨酸负染方法,AFM采用轻敲模式在大气常温下扫描成像,并对主要指标长度(直径)、Ra、Rq等进行测量.两种方法最终得到相似的形态学结果,流感病毒呈球状、丝状,并有一些形状介于两者之间.TEM提供了流感病毒二维图像,可见钉状突起,AFM则呈现了流感病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.与TEM观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具,其表征参数可以作为病毒形态学研究的量化指标.     

Beschreibung und Durchführung der Impfaktion bei Zoovögeln im Zoo am Meer Bremerhaven gegen Aviäre Influenza, hervorgerufen durch das Virus H5N1

The avian influenza A virus (H5N1), which causes the bird flu is latently present in populations of wild birds. The Bremerhaven Zoo is subject to an especially high infection risk owing to its close proximity to the North Sea coast and to resting places of birds. As an alternative to drastically killing all birds in an event of infection, a vaccination campaign was initiated in May 2006 as part of the “Program of the Federal Republic of Germany for the Vaccination of Birds in Zoos against Bird Flu”. The campaign is carried out in cooperation with the responsible authorities. This required formal approval of the authorities was subject to multiple conditions, the fulfilment of which required a large logistic effort. Some issues remained unresolved after the campaign had been completed. It is unclear, for instance, whether or not the applied inoculation is sufficient to immunize infected zoo birds and in which time intervals the vaccination has to be repeated in the future. The legal basis for the handing over of inoculated zoo birds to other institutions is published in the Federal Gazette.     

新的猪源性甲型H1N1流感病毒

2009年3月在美国和墨西哥流感样患者的呼吸道标本中鉴定出新的猪源性甲型H1N1流感病毒。该病毒可人一人传播,已蔓延到172个国家和地区。现就猪源性甲型H1N1流感病毒的鉴定、基因组结构特征做一综述。