流感病毒基因的密码子偏好性及聚类分析

流行性感冒病毒是一种造成人类及动物患流行性感冒的RNA病毒,它造成急性上呼吸道感染,并由空气迅速传播,在世界各地常有周期性的大流行。根据该病毒的基因组CDS序列,探讨了基因组序列密码子的使用模式和特性,并进行了病毒间的聚类分析。结果表明:流感病毒的G+C含量均低于A+U含量,偏向使用以A、U结尾的密码子的程度比使用以G、C结尾的较高,CUG、UCA、AGU、AGC、AGA、AGG、GUG、CCA、ACA、GGA、GCA、AUU、UGA、CAU、CAA、AAU、AAA、GAA等18个密码子为流感病毒共有的偏好性密码子,且以A结尾的居多,尤其偏爱AGA、GGA。聚类结果表明首先亚洲流感病毒H2N2和香港流感病毒H2N2聚为一类,亚洲流感病毒H1N1和俄罗斯流感病毒H1N1聚为一类,1997年和2003年~2004年发生的人禽流感聚为一类,说明它们的密码子使用的偏好性相似;而2009年爆发的甲型H1N1流感和任何一个流感的距离都比较远,说明甲型H1N1流感病毒是一种新型的病毒,不同于以往任何一种流感病毒。     

H3亚型鸭源流感病毒聚合酶PB1基因的序列分析

目的阐明H3亚型鸭流感病毒与其他亚型流感病毒的关系。方法对活禽市场分离的3株H3N8亚型鸭源流感病毒聚合酶PB1基因进行了序列分析。结果3株鸭源H3N8流感病毒聚合酶PB1基因核苷酸同源性为99.9%,与H9N2亚型流感病毒(DK/ST/2143/00)的同源性为96.31%~96.44%,而与H3N8亚型鸭流感病毒(Mal/Alberta/279/98)为88.65%~88.79%。系统进化树分析表明,本实验中的3株病毒属于相同的分支,且与A/duck/Hong Kong/Y439为代表的H9N2亚型禽流感病毒位于一进化分支,说明三株H3N8亚型流感病毒重排了H9N2亚型禽流感病毒的基因片段。结论不同亚型禽流感病毒在贮存宿主体内的重排以及重排病毒的新特点如鸭H3N8亚型流感病毒对禽的致病性,应当引起我们的高度重视。     

中东呼吸综合征冠状病毒结构蛋白与附属蛋白编码基因密码子偏爱性分析

中东呼吸综合征冠状病毒是一类对人类危害极重的RNA病毒,为了解其密码子使用特点和主要影响因素,该文采用CHIPS、CUSP、CodonW和SPSS软件对MERS-CoV 9条基因序列进行了偏爱性分析和多元统计分析,获得了每个基因密码子3个位置的GC含量和ENC值以及所有基因序列的RSCU值,并与大肠杆菌、酵母、人的密码子使用频率进行了对比分析。结果显示GC3明显低于GC2、GC1且小于50%,ENC值为50.59,表明该病毒密码子偏爱性偏低且偏爱以A、T碱基结尾的密码子。中性绘图和ENC绘图分析表明密码子的偏爱性主要受到选择作用的影响。通过和其它三种生物密码子使用频率进行比较,发现其与酵母密码子使用频率相差最小。在此基础上确定了MERS-CoV的19个最优密码子。研究结果对MERS-CoV寻找最佳宿主进行体外表达,从而研发基因工程疫苗及治疗性抗体具有一定的意义。     

甲型H1N1／2009流感病毒的种属跨越机制

在世界范围流行的甲型H1N1／2009流感病毒具有下述3个重要特征：可寄生于人体,易感人群很多,患者年龄偏低．本研究确定了病毒蛋白中的一块关键区域．该区域对病毒所寄生的物种的种属范围起决定性作用,并且是全球性流感病毒的一个标志性区域．正是该区域氨基酸的特性导致了上述3个特点．具体来说,对宿主的免疫系统而言,病毒蛋白质结构的变化会形成新的标靶结构,并且可以进一步导致宿主范围的变化．基于多肽链发生致病性结构转换的概率,本研究确定了甲型流感病毒中对控制宿主范围起决定性作用的氨基酸的位置．研究发现甲型H1N1／2009流感病毒中处于这些位点的多肽链在本质上可以在寄生于人的毒株中表达,而之前仅在宿主为禽、猪的毒株中被发现．其与另一氨基酸短串的协同构象改变对于甲型H1N1／2009流感病毒的种属跨越具有重要作用．人体对这些关键位点的免疫缺陷导致了甲型H1N1／2009流感病毒宿主人群多和青年人易致病的特点．     

047鸭子是否在高致病性H5N1流感病毒的亚洲地区性流行中起作用

野生水禽是甲型流感病毒的天然储存宿主，但自2002年来，H5N1对水禽、家禽和野生迁徙鸟类均具致死性，且其有潜在的从鸟类直接传播给人的可能性。2003年末和2005年的禽流感在亚洲10个国家传播，并在泰国、越南、柬埔寨引发53人死亡。所以作者对水禽是否为H5N1流感病毒的储存宿主以及了解H5N1在鸭子体内的特性进行研究。     

A型流感病毒NS1基因密码子去优化改造引起病毒毒力减弱

根据A型流感病毒密码子使用偏嗜性,选取稀有密码子对A/Puerto Rico/8/34(H1N1)病毒NS1基因内部110个氨基酸区域进行密码子同义突变改造,并全基因合成NS基因,利用反向遗传操作技术拯救出含有密码子去优化NS1基因的重组病毒(deoNS)。体外细胞噬斑形成实验和病毒生长曲线证明该病毒在MDCK细胞内的感染和复制能力比野生型病毒低约1000倍;BALB/c小鼠体内致病力实验证明deoNS病毒不能引起小鼠发病和死亡,该病毒在小鼠肺内的复制滴度比野生型病毒低100～1000倍。本研究探索了通过基因组密码子去优化改造途径降低A型流感病毒毒力的可行性,首次证明流感病毒NS1基因密码子去优化同义突变可以降低病毒毒力,为流感减毒活疫苗的研究提供了新的思路。     

甲型H1N1流感病毒HA蛋白抗原表位及受体结合位点突变特性的对比研究

人群中流行的H1N1病毒按其来源可分为两类:人感染的猪H1N1病毒与人类季节性H1N1流感病毒。这两类病毒在流行频率、易感性和致病性等方面存在明显差异。文章收集了1918～2009年间17株人感染的猪甲型H1N1毒株以及21株季节性H1N1毒株,通过序列比对、氨基酸残基保守性分析及3D结构对比等生物信息学方法,揭示造成这两类病毒流行病学和感染性差异的机制。研究发现这两类病毒HA蛋白的进化路径并不相同,且两者具有不同的突变特征,人感染的猪H1N1病毒中,Ca1、Ca2、Sa和Sb四个位点均较为保守,仅Cb位点的突变较快;季节性H1N1病毒仅有Ca1位点较为保守,其他四个抗原性位点均具有较快的突变速率,且较多的突变为新类型的氨基酸。另外,对受体结合位点的研究也显示,这两类病毒的该区域存在5个氨基酸水平的差异(ALA138SER、GLN192LYS、GLN196HIS、ALA198GLU和ALA227GLU),这些位点的差异使得人感染的猪H1N1流感病毒比人类季节性H1N1病毒的易感性更强。这些研究结果可为阐明两类H1N1流感病毒感染性及致病性差异提供更多的信息,并有助于进一步认识H1N1流感病毒的进化机制。     

寨卡病毒基因组密码子偏爱性分析

寨卡病毒是一类对人类危害极重的RNA病毒,为了解其密码子使用特点和主要影响因素,本研究采用CHIPS、CUSP、Codon W和SPSS软件对寨卡病毒所有基因序列进行了偏爱性分析和多元统计分析,获得了每个基因密码子3个位置的GC含量和ENC值以及所有基因序列的RSCU值,并与大肠杆菌、酵母、人的密码子使用频率进行了对比分析。结果显示,寨卡病毒GC3含量相对较高,为54.14%,ENC值为52.54,表明该病毒密码子偏爱性偏低且偏爱以G、C、A碱基结尾的密码子,这和一般病毒的偏爱性有所不同。中性绘图和ENC绘图分析表明密码子的偏爱性主要受到选择作用的影响。通过和其它3种生物密码子使用频率进行比较,发现其与人的密码子使用频率相差最小。在此基础上确定了寨卡的18个最优密码子。研究结果对寨卡病毒寻找最佳宿主进行体外表达,从而研发基因工程疫苗及治疗性抗体具有一定的意义。     

枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响

目前,有关同义密码子使用偏性对蛋白质折叠的影响研究中,样本蛋白均来源于不同的物种。考虑到同义密码子使用偏性的物种差异性,选取枯草杆菌的核蛋白为研究对象。首先,将每条核蛋白按二级结构截取为α螺旋片段、β折叠片段和无规卷曲（α-β混合）片段,并计算其蛋白质折叠速率。然后,整理每个片段相应的核酸序列信息,计算其同义密码子使用度。在此基础上,分析枯草芽孢杆菌核蛋白的同义密码子使用偏性与蛋白质折叠速率的相关性。发现对于不同二级结构的肽链片段,都有部分密码子的使用偏性与其对应的肽链折叠速率显著相关。进一步分析发现,与肽链片段折叠速率显著相关的密码子绝大部分为枯草杆菌全序列或核蛋白序列的每一组同义密码子中使用度最高的密码子。结果表明,在蛋白质的折叠过程中,枯草芽孢杆菌的同义密码子使用偏性起着重要作用。     

SARS冠状病毒的密码子偏爱性分析

为了分析SARS(Severe Acute Respiratory Syndrome)冠状病毒的密码子偏爱性(codon preference),为SARS冠状病毒基因表达中宿主系统的选择提供参考。运用EMBOSS(The European Molecular Biology Open Software Suite)的CHIPS(Codon Hetemzygosity in a Protein-codingSequence)和CUSP(Create a eodon usege table)程序对SARS冠状病毒的6个编码蛋白的基因进行分析,并将这6个编码序列拼接在一起进行全基因组的密码子偏爱性分析。分析结果与大肠杆菌、酵母及人的密码子偏爱性进行比较。结果显示SARS冠状病毒的CHIPS分析Nc(effective number of codons)值为53．338,S、E、M、N蛋白、3CL水解酶、RNA聚合酶的Nc值分别为45．733,61．000,59．040,46．618,46．924,51．902。编码SARS冠状病毒A,P,R,S,T,L等氨基酸的不同密码子使用频率有较大差异。大肠杆菌有25个、酵母有12个、人有20个密码子与SARS冠状病毒密码子使用偏爱性差异较大．因此可以得出结论：编码SARS冠状病毒氨基酸的密码子出现的频率较均一。SARS冠状病毒的密码子偏爱性与真核生物较接近,与原核生物相差较远,其基因表达选择在酵母等真核系统可能更为合适。     

甲型H1N1流感病毒NA、PB2和PA表位抗原区段的克隆和表达

目的:分析比对甲型H1N1流感病毒神经氨酸酶(NA)、聚合酶B2(PB2)和聚合酶A(PA)抗原的序列,克隆表达保守的和变异的表位抗原区段,为免疫学诊断试剂的研究提供候选抗原。方法:采用BioSun生物学软件比较新近公布的A/H1N1流感病毒和我国猪流感病毒浙江株NA、PB2和PA抗原序列,并预测筛选NA、PB2和PA抗原表位保守区段与变异区段,采用PCR逐步合成法合成NA、PB2和PA表位抗原区段基因序列,并利用原核表达载体pBVIL1进行克隆表达。结果:筛选的保守表位抗原区段和变异表位抗原区段为NA/135～180aa、NA/310～350aa、PB2/1～80aa、PA/41～90aa、PA/231～280aa和PA/341～400aa;获得6条表位抗原区段基因序列,且6条表位抗原区段均获得了高效表达,得到纯化后的抗原。结论:获得了A/H1N1流感病毒NA、PB2和PA表位抗原区段,为进一步研制特异的甲型流感病毒快速诊断试剂提供了抗原储备。     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

Analysis of Synonymous Codon Usage Bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended...     

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.     

Analysis of synonymous codon usage bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended, and the intra-genomic codon usage bias of 09H1N1 is quite low. Base composition constraint, dinucleotide biases and translational selection are the main factors influencing the codon usage bias of 09H1N1. At the genome level, we find that the codon usage bias of 09H1N1 is similar to H1N1 (A/swine/Kansas/77778/2007H1N1), H9N2 from Asia, H1N2 from Asia and North America and H3N2 from North America. Our results provide insight for understanding the processes governing evolution, regulation of gene expression, and revealing the evolution of 09H1N1.     

甲型H1N1流感病毒核酸荧光定量RT-PCR检测技术

目的:建立基于TaqMan荧光探针技术的甲型H1N1流感病毒实时定量RT-PCR方法。方法:分析H1N1流感病毒基因特性,根据新发甲型H1N1流感病毒突变基因片段设计检测引物和TaqMan荧光探针,建立荧光定量RT-PCR检测体系,体外转录制备RNA标准品,进行特异性、敏感性、重复性及盲样检测评价实验。结果:可特异性有效检测新发甲型H1N1流感病毒核酸,与H1～H16流感病毒基因无交叉反应;对RNA标准品的检测敏感性达103拷贝/μL;重复性实验中,阳性标准品Ct值变异系数(CV)10%,阴性标准品检测结果均呈阴性;12份盲样检测结果特异性好。结论:该荧光定量RT-PCR方法可作为甲型H1N1流感防控的病原快速诊断技术。     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

新甲型H1N1流感病毒小鼠致死模型的建立

建立新甲型H1N1流感病毒小鼠致死模型,为研究致病性、宿主适应性以及疫苗保护性提供动物模型,并寻找病毒在适应宿主过程中影响毒力和适应性的关键位点。将新甲型H1N1流感病毒A/四川/SWL1/2009 H1N1在小鼠中连续传15代,各代次毒株均在MDCK细胞上增殖后进行测序,根据序列分析结果选择6个传代毒株感染小鼠,连续监测14 d体重和死亡情况;并对第14代和15代病毒在噬斑实验纯化后克隆和测序分析。原代病毒不致死BABL/C小鼠,经动物体内连续传代适应宿主动物后,其毒力增强,具体表现为所选的6个传代毒株中第7、11、15代毒株可以100%致死试验小鼠;分析这6个传代毒株的全基因组表明这些毒株的部分氨基酸位点发生突变。新甲型H1N1流感病毒经小鼠体内连续传代后,建立了小鼠致死模型,病毒毒力增强可能与某些氨基酸位点的改变有关。     

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.