5株新疆出血热病毒分子流行病学研究

目的 从分子水平揭示中国新疆出血热（XHF）病毒基因结构与功能的关系。寻找传播途径及流行原因。方法 对分离自新疆的5株XHF病毒进行S基因片段的克隆和测序，与其他克里米亚-刚果出血热（CCHF）的S基因序列进行比较和分析。结果 5株病毒S基因全长均为1672个核苷酸，开放阅读框均为1449个核苷酸，编码一个含482个氨基酸的蛋白，新疆分离毒株的S基因核苷酸序列同源性（93.0%-99.5%)明显高于其他国家分离毒株的S基因核苷酸序列。系统发生树状图显示新疆毒株在一个分支之下形成独立的群体，明显区别于来自其他地区的CCHF病毒并且又进一步分为三组。结论 不同毒株的S基因序列差异不完全取决于病毒分离的宿主，地域和时间。     

湖北省新分离的二株乙型脑炎病毒全基因组序列特征分析

目的 对2008年湖北省新分离的2株乙型脑炎(简称乙脑)病毒进行全基因组序列测定和分析,了解当地乙脑病毒株的分子生物学特性.方法 病毒复苏鉴定后,使用覆盖乙脑病毒全基因组的16对特异性引物,RT-PCR法扩增乙脑病毒HBZG08-09株和HBZG08-55株的全基因组片段,直接测序拼接获得全基因组序列.使用ClustalX 1.83、MegAlign、Mega 4和Genedoc 3.2等生物学软件进行序列比对、核苷酸和氨基酸同源性分析、系统进化分析及氨基酸位点差异分析.结果 新分离的2株乙脑病毒基因组全长均为10 965个核苷酸,从97位到10 392位为开放阅读框,编码3432个氨基酸,2株间的全基因组核苷酸和氨摹酸间源性分别是98.2%和99.7%.进一步的基因型研究显示HBZG08-09株和HBZG08-55株均属于基因Ⅰ型乙脑病毒.2株病毒与我国近年来在河南及浙江的乙脑病毒分离株进化关系最近.与目前使用的减毒活疫苗株SA-14-14-2相比较,HBZG08-09株全基因组共存在82个氨基酸差异,HBZG08-55株存在84个氨基酸差异.但影响毒力或抗原的关键氨基酸位点未发生改变.结论 新分离的2株乙脑病毒均属于基因Ⅰ型,与疫苗株相比关键氨基酸位点未见变异.     

Analysis on the complete genome of a novel caprine parainfluenza virus 3

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals. One unique caprine PIV3 (CPIV3) strain named JS2013 was isolated in Chinese goat flocks with respiratory diseases in 2013. Now, the complete genome sequence of the strain JS2013 had been determined. A total of 15 overlapping DNA clones, covering the entire genome of the virus, were obtained by primer walking RT-PCR. The sequences of the 3′ and 5′ termini of the viral genome were amplified by 3′ and 5′ RACE. The viral genome was 15,618 nucleotides (nt) in length, which was consisted of six genes in the order 5′-leader-N-P/C/V-M-F-HN-L-tailer-3′. The junction sequences between two genes were highly conserved gene start and stop signal sequences, and trinucleotide intergenic regions (IGR) similar to those of other reported PIV3 strains. Phylogenetic analysis based on the complete genomes of JS2013 with other strains of genus Respirovirus demonstrated that the JS2013 obviously differed from HPIV1, Sendai virus, HPIV3 and other reported BPIV3 genotypes. Further analysis of HN genes of JS2013 along with two more CPIV3 strains isolated later indicated that CPIV3 strains formed a separate cluster. The results presented here suggested that CPIV3 is a new member of the genus Respirovirus.     

中山市6例疑似麻疹疫苗相关病例病原学特征分析

目的 分析中山市6例疑似麻疹疫苗相关病例的病原学特征。 方法 采集中山市2014－2015年6例疑似麻疹疫苗相关病例咽拭子样本并提取核酸,采用巢式PCR法扩增麻疹病毒核蛋白(N)基因羧基末端450个核苷酸片段并测序,分析其与世界卫生组织(WHO)参考株、麻疹病毒中国疫苗株沪₁₉₁(Shanghai₁₉₁,S₁₉₁)基因的亲缘关系、核苷酸与氨基酸序列相似度。 结果 6份样本中,2份未检出核酸,4份检出核酸,序列比对结果示3株病毒核苷酸、氨基酸序列相似度均为100%,与S₁₉₁株相比,核苷酸序列和氨基酸序列相似度也为100%,属于A基因型;1株病毒与Chin9322-H_1a株相比,核苷酸序列相似度为97.7%,氨基酸序列相似度为96.6%,为H_1a基因型。 结论 3例病例为疫苗株感染,1例为野毒株感染。     

Characterization of a new genotype of measles virus detected in China and England

We report the co-circulation of a new lineage of measles virus (MV) and an Edmonston-like (Ed-like) genotype of MV in China during 1995-7. Sequence analysis of 25 strains was performed on a 282 nucleotides (nt) region of the nucleoprotein (N) gene, a 450-nt region of the haemagglutinin (H) gene and a 152-nt region of the matrix (M) gene by direct sequencing of RT-PCR amplicons obtained from clinical specimens. The entire H gene was sequenced from two strains. The results showed that 24/25 Chinese strains belonged to a new genogroup and were distinct from the vaccine strains used in China and the UK, and also from MV strains previously described in Europe, Africa and the USA. The remaining strain was Ed-like. Two strains of the new genotype (IV) and one of the Ed-like genotype were also detected in the UK in 1996.     

济南市发热伴血小板减少综合征布尼亚病毒基因组特征分析

目的 分析济南市发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV)基因组特征。方法 通过参考文献并对引物进行矫正,获得SFTSV全基因组序列。应用DNAstar 7.1、MEGA等软件对基因组序列进行基因位点、遗传进化、同源比对分析,建立L、M、S片段基因系统进化树。结果 成功获得7株济南市SFTSV全基因组序列,每株分离株基因组全长均为11 490 bp,其中L片段6 368 bp, M片段3 378 bp, S片段1 744 bp。济南市SFTSV属C基因型C3分支。与湖北、河南及山东省泰安和青岛市基因序列同源性高,呈现出明显地域性特点。核苷酸变异以碱基转(颠)换为主,2018年SFTSV分离株共存在0.03%氨基酸突变位点,2019年存在0.02%氨基酸突变位点,2020年存在0.11%氨基酸突变,2020年较2018、2019年明显增多。结论 利用测序方法测定的济南市SFTSV,其基因组序列分析表明与我国流行的SFTSV相近,氨基酸序列突变有增多趋势,需密切关注其致病力、...     

A novel feline norovirus in diarrheic cats

By screening a collection of fecal samples from young cats housed in three different shelters in South Italy, noroviruses (NoVs) were found in 3/48 (6.2%) specimens of animals with enteritis signs while they were not detected in samples collected from healthy cats (0/57). Upon sequence analysis of the short RNA-dependent RNA polymerase (RdRp) region, the three strains displayed the highest nucleotide (nt) and amino acid (aa) identities to the prototype GIV.2 strain lion/Pistoia/387/06/ITA (91.0–93.0% nt and 97.0–98.0% aa). The sequence of ~ 3.4-kb portion at the 3′ end of the genome of a NoV strain, TE/77-13/ITA, was determined. In the full-length ORF2, encoding the VP1 capsid protein, the virus was genetically closest to the canine GVI.2 NoV strains C33/Viseu/2007/PRT and FD53/2007/ITA (81.0–84.0% nt and 93.0–94.0% aa identities), suggesting a recombination nature, with the cross-over site being mapped to the ORF1-ORF2 junction. Based on the full-length VP1 amino acid sequence, we classified the novel feline NoV, together with the canine strains Viseu and FD53, as a genotype 2, within the genogroup GVI. These findings indicate that, as observed for GIV NoV, GVI strains may infect both the canine and feline host. Unrestricted circulation of NoV strains in small carnivores may provide the basis for quick genetic diversification of these viruses by recombination. Interspecies circulation of NoVs in pets must also be considered when facing outbreaks of enteric diseases in these animals.     

浙江省部分地区2009--2010年蚊媒中乙型脑炎病毒分子流行病学特征研究

目的 了解浙江省部分地区蚊媒携带流行性乙型脑炎(乙脑)病毒(JEV)情况及其分子流行病学特征.方法 2009-2010年7-8月从浙江省慈溪市和仙居县乙脑监测点采集蚊13620只,利用荧光定量PCR方法检测蚊中JEV核酸,用BHK-21细胞分离病毒,扩增JEV分离株E基因,然后选择6株进行测序并进行同源性分析.结果 慈溪和仙居监测点蚊中JEV带病毒率分别为17.0％(27/159)和3.4％( 1/29);从2010年143批蚊中分离到22株JEV,分离率为15.4％.6株测序株E基因全长均为1500个核苷酸(nt),推导编码500个氨基酸(aa),未发现nt插入和丢失,nt和aa同源性分别为99.2％～ 99.8％和100.0％,与浙江省2007-2008年JEV分离株的nt和aa同源性分别为99.1％～99.3％和99.2％～ 99.8％,与乙脑疫苗株SA14-14-2同源性分别为87.6％～88.0％和97.8％.E基因进化树显示,6株测序株位于基因Ⅰ型分支上.结论 浙江省慈溪市和仙居县监测点蚊媒携带JEV,其中慈溪监测点蚊媒中乙脑带病毒率明显高于仙居监测点,JEV分离株均为基因Ⅰ型.     

河南省两株狂犬病毒核蛋白和糖蛋白基因的序列分析

目的分析河南省2株狂犬病毒核蛋白及糖蛋白的基因序列,了解狂犬病毒街毒株与人用及兽用狂犬病疫苗株间的差异。方法以免疫荧光法检测2004年采自河南省的100只犬脑标本,取阳性犬脑组织悬液接种鼠脑分离病毒。以RT-PCR法扩增病毒核蛋白及糖蛋白全基因,克隆测序后进行遗传学分析。结果分离到2株狂犬病毒,分别命名为Henan Hb1与Henan Sq1,序列分析表明2株病毒均为基因1型狂犬病毒,2株病毒的N基因与G基因核苷酸的同源性分别为99.3%和98.9%,氨基酸的同源性分别为98.7%和98.4%。2株病毒与CTN疫苗株同源性较高,N基因与G基因的核苷酸同源性分别为89.1%和85.6%～85.7%,氨基酸同源性分别为97.6%～98.0%和92.3%。与已知的基因1型狂犬病毒相比,2株病毒与印度尼西亚从犬中分离到的2株病毒同源性最高,N基因与G基因核苷酸同源性分别为92.1%～93.2%和91.9%～92.1%,氨基酸同源性分别为97.5%～98.6%和96.0%～96.2%。Henan Sq1株病毒在糖蛋白关键的毒力位点333位由W替换了R。系统发育分析表明Henan Hb1与Henan Sq1与印度尼西亚株、疫苗株CTN、中国分离株,以及泰国与马来西亚分离株进化关系最近,而与疫苗株3aG、PV、ERA及攻击毒CVS株等进化关系较远。结论2株狂犬病毒是基因1型狂犬病毒,但无论是N基因还是G基因的核苷酸序列,以及推导出来的氨基酸序列与已知的1型狂犬病毒株及疫苗株均有一定的差异。     

Emergence of multireassortant bluetongue virus serotype 4 in Hungary

The genome sequence and the phylogenetic relationships of a serotype 4 bluetongue virus (BTV-4) emerged during 2014 in Hungary are described in this study. Genome segment 2 encoding the major neutralization antigen, VP2, shared moderate sequence similarity (nt, ⩽94.3%) with the corresponding gene of contemporary and historic homotypic bluetongue viruses, whereas genome segments S1, S4, S5, S7–S10 were typically more closely related to the cognate genes of heterotypic isolates. Importantly, in many gene phylogenies the Hungarian BTV-4 strain showed genetic relationship to BTV strains identified in outbreaks in the western Mediterranean basin. Our results indicate the identified Hungarian bluetongue virus strain evolved through reassortment involving multiple genome segments from various heterotypic bluetongue viruses.     

Dating the divergence of the infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an economically significant rhabdovirus that can cause severe disease to most salmonid fish. Phylogenetic analyses of worldwide IHNV isolates have defined five major genogroups. Herein, to further the understanding of the molecular epidemiology and evolution of IHNV, Bayesian coalescent analyses were conducted to the time-stamped coding sequences of the N, G and NV genes. The nucleotide substitution rates and the divergence times were assessed. Among the three genes, NV, the smallest one coding for a non-virion protein, was conferred the highest mean rate. Estimates for the G subsets based upon the five genogroups indicated that L and U evolved much slower than the others. Age calculations suggested that the first diversification event of the IHNV isolates analyzed might have happened before the notification of the disease during the early 1950s. Selection analyses suggested that the three genes were mainly subject to purifying selection. In addition, surveys of codon usage variation showed that the three genes had influences other than mutational bias.     

Identification of a novel Aichivirus D in sheep

A novel kobuvirus was found in diarrheal fecal samples of Tibetan sheep using a viral metagenomics approach, and a full kobuvirus genome was successfully obtained by RT-PCR from a diarrheal fecal sample. The full genomic sequence was 8485 nucleotides (nt) in length with a standard picornavirus genome organization. The novel genome shares 62.9% and 77.8% nt homology with Aichivirus D1 genotype strain 1-22-KoV, and Aichivirus D2 genotype strain 2-44-KoV, respectively. According to the species classification criteria of the International Committee on Taxonomy of Viruses (ICTV), the new kobuvirus belongs to Aichivirus species D. Interestingly, compared with 2 known Aichivirus D genotype strains, the novel Aichivirus D has unique amino acid substitutions in the 5'untranslated region (-UTR), VP0, VP3, and VP1, with a recombination event in the 2C region.These characteristics make the novel Aichivirus D cluster into an independent branch in the phylogenetic tree, suggesting that strain may represent a novel genotype in Aichivirus D. Moreover, the novel Aichivirus D was detected in 9.2% (18/195) of the sheep diarrheal fecal samples from 4 farms in 3 counties of the Qinghai Tibet Plateau in China. In addition, full-length VP0, VP3, and VP1 genes were successfully obtained from 12 samples from 4 farms, and phylogenetic analysis based on these genes revealed a unique evolutionary pattern for this novel Aichivirus D strain.This study identified a novel Aichivirus D that is circulating in sheep in Qinghai Tibet Plateau in China and these findings provide a better understanding of the epidemiologic and genetic evolution of kobuviruses.     

吉林省2001～2008年麻疹病毒血凝素蛋白基因特征和氨基酸变异分析

目的研究吉林省2001～2008年麻疹病毒代表株的血凝素蛋白基因（Hemagglutinin,HA）特征和氨基酸变异。方法从吉林省2001～2008年流行的麻疹野病毒中选取5株代表株,提取核糖核酸,用逆转录-聚合酶链反应扩增HA基因片段,对扩增产物进行核苷酸序列分析,并与基因库中的中国麻疹疫苗病毒株沪191以及所有23种麻疹野病毒基因型代表株的H基因序列,进行基因亲缘关系、同源性、氨基酸变异以及糖基化位点变异比较。结果吉林省5株麻疹病毒分离株均属于H1基因型H1a基因亚型,5株病毒H基因之间有6～17个核苷酸差异（0.3%～0.9%）,3～9个氨基酸差异（0.5%～1.5%）。与中国现行疫苗病毒株沪191H基因相比,有89～95个核苷酸差异（4.8%～5.1%）,25～30个氨基酸差异（4.1%～4.8%）。与H1a基因亚型代表株China93-4H基因相比,有9～17个核苷酸差异（0.5%～0.9%）,3～7个氨基酸差异（0.5%～1.2%）。吉林省5株麻疹病毒分离株的H蛋白均保留了4个糖基化位点,第5个糖基化位点在氨基酸第240位由丝氨酸突变成天冬酰胺,导致一个潜在糖基化位点NLS238～240的缺失。结论吉林省2001～2008年流行的5株麻疹病毒,均丢失了一个可能影响抗原性的糖基化位点。5株麻疹病毒的核苷酸、氨基酸差异均不大,但无论与H1a基因亚型代表株China93-4相比,还是与疫苗株S191相比,核苷酸和氨基酸的同源性均呈逐年递减趋势,吉林省麻疹野病毒H基因的变异仍在逐年增加、逐年积累。     

浙江省2005-2010年风疹病毒分子流行病学研究

目的 分析2005-2010年浙江省风疹病毒流行株的病原学与分子流行病学特征。方法采集该期间风疹疫情中的疑似患者标本,于Vero细胞上分离病毒。采用巢式RT-PCR扩增流行株E1、E2和C三个结构基因片段,对扩增产物进行序列测定与分析。结果从52例临床标本中共分离到风疹病毒流行株7株,除Rvi/Zhejiang.CHN/23.08株为2B基因型外,其余6株均属于IE基因型。基因进化树上,IE基因型流行株分别与香港、海南地区流行株具有最近亲缘关系,而2B基因型流行株与BuenosAires.A RG/46.08位于同一分支。遗传距离分析表明,浙江2B型流行株与全球其他地区流行株间的遗传距离(0.011)小于与中国流行株(0.023)间的遗传距离。浙江风疹流行株与中国风疹疫苗株BRDⅡ在核苷酸水平上的同源性为C＞E2＞E1,而氨基酸水平上的同源性为E1 ＞C＞E2,对应存在3、11和23个氨基酸的变异。各位点氨基酸熵值表明,E1蛋白上仅存在一个易变位点（熵值＞0.600）,而E2蛋白上有7个易变异位点和1个高变异位点(熵值＞1.000)。结论2005-2010年浙江省存在两种基因型风疹病毒流行,IE为优势基因型,2B可能为境外输入株。流行株E1基因氨基酸序列相对保守,而E2基因氨基酸变异较大,在风疹病毒流行病学监测中除关注E1基因外,应开展对结构基因E2和C的研究。     

3株TT病毒的基因组结构特点及其在肝炎病人中感染情况

目的：了解TT病毒(TTV)分离株基因组结构特点及其在各型病毒性肝炎患中感染情况。方法：对49株TTV相关病毒全基因组结构进行分子进化树分析，比较从我国分离的3株TTV全基因结构与原型株的同源性，并采用巢式聚合酶链反应法(nPCR)对各型病毒性肝炎患血清中TTV DNA进行检测。结果：3株TTV分离株基因组全长为3739bp，有2个读码框架（ORFs），ORF1编码770个氨基酸，位于589-2901核苷酸；ORF2编码202个氨基酸，位于107-715核苷酸。与TTV原型株TA278的核苷酸同源性为86％-96％，ORF1氨基酸同源性为88％-97％，ORF2氨基酸同源性为79％-96％。全基因组序列分子进化树分析表明，从我国分离的3株TTV属于原型组。在180例各型病毒性肝炎患中，检出TTV DNA阳性40例，阳性率为22.2％。在甲-戊型肝炎、非甲、戊型肝炎、慢性乙型肝炎患和正常人中，TTV DNA流行率分别为24.2％（29/120），18.3％（11/60），17.8％（106/595）和19.8％（19/96），4组无显性差异。结论：从我国分离的3株TTV的全基因结构特点与原型株相似，各型病毒性肝炎、慢性乙型肝炎和正常人群TTV DNA流行率相对较高。     

1998-2009年浙江省H3N2亚型流行性感冒病毒全序列变异的研究

目的 比较1998-2009年浙江省H3N2亚型流行性感冒(简称流感)流行株HA基因进化与全基因进化状况的一致性,并探讨全基因组序列上可能存在的潜在抗原区域.方法 选择浙江省CDC流感实验室于1998-2009年流感疫情中分离保存的H3N2亚型流感流行代表株19株,采用RT-PCR法进行全基因组序列扩增,并将这些毒株与10株同期H3N2亚型流感疫苗株进行全序列及HA1基因的系统进化比较;通过氨基酸替换比较、熵值计算及正向选择位点的筛选,确定各基因上的氨基酸易变位点.结果 H3N2亚型流感病毒的全序列长度为4466个氨基酸,其中有137个氨基酸位点发生稳定变异.在HA基因上第144和158位氨基酸分别经历了4次和3次替换,NA基因的第93、143、307、370、372位氨基酸和NP基因的第450位氨基酸经历了2次替换,且HA基因和NA基因上分别有29%(12/41)和77%(24/31)的变异位点位于已知抗原决定簇之外的区域.氨基酸位点熵值分析显示,HA基因非抗原决定簇的3、225、361位,NA基因非抗原决定簇的93、143、147、150、372位,PB1基因的113、576、586位,PA基因的101、256、382、421、437位,NP基因的377、450位,M1基因的218位及M2基因的31位氨基酸均为易变位点.结论 浙江省1998-2009年H3N2亚型流感病毒在HA和NA已知抗原决定簇之外的区域与部分内部基因上,可能存在着一些尚未被发现或者新形成的抗原位点.与HA基因的系统进化比较,全序列能更为全面地反映出流感流行株之间的亲缘关系与进化规律.

Abstract：

Objective To analyze the consistency of evolution condition between HA gene and the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province from 1998 to 2009,and to study the potential antigenic region on the whole genome.Methods The sequences of whole genome of 19 Zhejiang influenza virus isolates circulated from 1998 to 2009,which conserved by influenza laboratory of Zhejiang Provincial Centre for Disease Prevention and Control,were amplified using RT-PCR assays.The obtained sequences were used to conduct phylogenetic analysis with 10 contemporaneous vaccine strains.Three methods,including comparison of the amino acid substitutions,calculation of the entropy value and the filtering of positive selection sites,were used to confirm the mutable sites on each gene.Results The whole genome of influenza virus subtype A/H3N2 was 4466 amino acids in length,with 137 stable mutations.The 144,158 aa of HA gene mutate four and three times respectively; 93,143,307,370,372 aa of NA gene and 450 aa of NP gene mutate twice,and there were 29%(12/41) and 77%(24/31) mutations of HA and NA genes occurred on the non-epitope regions respectively.Analysis of the entropy value suggest that many amino acid sites on the non-epitope regions were prone to mutation,including 3,225,361 aa of HA gene; 93,143,147,150,372 aa of NA gene; 113,576,586 aa of PB1 gene; 101,256,382,421,437 aa of PA; 377,450 aa of NP gene; 218 aa of M1 gene and 31 aa of M2 gene.Conclusion Based on the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province in 1998 to 2009,there may be several unknown or new antigen sites existing on the non-epitope regions of HA and NA genes and parts of internal genes.The phylogenetic tree constructed based on the complete sequence was more comprehensive than on the HA gene to reflect the genetic relationship and law of evolution among the influenza virus strains.     

河北省2株汉城病毒的遗传特征分析

目的 对河北省2株汉城病毒(SEOV)进行基因分型及系统进化分析.方法 用RT-PCR法扩增SEOV Li株和LF18株的S与M全基因片段;利用DNAStar软件分析S与M基因片段核苷酸序列同源性;利用Phylip软件构建系统进化树.结果 Li株与LFl8株的全S基因片段均由1772个核苷酸组成,开放阅读框(ORF)的位置均为43～1332核苷酸,编码429个氨基酸的核蛋白.2株病毒的M片段全基因序列均由3653个核苷酸组成,ORF的位置均为49～3450核苷酸,编码1133个氨基酸的糖蛋白前体.2株病毒的糖蛋白前体有62个半胱氨酸残基以及6个糖基化位点.2株病毒的全S、M基因片段的核苷酸序列与包括疫苗株L99、Gou3、Z37在内的已知SEOV的同源性分别为87.6%～99.2%和83.6%～97.3%.全S和全M基因片段构建的系统进化树显示2株病毒均为SEOV第Ⅲ亚型.结论 Li株和LF18株都属于SEOV,为SEOV的第Ⅲ亚型.河北省汉坦病毒的流行毒株的遗传特征与疫苗株Z37相似.