5株呼吸道合胞病毒地方株F蛋白基因序列分析

目的呼吸道合胞病毒（ＲＳＶ）Ｆ蛋白是ＲＳＶ感染免疫中最重要的病毒蛋白，为了解我国ＲＳＶ地方株Ｆ蛋白的基因状况和变异特征，随机选取北京、广州、长春和河北四个地区具有不同流行特征的ＲＳＶ地方株（Ａ亚型）５株，进行ＲＳＶＦ蛋白全基因的核苷酸序列分析。方法以提取的病毒ｍＲＮＡ为模板进行ＲＴ－ＰＣＲ扩增、目的基因的克隆及序列测定，对地方株及原型株的序列进行比较分析。结果地方株Ｆ蛋白基因与原型株Ａ２株有很高的同源性，核苷酸全序列的同源性为９５．１％～９６．１％，氨基酸同源性为９６．７％～９７．４％。核苷酸有义突变率为２２．６％～２５．９％。３非编码区的核苷酸序列比蛋白编码区变异显著。河北地方株（Ｅ７３株）在３非编码区有６个核苷酸的插入。Ｆ２亚单位的氨基酸变异高于Ｆ１亚单位。在北京地方株（ＺＨＳ１３株）Ｆ１亚单位内，由具有中和能力单克隆抗体所识别的抗原表位区中存在一个氨基酸的变异。结论我国ＲＳＶ地方株与原型株之间的Ｆ蛋白基因尽管存在一定的变异，但仍有很高的同源性。地方株间Ｆ蛋白的核苷酸、氨基酸变异的位置及形式很相似，提示我国ＲＳＶ的不同流行特征可能并非由于Ｆ蛋白的基因变异所致。     

呼吸道合胞病毒G蛋白胞外区基因克隆及序列分析

目的 对呼吸道合胞病毒Ｇ蛋白胞外区基因进行克隆及序列分析。方法 从ＲＳＶ感染的喘息型支气管炎患儿咽试子分离的病毒培养物中提取病毒ＲＮＡ,进行逆转录及ＰＣＲ扩增,克隆到Ｇ蛋白胞外区基因ＤＮＡ序列分析。结果 克隆片段全长６９０ｂｐ,编码一个含有２２９个氨基酸的多肽。与５个ＧｅｎＢａｎｋ中不同的ＲＳＶ分离株（包括Ａ,Ｂ亚型）的Ｇ蛋白胞外区的氨基酸序列相比,Ａ亚型的ＲＳＶ毒株氨基酸水平的同源率为９５．２％     

丙型肝炎病毒1b型NS5 A区基因结构变异与α干扰？…

目的 观察丙型肝炎患者丙型肝炎病毒（ＨＣＶ）１ｂ型基因组部分ＮＳ５ Ａ区核苷酸,氨基酸的变异情况并探讨其与α干扰素疗效的相关性。方法 患者干扰素治疗前,中,后留血标本,用聚合酶链反应（ＰＣＲ）扩增ＨＣＶ病毒ＮＳ５ Ａ区部分基因片段并用直接测序法测序。与ＨＣＶ－Ｊ株及ＨＣＶ－河北株（ＨＣＶ－ＨＢ）比较核苷酸及氨基酸序列的同源性,根据α干扰素疗效分析ＨＣＶ１ｂ是否存在干扰素敏感决定区。     

呼吸道合胞病毒的分型研究

目的 了解北京地区１９９０￣１９９１年和１９９７￣１９９８年两个非连续的流行年中呼吸道合胞病毒（ＲＳＶ）Ａ、Ｂ亚型和基因型的流行情况。方法 用间接免疫荧光法（ＩＩＦ）检测ＲＳＶ阳性鼻咽分泌物（ＮＰＳ）标本或ＲＳＶ分离株,划分Ａ、Ｂ亚型。根据Ｎ基因片段的限制性酶切图型将ＲＳＶ分离株分成至少６个基因型ＮＰＩ－６ＮＰＩ,３和６属于Ｂ亚型,ＮＰ２４和５属于Ａ亚型。根据ＳＨ基因片段的核苷酸序列将Ａ亚型分离株     

甲3(H3N2)亚型流感病毒相变异分子生物学基础的研究

目的弄清甲３（Ｈ３Ｎ２）亚型流感病毒相变异的分子生物学基础。方法病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ）扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果３５株甲３（Ｈ３Ｎ２）亚型流感病毒的ＨＡ１区基因长度均为９８４个核苷酸,它们间无发生任何核苷酸丢失或插入并发现ＨＡ１蛋白分子上氨基酸序列多变点主要在ＨＡ蛋白的顶部,尤其抗原决定簇Ｂ区和受体结合部位（ＲＢＳ）,这进一步证实了,ＨＡ蛋白分子上氨基酸替换主要是人群免疫压力所造成。同时还发现了半胱氨酸和脯氨酸具有高保守性及糖基化位点主要集中在ＨＡ１区的Ｎ和Ｃ端,尤其Ｎ端。糖基化位点如此分布在病毒基因进化和流行病学上意义至今不清楚。结论Ｈ３Ｎ２亚型病毒“Ｏ”相毒株的出现与其蛋白分子上第２２６位氨基酸发生替换密切相关并推测“Ｏ”相毒株ＨＡ蛋白三维结构与“Ｄ”相的不同。     

痘苗病毒天坛株基因组病毒生长因子基因结构的研究

克隆了位于我国痘苗病毒天坛株基因组左端的病毒生长因子（ＶＧＦ）基因并对其编码多肽的结构特点进行了分析。结果表明，由天坛株ＶＧＦ基因所推导的氨基酸序列具有典型的上皮生长因子（ＥＧＦ）超家族成员的特征序列和结构特点，在核苷酸及氨基酸水平上天坛株ＶＧＦ与其他正痘病毒ＶＧＦ的同源性在８５％～９５％之间，氨基酸残基的缺失和插入等变异主要集中出现在多肽的信号肽和穿膜区，推测对功能的影响不大。此外，我们发现由天坛株基因组右末端的开放读码框架（ＯＲＦ）ＴＢ２２Ｌ推导的氨基酸序列与天坛株ＶＧＦ多肽第６７位至１４０位完全相同，提示ＯＲＦＴＢ２２Ｌ可能是天坛株基因组第二个ＶＧＦ基因的变异产物。     

传染性法氏囊病病毒HZ96VP2cDNA的结构分析及在大肠杆菌中的表达

以随机引物反转录传染性法氏囊病病毒（ＩＢＤＶ） 杭州分离株ＨＺ９６ 基因组合成第１ 链ｃＤＮＡ。以第１ 链ｃＤＮＡ为模板,ＰＣＲ 扩增ＨＺ９６ ＶＰ２ ｃＤＮＡ ;Ｓａｎｇｅｒ 双脱氧法测序ＨＺ９６ ＶＰ２ ｃＤＮＡ, 并对其基因结构氨基酸序列进行计算分析; 构建非融合表达质粒, 在大肠杆菌中表达ＶＰ２ 蛋白。结果表明, 克隆的ＨＺ９６ＶＰ２ ｃＤＮＡ 全长为１４３１ 个核苷酸对（ｂｐ） , 含起始密码子ＡＴＧ 和终止密码子ＴＡＡ, 编码ＶＰ２ 第２０ 至第４９５ 共４７６ 个氨基酸; ＨＺ９６ ＶＰ２ 在核苷酸和氨基酸水平上, 与其它Ｉ 型ＩＢＤＶ ＶＰ２ 的同性均在９０ ％ 以上; 对ＨＺ９６ 分离株与强毒株ＶＰ２ ｃＤＮＡ 编码的氨基酸序列进行二级结构和亲水性分析发现,ＨＺ９６ 分离株ＶＰ２ 第２７９ 和２８４ 位氨基酸的变异,可导致附近区域（２７４ ～２９０ 位氨基酸） 的亲水性降低和α－ 螺旋的消失。ＨＺ９６ ＶＰ２ 在大肠杆菌中的表达量为８ ％ 。     

中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析

目的 分析中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点.方法 收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构-非结构蛋白VP3-VP1-2A区序列,进行序列同源性比较并分析其基因特点.结果 42株HAV病毒株在VP1-2A连接处核苷酸和氨基酸序列同源性分别为89.1%～100%和97.3%～100%;在全长结构蛋白VP3-VP1区的核苷酸和氨基酸序列同源性分别为87.6%～100%和98.8%～100%.VP1-2A连接处序列相同的病毒株在全长结构蛋白VP3-VP1区的核苷酸同源性为98.4%～100%,0～2个氨基酸位点不同.本实验所得序列在中和抗原位点处氨基酸序列均未变异.结论 42株病毒株均属于I型,40株是IA亚型,2株IB亚型.本实验所用HAV流行株在结构蛋白VP3-VP1区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异.VP1-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VP1区核苷酸序列相同或相近,氨基酸序列保守.     

鹅流感病毒2/96-H5N1亚型毒株RNA1-3及RNA5节段核苷酸全序测定

目的　明确广东鹅流感病毒２９６Ｈ５ Ｎ１ 亚型毒株ＲＮＡ１３ 和ＲＮＡ５ 节段核苷酸全序列及其所编码蛋白的氨基酸序列,以及这些基因节段与香港禽流感病毒１５６９７Ｈ５ Ｎ１ 亚型毒株相应节段间的关系。方法　病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ） 扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　广东鹅流感病毒２９６Ｈ５ Ｎ１ 亚型毒株ＲＮＡ１３ 和ＲＮＡ５ 节段长度分别为２３４１,２ ３４１ ,２ ２３３ 和１５６５ 个核苷酸。它们分别编码ＰＢ２（ 含７５９ 个氨基酸）,ＰＢ１（ 含７５７个氨基酸） ,ＰＡ（ 含７１６ 个氨基酸） 和ＮＰ蛋白（ 含４９８ 个核苷酸） 。这些蛋白与香港禽流感病毒１５６９７Ｈ５ Ｎ１ 亚型毒株相应蛋白氨基酸序列的同源性分别为９６－４％ ,９７－２％ ,９７－３ ％ 和９７－０％ 。结论　本毒株ＲＮＡ１３ 和ＲＮＡ５ 节段长度分别为２ ３４１,２ ３４１,２ ２３３ 和１ ５６５ 个核苷酸,它们与香港１５６９７Ｈ５ Ｎ１ 亚型毒株间存在着差异     

中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析

目的 分析中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点.方法 收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构-非结构蛋白VP3-VP1-2A区序列,进行序列同源性比较并分析其基因特点.结果 42株HAV病毒株在VP1-2A连接处核苷酸和氨基酸序列同源性分别为89.1%～100%和97.3%～100%;在全长结构蛋白VP3-VP1区的核苷酸和氨基酸序列同源性分别为87.6%～100%和98.8%～100%.VP1-2A连接处序列相同的病毒株在全长结构蛋白VP3-VP1区的核苷酸同源性为98.4%～100%,0～2个氨基酸位点不同.本实验所得序列在中和抗原位点处氨基酸序列均未变异.结论 42株病毒株均属于I型,40株是IA亚型,2株IB亚型.本实验所用HAV流行株在结构蛋白VP3-VP1区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异.VP1-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VP1区核苷酸序列相同或相近,氨基酸序列保守.     

重庆地区急性呼吸道感染患儿呼吸道合胞病毒流行特征及其G基因序列分析

目的 分析重庆地区2008-2009年度急性呼吸道感染住院患儿呼吸道合胞病毒(respiratory syncytial virus,RSV)的亚型流行情况,并了解优势流行株BA株的G蛋白基因特征.方法 采集2008年4月-2009年3月全年于重庆医科大学附属儿童医院因急性呼吸道感染住院的508例患儿鼻咽深部分泌物,用RT-PCR方法检测RSV并进行亚型鉴定,选取29例B亚型和10例A亚型RSV阳性标本,用RT-PCR的方法扩增全长G蛋白并测序.结果 在508例标本中,RSV阳性126例(24.8%),其中检测出A亚型43例(34.1%),B亚型80例(63.5%),A、B亚型混合感染3例(2.4%).所测的10株A亚型的G基因与标准株A2的核苷酸同源性为91.4%～92.0%,均属GA2基因型;29株B亚型的G基因与标准株CH18537的核苷酸同源性为92.0%～93.0%,其中19株均为具有60个高度重复核苷酸插入的BA株.B亚型流行株与CH18537标准株相比,G基因有多种核苷酸变异如缺失、插入等,尤其在G蛋白近C端1/3处的高变区.结论 2008-2009年RSV仍是重庆地区儿童急性呼吸道感染的主要病原,与既往两年A亚型优势流行不同,2008-2009年度B亚型毒株流行占优;近年新发现的BA株可能已成为本地区优势流行株,BA株G基因变异是否导致G蛋白功能增强,进而促进其优势流行尚有待研究.     

Inhibition of respiratory syncytial virus of subgroups A and B using deoxyribozyme DZ1133 in mice

Respiratory syncytial virus (RSV) commonly infects the upper and lower respiratory tracts. Currently, there is no effective treatment available. Deoxyribozymes are a potential therapeutic for RSV and their activity is based on the ability to bind and cleave complementary RNA sequences to inhibit protein expression. DZ1133 is a deoxyribozyme that targets the conserved genomic RNA sequence of the RSV nucleocapsid protein and has been shown to significantly inhibit various strains of RSV including subgroups A and B, standard A2 and CH18537 strains, and CQ381513, CQ381170, BJ01 and BJ04 strains. Treatment with DZ1133 decreased viral plaque formation in lungs of RSV-infected BALB/c mice. In addition, viral mRNA expression was reduced, airway inflammation was alleviated, and leukocyte counts were reduced in bronchoalveolar lavage fluid of RSV-infected mice. The antiviral effect of DZ1133 was dose-dependent (0.2–0.8 mg) and more efficient than antisense oligonucleotide inhibition of gene expression. However, levels of cytokines TNF-, IFN-γ, IL-12, and IL-10 induced by RSV infection were not affected by DZ1133 treatment. Our data demonstrate that DZ1133 is a potential therapeutic agent against both subgroups A and B RSV infection in vivo.     

Infectious bronchitis virus: S1 gene characteristics of vaccines used in China and efficacy of vaccination against heterologous strains from China.

The entire S1 protein genes of eight infectious bronchitis (IB) vaccine strains used in China were compared with those of the IB virus isolates present in the field in China. The nucleotide and amino acid similarities between the eight IB vaccine strains and the field strain, tl/CH/LDT3/03, which was isolated from a teal (Anas sp.), were not more than 81.1% and 79.2%, respectively. Phylogenetic analysis based on the S1 genes showed that the vaccines and field strains belonged to different clusters and showed larger evolutionary distances, and indicated that they were of different genotypes. Four out of the eight vaccines, in addition to the Massachusetts type vaccine H120, were used for protection tests against challenge by the IB virus isolate tl/CH/LDT3/03. This revealed that each of the five IB vaccines induced poor protection against the teal isolate, as assessed by respiratory protection, clinical signs and mortality, indicating the necessity of developing vaccines from local strains for IB control in China.     

Phylogenetic analysis of the S1 glycoprotein gene of infectious bronchitis viruses isolated in China during 2009–2010

As part of our ongoing surveillance program, 40 field strains of avian infectious bronchitis virus (IBV) were isolated from dead or diseased chicken flocks in different areas of China between 2009 and 2010. S1 glycoprotein genes of these strains were sequenced and analyzed with 38 strains published in GenBank. S1 genes of these isolated strains and the vaccine strains showed nucleotide homologies ranging from 65.2 to 82% and amino acid homologies ranging from 58.4 to 81.9%. Meanwhile, Chinese IBV strains isolated in this study, which were mainly nephropathogenic, could be separated into six variant lineages (CH I–CH VI), and current vaccine strains used in China formed Mass variant lineage that is evolutionarily distant from Chinese isolates. Moreover, CK/CH/GD/NC10, CK/CH/GD/KP10, and our previous isolates TC07-2 formed the CH VI lineage, showing larger evolutionary distances from other strains. Taken together, these findings suggested that various variant lineages were co-circulating in China now, and appeared to be continuously evolving, alternative indigenous vaccines indeed need for effective control of IB in China.     

Molecular epidemiology of subgroup C avian pneumoviruses isolated in the United States and comparison with subgroup a and B viruses

The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.     

中国分离乙脑病毒与灭活疫苗株(P3株)E基因差异分析

目的 分析我国近年来从蚊虫及患者标本中分离的乙脑病毒与灭活疫苗株（P3株）之间在E基因区段核苷酸及氨基酸差异。方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列，通过Clustal X（1．8）、DNASTAR、GENEDOC（3．2）等生物学软件进行分析。结果 P3株与福建分离株之间核苷酸同源性在98．3％-98．5％之间、氨基酸同源性在98．2％-98．6％之间；P3株与上海分离株之间核苷酸同源性在88．0％-88．5％之间、氨基酸同源性在98．0％-98．4％之间。E基因区段500个氨基酸中P3株与所有新分离乙脑病毒株之间共存在19个位点的差异，其中在E-76、E-306、E-408处所有新分离毒株与P3株存在共同的差异；在E-160、E-487处福建分离株与P3株存在共同差异；上海分离株与P3株在E-129、E-222、E-227、E-366存在共同差异。结论 上海蚊虫中分离的Ⅰ型乙脑病毒和福建省脑炎患者中分离的Ⅲ型乙脑病毒与P3株在E基因的部分氨基酸位点存在差异，但均不处在影响病毒生物学特性的关键位点。     

Deduced sequences of the membrane fusion and attachment proteins of canine distemper viruses isolated from dogs and wild animals in Korea

Canine distemper virus (CDV) causes highly contagious respiratory, gastrointestinal, and neurological diseases in wild and domestic animal species. Despite a broad vaccination campaign, the disease is still a serious problem worldwide. In this study, six field CDV strains were isolated from three dogs, two raccoon dogs, and one badger in Korea. The full sequence of the genes encoding fusion (F) and hemagglutinin (H) proteins were compared with those of other CDVs including field and vaccine strains. The phylogenetic analysis for the F and H genes indicated that the two CDV strains isolated from dogs were most closely related to Chinese strains in the Asia-1 genotype. Another four strains were closely related to Japanese strains in the Asia-2 genotype. The six currently isolated strains shared 90.2–92.1 % and 88.2–91.8 % identities with eight commercial vaccine strains in their nucleotide and amino acid sequences of the F protein, respectively. They also showed 90.1–91.4 % and 87.8–90.7 % identities with the same vaccine strains in their nucleotide and deduced amino acid sequences of the H protein, respectively. Different N-linked glycosylation sites were identified in the F and H genes of the six isolates from the prototype vaccine strain Onderstepoort. Collectively, these results demonstrate that at least two different CDV genotypes currently exist in Korea. The considerable genetic differences between the vaccine strains and wild-type isolates would be a major factor of the incomplete protection of dogs from CDV infections.     

Comparative sequence analysis of the VP7 genes of G6, G8 and G10 bovine group A rotaviruses and further characterization of G6 subtypes

Summary.  We previously reported the relatively high prevalence (15%) of bovine G6 subtypes (G6s) in the field using RT-PCR and restriction fragment length polymorphism (RFLP) analysis (Chang et al., Arch. Virol. 141: 1727–39). In the present study, we report the nucleotide and antigenic characterization of a G6s strain (C-8336). We also sequenced the VP7 genes of four additional bovine rotavirus (BRV) strains: another G6s (MC27), G6 (IND), G8 (C-8008) and G10 (2292B) and compared these with other bovine and human rotavirus strains. The C-8336 and MC27 strains were confirmed as P[11]G6s by RT-PCR and RFLP analysis. The VP7 genes of the C-8336 and MC27 strains showed high homology to each other (∼98%) and with other bovine G6s strains (greater than 95% homology in nucleotide and amino acid sequence with KN-4{P[11]G6s}) and also showed lower, but substantial sequence homology with human G6s strains and prototype G6 BRV (79–87% in nucleotide and 88–91% in amino acid). Serologic analysis of the cell culture adapted C-8336 strain showed that it was neutralized by a G6 monoclonal antibody (MAb IC3) to similar titers as the reference NCDV and IND G6 strains. In two-way cross-neutralization tests, strain C-8336 showed 4- to 16-fold differences in antibody titers with NCDV and IND G6 BRV. Moreover polyclonal antiserum against strain C-8336 neutralized the NCDV and IND strains weakly. Genetic variability was also observed among G8 and G10 bovine and human group A rotaviruses: the VP7 genes of the bovine C-8008 (P[5]G8) and 2292 B (P[11]G10) strains showed from 10 to 17% nucleotide divergence with those of Cody I801 (P[1]G8, bovine), A5 (P[1]G8, bovine), 69 M (P[10]G8, human) and Hal 1166 (P[14]G8, human), and I321(P[11]G10, human) and MC35 (P[14]G10, human) rotaviruses, respectively. The divergence of VP7 genes among bovine and human G6, G8 and G10 strains appears related to host species origin and their combination with VP4 (P type). The data presented in this report confirms the genetic variability among homotypic bovine and human strains and highlights the importance of continued monitoring of BRV G and P types circulating in the field for the future development and monitoring of effective vaccines. Received July 4, 1999/Accepted October 31, 1999     

Genetic Variability among Group A and Group B Respiratory Syncytial Viruses in a Children’s Hospital

Respiratory syncytial (RS) viruses isolated over three epidemic periods in a children’s hospital in the United States were analyzed. The viruses (n = 174) were characterized as to major antigenic group (group A or B) by a PCR-based assay. Group A RS viruses were dominant the first 2 years, followed by a year with group B dominance (ratios of group A to group B viruses for epidemic periods, 56/4 for 1993–1994, 42/3 for 1994–1995, and 19/50 for 1995–1996). Genetic variability within the groups was assessed by restriction fragment analysis of PCR products; 79 isolates were also analyzed by nucleotide sequence determination of a variable region of the glycoprotein G gene. Among the group A RS virus isolates, this G-protein variable region had amino acid differences of as great as 38%. The G-protein amino acids of the group A viruses differed by up to 31% from the G-protein amino acids of a prototype (A2) group A virus. Among the group B RS virus G proteins, amino acid differences were as great as 14%. The G-protein amino acids of the group B viruses differed by up to 27% from the G-protein amino acids of a prototype (18537) group B virus. The group A and group B RS viruses demonstrated genetic variability between years and within individual years. Phylogenetic analysis revealed that there were multiple evolutionary lineages among both the group A and group B viruses. Among the recent group B isolates, variability was less than that seen for the group A viruses. However, comparisons to prototype strains revealed that the group B RS viruses may vary more extensively than was observed over the 3 years studied in the present investigation.