贵州及周边省市狂犬病病毒的分群和进化研究

目的 全面掌握贵州及周边省市狂犬病病毒(RABV)的遗传进化特征和分群特点,了解狂犬病疫苗株与流行株的分子遗传差异,为贵州狂犬病疫情的预防和控制提供指导依据.方法 搜索GenBank数据库中贵州及周边5个省市具有明确时间、地区及宿主信息的流行毒株,对其糖蛋白基因(G)全序列进行汇总,并结合本实验室获得的贵州毒株和国内现役疫苗株G序列,利用MEGA和BEAST软件分别进行系统进化和时间进化分析.结果 贵州及周边省市79株RABV均属于基因1型,9个类群的RABV流行株具有明显的地域特征,11株疫苗株分化为2个类群;RABV流行株与疫苗株G基因核苷酸和氨基酸同源性分别为0.827～0.999和0.713～0.998;贵州及周边省市RABV流行毒株的最近共同祖先(TMRCA)可追溯至150年前.结论 贵州及周边省市流行的RABV优势毒株群仍属于基因1型,但毒株经过多年流行已经出现了分化,有必要对流行毒株与疫苗株的遗传变异一致性进行深入研究.     

山东省首次分离出基因1型流行性乙型脑炎病毒

目的从山东省济南市采集的蚊虫标本中分离流行性乙型脑炎（乙脑）病毒（Japanese Encephalitis Virus,JEV）,并确定其基因型别及前膜蛋白（Precursor Membrane,PrM）基因片段和包膜蛋白（Envelope,E）基因区段分子特征。方法对2008年采集的蚊虫标本进行病毒分离,对新分离的病毒进行血清学及分子生物学鉴定。逆转录-聚合酶链反应扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X（1.83）软件做碱基配对分析,MEGA3.1完成病毒进化分析,GENEDOS（3.2）软件完成氨基酸位点分析。结果共采集15700只蚊虫标本,包括库蚊、骚扰阿蚊、伊蚊、按蚊。随机抽取20批标本,从其中的1批三带喙库蚊标本中分离到1株病毒,经过血清学及分子生物学鉴定属于基因1型JEV。新分离JEV与减毒活疫苗株SA14-14-2株E区段核苷酸和氨基酸序列的同源性分别为88.0%和97.2%,存在14处氨基酸位点差异。结论从山东省首次分离到基因1型JEV。     

Human Papillomavirus Type 16 L2 Gene Sequence Variation Analysis in Indonesian Cervical Cancer Specimens

Background: Human papillomavirus type 16 (HPV16) is the most prevalent etiology of cervical cancer in Indonesian women. The L2 minor capsid protein has considerable potential as a broad-protective antigen target of the cervical cancer vaccine strategies, yet the data on L2 gene variation is still minimal. In this research, we determined the variations of the HPV16 L2 gene sequences in Indonesian cervical cancer specimens. Methods: We cross-sectionally observed 23 DNA isolates of HPV16 positive cervical cancer specimens stored in the laboratory of the Center for Diagnostic and Research on Infectious Diseases (PDRPI Lab), Faculty of Medicine, Universitas Andalas, Padang, Indonesia. We detected and amplified the HPV16 L2 gene sequences in the samples, followed by sequencing, DNA alignment, single nucleotide polymorphisms (SNPs) analysis, and phylogenetic tree reconstruction. Results: As many as 35 SNPs were found, consist of 18 synonymous SNPs (sSNPs) and 17 non-synonymous SNPs (nsSNPs). Amino acid variations were mostly detected at S269P (100%) and L330F (43.48%) with no variation in the immuno-protective region near L2 N-terminus. A total of 5 HPV16 phylogenetic sub-lineages were found closely related to A1 (n=5), A2 (n=12), A3 (n=2), A4 (n=3), and C (n=1). Conclusion: The variations of HPV16 L2 gene sequences are mainly located in the central region of the L2 sequences, and the cross-protective region near the L2 N-terminus is remarkably conserved. This study should enhance the information about HPV16 L2 gene variation in Indonesia.     

手足口病患儿柯萨奇病毒A4的鉴定及其5’UTR—VP4一VP2区序列分析

目的分析2009年北京市西城区手足口病患儿柯萨奇病毒A4（CoxA4）5’UTR-VP4-VP2区进化特征,探讨其与国内外其他地区CoxA4分离株的关系。方法提取病毒RNA,进行半巢式RT-PCR扩增肠道病毒（EV）5’UTR-VP4-VP2区。通过测序和Blast比对确定EV型别;并对其进行序列和进化分析。结果共鉴定出两株CoxA4,Genbank登录号为JQ429434和JQ429435。进化分析表明,CoxA4包括3个进化树分支：原型株AY421762位于独立的分支,其余CoxA4构成两个基因型（I型和II型）,每个基因型包括两个亚型（A和B）。在5’UTR-VP4-VP2区．两株CoxA4核苷酸序列同源性为96．00％;它们与原型株、基因Ⅰ型及Ⅱ型毒株序列同源性分别为87．00％～88．00％,85．00％～87．00％,92．00％～98．OO％。两株CoxA4与其他中国株均位于基因Ⅱ型分支上。结论北京市西城区CoxA4株与我国近年CoxA4流行株在5’UTR—VP4一VP2区具有较高的同源性,且进化关系密切。     

江西省流行性乙型脑炎病毒的分离与鉴定

目的调查江西省的虫媒病毒分布状况,在当地采集蚊虫标本进行病毒分离与鉴定。方法 2008-2009年夏季在江西省共选择8个标本采集点,使用诱蚊灯采集蚊虫标本,通过组织培养法分离病毒,并对病毒分离物进行血清学和分子生物学鉴定;利用生物信息学技术对新分离病毒的序列进行分析,完成同源性和系统发生分析。结果 2008年采集到3属3种共计11916只蚊虫标本;2009年采集到4属5种共计5905只蚊虫标本;共分离到4株病毒,经鉴定均为流行性乙型脑炎(乙脑)病毒;新分离乙脑病毒与基因Ⅰ型乙脑病毒的同源性最高,系统进化结果显示,4株乙脑病毒均属于基因Ⅰ型。结论在江西省分离到基因Ⅰ型乙脑病毒,与上海市和浙江省分离株进化关系较近。     

Epidemiology and phylogenetic analysis of VP7 and VP4 genes of rotaviruses circulating in Rawalpindi,Pakistan during 2010

Human group A rotaviruses (RVAs) possess a large genetic diversity and new RVA strains and G/P genotype combinations are been identified frequently. Only a few studies reporting the distribution and co-circulation of RVA G and P genotypes are available for Pakistan. This hospital based study showed a RVA prevalence rate of 23.8%, which is similar to RVA detection rates estimated in other Eastern Mediterranean countries. During 2010, the following RVA strains were found to co-circulate: G1P[8] and G2P[4] (both 24.3%), G1P[6] (12.1%), G9P[8] (10.8%), G9P[6] (5.4%), G12P[6] (6.7%), G6P[1] (2.7%) and mixed infections (6.7%). Sequence analyses of selected G1, G2, G9 and G12 RVA strains revealed a close evolutionary relationship with typical human RVA strains. Sequence identities among the Pakistani VP7 RVA genes encoding G1, G2, G9 and G12 ranged between 91.5–98.7%, 99.6–98.9%, 97.7–99.5% and 99.2–99.9%, respectively. Analysis of the VP4 genes revealed co-prevalence of distinct lineages of the P[8] genotype. P[6] and P[4] showed a close relationship with typical human RVA strains detected in several Asian countries. The two G6P[1] RVA strains were closely related to typical bovine RVA strains, suggesting one or multiple interspecies transmission events. Our data provide important baseline data on the burden of RVA disease and genotype distribution in Rawalpindi, Pakistan, which is important with respect to vaccine introduction in national immunization programs.     

SNP typing reveals similarity in Mycobacterium tuberculosis genetic diversity between Portugal and Northeast Brazil

Human tuberculosis is an infectious disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Although spoligotyping and MIRU-VNTR are standard methodologies in MTBC genetic epidemiology, recent studies suggest that Single Nucleotide Polymorphisms (SNP) are advantageous in phylogenetics and strain group/lineages identification. In this work we use a set of 79 SNPs to characterize 1987 MTBC isolates from Portugal and 141 from Northeast Brazil. All Brazilian samples were further characterized using spolygotyping. Phylogenetic analysis against a reference set revealed that about 95% of the isolates in both populations are singly attributed to bacterial lineage 4. Within this lineage, the most frequent strain groups in both Portugal and Brazil are LAM, followed by Haarlem and X. Contrary to these groups, strain group T showed a very different prevalence between Portugal (10%) and Brazil (1.5%). Spoligotype identification shows about 10% of mis-matches compared to the use of SNPs and a little more than 1% of strains unidentifiability. The mis-matches are observed in the most represented groups of our sample set (i.e., LAM and Haarlem) in almost the same proportion. Besides being more accurate in identifying strain groups/lineages, SNP-typing can also provide phylogenetic relationships between strain groups/lineages and, thus, indicate cases showing phylogenetic incongruence.Overall, the use of SNP-typing revealed striking similarities between MTBC populations from Portugal and Brazil.     

Current perspectives on the phylogeny of Filoviridae

Sporadic fatal outbreaks of disease in humans and non-human primates caused by Ebola or Marburg viruses have driven research into the characterization of these viruses with the hopes of identifying host tropisms and potential reservoirs. Such an understanding of the relatedness of newly discovered filoviruses may help to predict risk factors for outbreaks of hemorrhagic disease in humans and/or non-human primates. Recent discoveries such as three distinct genotypes of Reston ebolavirus, unexpectedly discovered in domestic swine in the Philippines; as well as a new species, Bundibugyo ebolavirus; the recent discovery of Lloviu virus as a potential new genus, Cuevavirus, within Filoviridae; and germline integrations of filovirus-like sequences in some animal species bring new insights into the relatedness of filoviruses, their prevalence and potential for transmission to humans. These new findings reveal that filoviruses are more diverse and may have had a greater influence on the evolution of animals than previously thought. Herein we review these findings with regard to the implications for understanding the host range, prevalence and transmission of Filoviridae.     

成都机场口岸常见媒介蝇类的分子鉴定

目的:建立以线粒体COI基因作为分子标记的蝇类分子鉴定方法。方法:设计COI基因引物对成都双流机场口岸常见4科10属14种卫生蝇类进行PCR扩增和序列测定,并与不同地区蝇类的COI基因片段比较,分析其碱基差异和系统发育关系。结果:部分不同地理来源的种类种内碱基差异变化较大,中国四川与马来西亚棕尾别麻蝇碱基差异为3.4%,中国四川与中国广东巴浦绿蝇碱基差异为3.3%;其他种类种内碱基差异较小(<1.0%)。系统发育分析显示所有种类的COI基因分子鉴定结果和形态学结果相一致。麻蝇科种类聚为一个分支,而丽蝇科、花蝇科和蝇科种类没有聚集形成单系。结论:可以将COI基因片段作为分子标记,建立一种适用于口岸的蝇类种类之间分子鉴定方法。     

A multi-national European cross-sectional study of feline calicivirus epidemiology,diversity and vaccine cross-reactivity

BackgroundFeline calicivirus (FCV) is an important pathogen of cats for which vaccination is regularly practised. Long-term use of established vaccine antigens raises the theoretical possibility that field viruses could become resistant. This study aimed to assess the current ability of the FCV-F9 vaccine strain to neutralise a randomly collected contemporary panel of FCV field strains collected prospectively in six European countries.MethodsVeterinary practices (64) were randomly selected from six countries (UK, Sweden, Netherlands, Germany, France and Italy). Oropharyngeal swabs were requested from 30 (UK) and 40 (other countries) cats attending each practice. Presence of FCV was determined by virus isolation, and risk factors for FCV shedding assessed by multivariable logistic regression. Phylogenetic analyses were used to describe the FCV population structure. In vitro virus neutralisation assays were performed to evaluate FCV-F9 cross-reactivity using plasma from four vaccinated cats.ResultsThe overall prevalence of FCV was 9.2%. Risk factors positively associated with FCV shedding included multi-cat households, chronic gingivostomatitis, younger age, not being neutered, as well as residing in certain countries. Phylogenetic analysis showed extensive variability and no countrywide clusters. Despite being first isolated in the 1950s, FCV-F9 clustered with contemporary field isolates. Plasma raised to FCV-F9 neutralized 97% of tested isolates (titres 1:4 to 1:5792), with 26.5%, 35.7% and 50% of isolates being neutralized by 5, 10 and 20 antibody units respectively.ConclusionsThis study represents the largest prospective analysis of FCV diversity and antigenic cross-reactivity at a European level. The scale and random nature of sampling used gives confidence that the FCV isolates used are broadly representative of FCVs that cats are exposed to in these countries. The in vitro neutralisation results suggest that antibodies raised to FCV-F9 remain broadly cross-reactive to contemporary FCV isolates across the European countries sampled.