三株新城疫广西分离株全基因组序列的测定与分析

根据GenBank上所公布的新城疫病毒(NDV)的全基因组序列,设计了8对引物,运用RT-PCR方法获取了3株广西地方强毒株GX7/02、GX9/03和GX11/03的全基因组序列,并对其进行了比较分析。此3株病毒的全基因组序列均由15192个碱基组成,与GenBank公布的ZJ1、U.S/Largo/71、Italy/2736/00等7个毒株的全基因组序列长度相同,比LaSota、Clone-30和B1的全基因组序列多出6个核苷酸,此6个核苷酸位于np基因的非编码区内,相对与NDV毒株LaSota、Clone-30和B1序列的1647~1648nt位。通过序列的比较分析,发现GX7/02、GX9/03和GX11/03与ZJ1毒株的同源性较高,而与LaSota、Clone-30和B1等毒株的同源性较低。     

Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia

Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.     

新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

Complete-genome phylogenetic approach to varicella-zoster virus evolution: genetic divergence and evidence for recombination

Recent studies of varicella-zoster virus (VZV) DNA sequence variation, involving large numbers of globally distributed clinical isolates, suggest that this virus has diverged into at least three distinct genotypes designated European (E), Japanese (J), and mosaic (M). In the present study, we determined and analyzed the complete genomic sequences of two M VZV strains and compared them to the sequences of three E strains and two J strains retrieved from GenBank (including the Oka vaccine preparation, V-Oka). Except for a few polymorphic tandem repeat regions, the whole genome, representing approximately 125,000 nucleotides, is highly conserved, presenting a genetic similarity between the E and J genotypes of approximately 99.85%. These analyses revealed that VZV strains distinctly segregate into at least four genotypes (E, J, M1, and M2) in phylogenetic trees supported by high bootstrap values. Separate analyses of informative sites revealed that the tree topology was dependent on the region of the VZV genome used to determine the phylogeny; collectively, these results indicate the observed strain variation is likely to have resulted, at least in part, from interstrain recombination. Recombination analyses suggest that strains belonging to the M1 and M2 genotypes are mosaic recombinant strains that originated from ancestral isolates belonging to the E and J genotypes through recombination on multiple occasions. Furthermore, evidence of more recent recombination events between M1 and M2 strains is present in six segments of the VZV genome. As such, interstrain recombination in dually infected cells seems to figure prominently in the evolutionary history of VZV, a feature it has in common with other herpesviruses. In addition, we report here six novel genomic targets located in open reading frames 51 to 58 suitable for genotyping of clinical VZV isolates.     

江西省2010年柯萨奇病毒A组24型变异株的全基因组序列分析

急性出血性结膜炎(Acute hemorrhagic conjunctivitis,AHC)是目前人类最常见的眼病之一,柯萨奇病毒A组24型变异株(Coxsackievirus A24 variant,CV-A24v)是近年来报道引起该病的主要病原体。本研究选取10株来自江西省2010年AHC暴发疫情的CV-A24v,采用特异性引物扩增并测定其全基因组序列。对该10条CV-A24v的全基因组序列进行系统发育分析以及重组分析,计算本研究测定的江西10条以及GenBank中所有22条CV-A24v的全基因组序列的氨基酸置换熵值,并预测其正向选择位点。结果表明,在江西10条CV-A24v基因组序列中未检测到重组。基于全基因组序列构建的最大似然树表明江西10株CV-A24v属于GIV基因型,且分处于两条传播链。对上述32条CV-A24v序列的氨基酸置换熵值计算,共得到25个易突变位点(熵值>0.6),易突变概率最高的区段为2A区。基于Datamonkey中FUBAR和FEL模型分析,发现位于结构蛋白VP2区的234位氨基酸为两种模型共同获得的CV-A24v的正向选择位点。本研究分析了江西10株CV-A24v的全基因组序列特征,为CV-A24v引起的AHC防控工作提供了基础资料。     

新城疫病毒分离株HN和P基因分子进化及其相关研究

为探讨新城疫病毒(Newcastle disease virus,NDV)血凝素-神经氨酸酶(HN)和磷蛋白(P)基因遗传特性以及相互关系,将1997～2005年间国内分离到12株NDV毒株,分别进行HN和P基因克隆测序,结合15个已发表的国内外不同时期的NDV毒株HN和P基因,计算所有毒株HN和P基因的不同核苷酸和氨基酸片段进化距离,利用统计软件进行了不同片段间进化距离的方差分析,HN或P基因核苷酸进化距离与毒株分离时间、HN或P基因片段与其全长间以及HN和P基因全长间的相关分析.统计分析显示:NDV HN或P基因不同核苷酸和氨基酸序列片段变异程度不一样;不同毒株间HN或P基因片段与其全长间以及HN和P基因全长间无论是核苷酸还是氨基酸遗传变异高度相关.以上说明,NDV HN和P基因虽以不同的方式进化,但是HN和P基因遗传变异的趋势是相同的.HN和P基因的变异与分离时间有一定的联系.     

Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene

The entire nucleotide sequences of 70 hepatitis B virus (HBV) isolates of genotype B (HBV/B), including 38 newly determined and 32 retrieved from the international DNA database (DDBJ/EMBL/GenBank), were compared phylogenetically. Two subgroups of HBV/B were identified based on sequence divergence in the precore region plus the core gene, one with the recombination with genotype C and the other without it. The analysis over the entire genome of HBV/B by the SimPlot program located the recombination with genotype C in the precore region plus the core gene spanning nucleotide positions from 1740 to 1838 to 2443 to 2485. Within this genomic area, HBV/B strains with the recombination had higher nucleotide and amino acid homology to genotype C than those without the recombination (96.9 versus 91.1% in nucleotides and 97.0 versus 92.9% in amino acids). There were 29 HBV/B strains without the recombination, and they were all recovered from carriers in Japan. The remaining 41 HBV/B isolates having the recombination with genotype C were from carriers in China (12 strains), Hong Kong (3 strains), Indonesia (4 strains), Japan (3 strains), Taiwan (4 strains), Thailand (3 strains), and Vietnam (12 strains). Due to the frequency of the distribution of HBV/B without the recombination (29 of 32 isolates, or 91%) and the fact that it was exclusive to Japan, it was provisionally classified into the Bj (j standing for Japan) subgroup, and HBV/B with the recombination was classified into the Ba (a for Asia) subgroup. Virological differences between HBV/Bj and HBV/Ba may be reflected in the severity of clinical disease in the patients infected with HBV of genotype B, which seems to be under strong geographic influences in Asia.     

基于相对密码子频率的芜菁花叶病毒基因组序列的进化分析

现有92株芜菁花叶病毒(TuMV)的全基因组序列已在GenBank报道,据分析报道其中58株不含重组序列。利用系统聚类法对92株TuMV的全基因组序列和58株TuMV全基因组序列的相对密码子频率RSCU值进行聚类分析。同时利用系统发育分析方法分析了这92株和58株TuMV全基因组序列。结果发现,92株芜菁花叶病毒株的密码子偏性聚类树与其系统进化树的一致度很低;而不含重组序列的58株芜菁花叶病毒株的密码子偏性聚类树与其系统进化树的一致度却非常高,且与寄生宿主类型基本对应。这表明在不存在重组的情况下,TuMV密码子频率的偏性可能是宿主内的一种选择压力,影响TuMV基因组的点突变进化方向,促使TuMV适应宿主内环境。     

Evidence of recombination in the norovirus capsid gene

Noroviruses are single-stranded RNA viruses with high genomic variability. They have emerged in the last decade as a major cause of acute gastroenteritis. It remains so far unclear whether norovirus evolution is driven by sequence mutation and/or recombination. In this study, we have assessed the occurrence of recombination in the norovirus capsid gene. For this purpose, 69 complete capsid sequences of norovirus strains accessible in GenBank as well as 25 complete capsid sequences generated from norovirus-positive clinical samples were examined. Unreported recombination was detected in about 8% of norovirus strains belonging to genetic clusters I/1 (n = 1), II/1 (n = 1), II/3 (n = 1), II/4 (n = 3), and II/5 (n = 1). Recombination breakpoints were mainly located at the interface of the putative P1-1 and P2 domains of the capsid protein and/or within the P2 domain. The recombination region displayed features such as length, sequence composition (upstream and downstream GC- and AU-rich sequences, respectively), and predicted RNA secondary structure that are characteristic of homologous recombination activators. Our results suggest that recombination in the norovirus capsid gene may naturally occur, involving capsid domains presumably exposed to immunological pressure.     

中国禽源新城疫病毒(NDV)流行株F和HN基因的遗传演化和变异频率

【目的】新城疫(ND)是中国流行最严重的疫病之一,对家禽业可造成巨大的经济损失,疫苗防控是控制ND的重要措施。新城疫病毒(NDV)流行株的遗传演化一直是研究NDV的焦点。本文利用分子信息学手段,通过比较近20年间NDV流行株不同基因型F和HN基因的分子特征和遗传变异频率,解析免疫压力下NDV的演化规律。【方法】利用Lasergene 7.1和MEGA5.1软件,选取本实验室89株NDV分离株,结合从Gen Bank下载的364株NDV流行株以及15株NDV经典毒株的基因序列,对其进行系统发育、分子特征和替代频率分析。【结果】系统发育表明,NDV已经演化为15个基因型。一致性比较显示,NDV流行株相同基因型之间核苷酸(氨基酸)高度同源,而不同基因型之间差异较大且存在明显的氨基酸变异积累。NDV基因型的分布与时间、地域密切相关,VII d亚型为中国NDV优势流行株。为评估NDV变异的频率,以Go/GD/QY/1997株(中国较早发生的基因VII亚型)为参照,1997-2015年间NDV的F/HN基因的年平均核苷酸(氨基酸)替代率为2.31×10~(-3)(2.26×10~(-3))/3.37×10~(-3)(2.35×10~(-3))。其中,1997-2001年(未使用基因VII型疫苗)F/HN基因核苷酸年平均替代率为4.72×10~(-3)/8.28×10~(-3);2002-2015年(疫苗使用后)为1.6×10~(-3)/1.84×10~(-3),显示出基因VII型疫苗在控制NDV变异速度方面具有明显的效果。【结论】生物信息学分析证实:研制出与NDV流行毒株相匹配的新型疫苗是控制当前NDV变异的关键。     

四株不同宿主来源的Class I基因3型新城疫病毒全基因组序列测定及比较分析

为了解析基因型相同但宿主来源不同的新城疫病毒的全基因组差异。本文采用RT-PCR方法分别获得4株（JS/3/09/Ch,ZJ/3/10/Ch,AH/2/10/Du,JS/9/08/Go）Class I 基因3型病毒的全基因组核苷酸序列,并与GenBank中已公布的Class I基因3型病毒全基因组序列进行比对分析。本实验4株病毒的基因组长度均为15198bp,在基因组1607～1608位有6碱基的缺失,在2381～2382位有12碱基的插入,裂解位点为112EQ/RQE/GRL117是标准弱毒特征。5株Class I基因3型病毒之间全基因组同源性超过93%;而与Class II弱毒株同源性最低只有72.2%;比较6个结构蛋白基因的同源性,NP基因的同源性最高(98.3%～96.4%),而P基因最低(96.1%～91.9%)。结果表明不同宿主来源的Class I基因3型新城疫病毒在遗传信息方面差异不大,但NP/F/L基因的变异幅度较P/M/HN基因明显。     

Genomic organization and recombination analysis of human norovirus identified from China

Noroviruses (NoVs) are one of the major causal agents of acute gastroenteritis in both industrial and developing countries including China. Recent studies have revealed that NoV genome is highly prone to mutation and recombination which may lead to emergence of new strains. In the present study, three full-length genomes of human NoV from China were determined and the genomic organization and recombination were analyzed. They had similar genome organization and contained three predicted ORFs, though the 5′UTR of those three strains were 2, 4 and 8 nucleotides, respectively. Phylogenetic analysis showed that the HU/GII/SHANGHAI/SH312/2008/CHN strain may be a recombinant of GII-3 capsid and GII-4 polymerase. To confirm the finding and detect the breakpoints where the recombination event occurred, we performed recombination analysis based on the genomic sequences of HU/GII/SHANGHAI/SH312/2008/CHN as the query sequence, and AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3 as the background sequences, using RPD software. Results indicated that the two parental strains were AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3. The breakpoint for this recombination event located at position 5,107 nt of the genome (in the ORF1 and ORF2 overlap).     

简化cDNA末端快速扩增技术测定基因Ⅲ、Ⅵb和Ⅶ d型新城疫病毒基因组末端序列及分析

[目的]简化cDNA末端快速扩增技术(Rapid amplification of cDNA ends,fLAcE)流程,测定基因Ⅲ、VIb)和VIId型新城疫病毒(Newcastle disease virus,NDV)基因组两侧末端序列,并对NDV的leader和trailer进行分析.[方法]利用T4 RNA连接酶将特定寡聚核苷酸片段的连接于病毒基因组RNA和eDNA,再利用RT-PCR或PCR方法对病毒基因组的末端进行快速的扩增.[结果]建立一套操作简单、低成本、可重复性高的RACE方法,测定了三种基因型5株NDV 3'末端leader和5'末端trailer序列比对分析.[结论]本实验测定的鹅源VII型毒株JS/7/05/Ch基因组的15,184 nt由一个T变为了C,5'端trailer与3'端leader的连续互补序列由8 nt变为12 nt,而其它4株基因Ⅲ型和VI型NDV均未发现该突变.通过RNA的二级结构分析,NDV基因组和反向基因组RNA的3'末端形成一个发卡结构.JS/7/05/Ch等3株NDV U→C(T→C)的突变位于发卡环上,不影响二级结构的形成,发卡环的RNA序列突变为3'-UCUC-5',与基因组3'端发卡环的3'-UCUUA-5'相似,推测可能影响了基因组RNA的复制速度.     

Complete genome sequencing and analysis of an anti-tumor Newcastle disease virus strain

HBNU/LSRC/F3, a Newcastle disease virus (NDV) strain stored in our lab, exhibited an anti-tumor ability in our previous studies. Nonetheless, very little is known about its genome sequence, which is vital for further study. Here, the complete HBNU/LSRC/F3 genome was fully sequenced and compared with other NDV sequences. Its genome contained 15,192 nucleotides (nt) consisting of two termini and six genes in the following order: 3′-Le-NP-P-M-F-HN-L-Tr-5′. Phylogenetic analysis indicated that this NDV strain belonged to the Class II genotype IX group. A multibasic amino acid (aa) sequence was found at the cleavage site (¹¹²RRQRR↓F¹¹⁷) within the fusion (F) protein, and a 6 nt insertion was present in the 5′ non-coding region of the NP gene. The whole genome sequence was highly similar to other genotype IX NDV genomes reported in China. Overall, this study provides insight into the sequence characteristics of genotype IX NDVs, which will be useful for subsequent investigations.     

Escherichia coli molecular phylogeny using the incongruence length difference test

Molecular phylogeny of the species Escherichia coli using the E. coli reference (ECOR) collection strains has been hampered by (1) the absence of rooting in the commonly used phenogram obtained from multilocus enzyme electrophoresis (MLEE) data and (2) the existence of recombination events between strains that scramble phylogenetic trees reconstructed from the nucleotide sequences of genes. We attempted to determine the phylogeny for E. coli based on the ECOR strain data by extracting from GenBank the nucleotide sequences of 11 chromosomal structural and 2 plasmid genes for which the Salmonella enterica homologous gene sequences were available. For each of the 13 DNA data sets studied, incongruence with a nonnucleotide whole-genome data set including MLEE, random amplified polymorphic DNA, and rrn restriction fragment length polymorphism data was measured using the incongruence length difference (ILD) test of Farris et al. As previously reported, the incongruence observed between the gnd and plasmid gene data and the whole-genome data was multiple, indicating numerous horizontal transfer and/or recombination events. In five cases, the incongruence detected by the ILD test was punctual, and the donor group was identified. Congruence was not rejected for the remaining data sets. The strains responsible for incongruences with the whole-genome data set were removed, leading to a "prior-agreement" approach, i.e., the determination of a phylogeny for E. coli based on several genes, excluding (1) the genes with multiple incongruences with the whole genome data, (2) the strains responsible for punctual incongruences, and (3) the genes incongruent with each other. The obtained phylogeny shows that the most basal group of E. coli strains is the B2 group rather than the A group, as generally thought. The D group then emerges as the sister group of the rest. Finally, the A and B1 groups are sister groups. Interestingly, the most primitive taxon within E. coli in terms of branching pattern, i.e., the B2 group, includes highly virulent extraintestinal strains with derived characters (extraintestinal virulence determinants) occurring on its own branch.      

天津市致病毒性脑炎柯萨奇病毒B组5型全基因组序列分析

肠道病毒是我国病毒性脑炎(Viral encephalitis,VE)的主要病原体。本文研究对4株引起VE的天津柯萨奇病毒B组5型(Coxsackievirus B5,CV-B5)分离株进行Illumina MiniSeq高通量测序,并对其全基因组特征、进化及重组特点进行分析。结果提示,4株CV-B5天津分离株的全基因组核苷酸和氨基酸序列同源性分别为84.5%~100.0%和98.1%~100.0%,与国内流行株的全基因组核苷酸序列同源性为83.2%~96.5%,氨基酸序列同源性为96.4%~99.4%。基于全基因组的系统进化分析将CV-B5流行株分为A-D四个基因型,其中天津与国内流行株均属于C基因型。C基因型进一步分为3个进化分支,而天津分离株处在两个不同的分支上。基于基因组各区段序列的系统进化与SimPlot重组分析结果显示,天津分离株15-39N、15-41N与埃可病毒30型(Echovirus 30,E-30)原型株在P3区3B、3C、3D区域均检测到重组信号。本研究有助于了解CV-B5的全基因组特点和重组规律,为相关疾病的防控提供依据。     

Molecular Epidemiology of Echovirus 18 Circulating in Mainland China from 2015 to 2016

Echovirus 18(E18), a serotype of Enterovirus B(EV-B) species, is an important pathogen in aseptic meningitis. E18 had rarely been detected in mainland China, but became the predominant pathogen associated with viral encephalitis(VE) and meningitis in Hebei province for the first time in 2015. To investigate the molecular epidemiology and genetic characteristics of E18 in mainland China, sixteen E18 strains from patient throat swabs with hand, foot, and mouth disease(HFMD) in six provinces in China collected between 2015 and 2016, and four E18 strains isolated from 18 patient cerebrospinal fluid specimens with VE in Hebei Province in 2015 were obtained and sequenced. Combined with the sequences from the GenBank database, we performed an extensive genetic analysis. Phylogenetic analysis of VP1 gene sequences revealed that all E18 strains from mainland China after 2015 belonged to subgenotype C2. There were no obvious specific differences in phylogenetic and variation analyses of E18 genome sequences between HFMD and VE/meningitis strains. Potential multiple recombination may have occurred in the 50-untranslated region and in the P2 and P3 nonstructural protein-encoding regions of E18 strains from China. The current E18 strains were potential multiplerecombinant viruses. Overall, these findings supported that E18 caused HFMD, VE, and meningitis, although there were no significant associations between clinical features and viral genomic characteristics.     

Genotypic and pathotypic characterization of Newcastle disease viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3' leader and 5' trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have "died out" after the first panzootic (1926-1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India.     

Evidence for frequent recombination within species human enterovirus B based on complete genomic sequences of all thirty-seven serotypes

The species Human enterovirus B (HEV-B) in the family Picornaviridae consists of coxsackievirus A9; coxsackieviruses B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. We have determined complete genome sequences for the remaining 22 HEV-B serotypes whose sequences were not represented in public databases and analyzed these in conjunction with previously available complete sequences in GenBank. Members of HEV-B were monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5'-nontranslated region (NTR), where they are known to cluster with members of HEV-A. Within HEV-B, phylogenies constructed from the structural (P1) and nonstructural regions of the genome (P2 and P3) are incongruent, suggesting that recombination had occurred. Similarity plots and bootscanning analysis across the complete genome identified multiple sites at which the phylogeny of a given strain's sequence shifted, indicating potential recombination points. These points are distributed in the 5'-NTR and throughout P2 and P3, but no sites with >80% bootstrap support were identified within the capsid. Individual sequence comparisons and phylogenetic analyses suggest that members of HEV-B have recombined with one another on multiple occasions, resulting in a complex mosaic of sequences derived from multiple parental viruses in the nonstructural regions of the genome. We conclude that RNA recombination is a common mechanism for enterovirus evolution and that recombination within the nonstructural regions of the genome (P2 and P3) has been observed only among members of the same species.