我国广东、福建地区2000～2001年手足口病肠道病毒71型分离株的种系进化分析

目的 采用逆转录聚合酶链反应(RT-PCR)检测我国广东、福建地区2000-2001年手足口病(HFMD)散发病例中的肠道病毒71型(EV71)，进而通过扩增VP1节段的核苷酸序列，进行毒株的种系进化分析。方法以肠道病毒特异引物对EV-1、EV-2进行RT-PCR，经琼脂糖凝胶电泳鉴定为阳性的标本，进一步用EV71特异引物对159S、162A进行RT-PCR，扩增的VP1节段经纯化后与测序载体pGEM-T连接，转化大肠埃希菌DH5a，筛选后测序。所得序列与我国深圳、上海、武汉地区的EV71流行株，中国台湾1998年4例HFMD暴发分离的EV71毒株，及来自美国、日本、匈牙利等国家地区的EV71毒株的核苷酸序列，用ClustalX1．8和PHYuP3．5进行比对分析，构建种系进化树。结果 25份标本检测EV71的阳性率为20％，所得序列经种系进化分析，与肠道病毒71型的其他毒株同源，与深圳1998年HFMD散发分离的EV71毒株同源性为94％，与上海2000年HFMD暴发分离株同源性为94％～96％，与武汉1987年HFMD病例中分离的毒株同源性为91％，而与国外EV71毒株同源性仅为82％～84％。结论 EV71是我国南方地区HFMD的主要病原之一；我国大陆地区的EV71毒株在种系进化上有较高的同源性；与我国台湾地区大部分分离株亦有90％～91％的核苷酸同源性，但大陆EV71引起的HFMD发病症状较轻，有别于1998年台湾地区EV71的大暴发。     

An outbreak of enterovirus 71 infection in Taiwan, 1998. II. Laboratory diagnosis and genetic analysis.

BACKGROUND: An epidemic of enterovirus 71 (EV71) occurred in Taiwan from April to December of 1998, with two peaks, one in June and the other in October. Many enteroviruses were isolated in our laboratory from 258 cases during this outbreak. Approximately half of the enteroviruses isolated were EV71 and one fifth were coxsackievirus A16. OBJECTIVES: To analyze laboratory findings in the EV71 epidemic of 1998 in Taiwan, various EV71 specimens in different cell lines were examined. In addition, genetic analysis of 5' non-coding region (NCR) was performed to analyze the strain variation in this outbreak. RESULTS: The cytopathic effect induced by EV71 was observed 2-13 (mean of 4.5) days post-inoculation in Vero cells and 4-15 (mean of 6.6) days in green monkey kidney (GMK) cells inoculated with throat swabs. Of the total positive EV71 cases, virus was most frequently obtained from throat swabs (91.7%), less from stools (64.8%), and none from cerebral spinal fluid (CSF). Molecular analyses of EV71 by sequencing the 5' NCR of 34 strains obtained from different clinical categories and various geographic areas showed that their sequences differed (0-13 bp in 681 bp sequenced) by approximately 0-2%. The sequences of these isolates differed from EV71 prototype BrCr or MS strain by 17.5-19%, with the exception of two samples which exhibited nucleotide variation by only 8.9 and 8.2%, when compared to the MS strain. CONCLUSION: EV71 was most frequently isolated from throat swab specimens in Vero cells. The molecular analyses of the 5' NCR of EV71 revealed that most isolates from this epidemic belonged to a group of closely related clones and only two were in a different group which was clustered with the EV71 MS strain.     

Isolation and identification of an enterovirus 77 recovered from a refugee child from Kosovo, and characterization of the complete virus genome

The complete nucleotide sequence of an enterovirus 77 isolate is reported. The virus designated FR/CF496-99 (France/Clermont-Ferrand 496-1999) was recovered from the feces of a 4-year-old child hospitalized for Salmonella gastroenteritis. The virus was identified by a molecular typing assay based on the genomic sequence encoding the VP1 capsid protein. The phylogenetic analysis based on the VP1 sequence demonstrated that the enterovirus isolated in the child clustered with viruses included in the human enterovirus B species (HEV-B) and was most closely related to enterovirus 77. A sliding window analysis of the complete genome showed an overall nucleotide similarity >80% between the P3 genomic region of the FR/CF496-99 isolate and that of the echovirus 30 prototype strain. A comparative analysis based on partial 3D(pol) sequences showed that the FR/CF496-99 virus was more closely related to recent enteroviruses from different serotypes and different geographical areas than to the prototype strains collected in the 1950s. This suggests that, in this enterovirus, the 3D(pol) encoding sequence is of recent origin.     

An outbreak of enterovirus 71 infection in Taiwan 1998: a comprehensive pathological, virological, and molecular study on a case of fulminant encephalitis.

BACKGROUND: In a recent enterovirus outbreak in Taiwan, serotype 71 was the culprit of encephalitis causing rapid clinical deterioration and death among young children. OBJECTIVES: Since knowledge of enterovirus 71 (EV71) infection in the central nervous system is still limited, the purpose of the present case study was attempted to uncover the pathogenesis of the virus. STUDY DESIGN: We performed a detailed pathological examination, virological and molecular studies on a case of EV71 infection with a rapidly fatal outcome. In addition, the whole genome of the virus was sequenced to determine the genetic relationships to other enteroviruses and two other EV71 strains (a prototype BrCr and a neurovirulent MS strain), and to provide the genetic basis of its neurovirulence of the new isolate, NCKU9822 strain. RESULTS: Characteristic features of acute encephalomyelitis were observed, with most prominent lesions in the spinal cord and brain stem. Mild myocarditis and pancreatitis were also noticed. EV71 antigen was localized to neurons on immunohistochemical staining. EV71 was recovered from all organs with inflammatory reaction. Sequence analysis showed that overall NCKU9822 and the two EV71 strains shared 80% nucleotide identity and 95% amino acid identity. It had only 45% amino acid and 52% nucleotide identities with polioviral P1 capsid region. CONCLUSION: The spinal cord and brain stem were the main targets of EV71 in the fatal cases in this outbreak, however, heart and pancreas might also be involved. Since the amino acid sequences in the P1 region are conserved (97% identity) among the three EV71 strains as compared to other enteroviruses and polioviruses, these EV71 neurovirulent strains might share the same mechanisms of neurovirulence, and the mechanisms might be different from those in polioviruses.     

Genetic analysis of the VP1 region of enterovirus 71 reveals the emergence of genotype A in central China in 2008

Enterovirus 71 (EV71) strains from children were characterized by full-length VP1 nucleotide sequencing. Out of 22 clinical specimens, five isolates identified as EV71 were recovered by virus isolation. The VP1 sequences of the five isolates had more than 97.4% sequence identity with prototype virus BrCr, clustering in the genotype A lineage. This represents the first record of genotype A EV71 in China since the BrCr prototype strain was discovered in the USA in 1969.     

肠道病毒71型山东临沂分离株全基因组序列分析

目的 自1例死亡患儿的标本中分离EV71,并分析其全基因组序列特点,研究其基因序列的改变是否与其神经毒力有关.方法 咽拭子标本采自山东临沂市人民医院1例死亡患儿,在人横纹肌瘤细胞(RD)上分离EV71,分段扩增获得EV71的全序列,用BLAST、Bioedit和MEGA 4进行序列分析.结果 获得1株EV71分离株SDLY107,基因组全长7405 bp,全基因组核苷酸序列与2008年的阜阳株Fuyang.Anhui.P.R.C/17.08/2同源性最高,为98.6%,与原型株BrCr/70的同源性为80.0%,与神经毒型株MS/87的同源性为86.5%.系统进化分析表明,SDLY107与中国大陆的北京株、河南株、广西株、深圳株、兰州株、阜阳株、重庆株、浙江株的亲缘关系较近,按照传统的VP1基因分型方法,可归为C4亚型,是近年来我国大陆流行的主要基因亚型.氨基酸序列分析发现,SDLY107与其他毒株相比,有2个特有的突变(E947D,K1873R).结论 SDLY107分离株属于C4亚型,氨基酸突变E947D和K1873R可能与EV71的致病性有关.

Abstract：

Objective To isolate enterovirus 71 from a death children,and analyze whether the neurovirulence was related to the variation of nucleotide and amino acid. Methods Enterovirus 71 was isolated from throat swabs which were colleted from Shandong Linyi People's Hospital. The full length genome was sequenced by amplification with RT-PCR and sequencing of 9 overlapped gene fragments covering full length of the genomes. The nucleotide and amino acid sequenced was aligned by BLAST, Bioedit and MEGA 4. Results A strain of enterovirus 71 was isolated and named as SDLY107. The full length was 7405 bp. The results of homology analysis of overall nucleotide sequence showed that strain Fuyang. Anhui. P. R. C/17.08/2 had highest homology (98.6%)with strain SDLY107, and the homology was 80.0% between strain SDLY107 with prototype strain BrCr/70,and 86. 5% between strain SDLY107 with nerve strain MS/87. Phylogenetic analysis showed that the phylogeny was close between SDLY107 with some isolated strains from Chinese Mainland, such as Beijing, Henan, Guangxi, Sbenzhen, Lanzhou, Fuyang, Chongqing and Zhejiang strains, which was clustered for C4 subtype. The results of amino acid sequence analysis showed that there were 2 mutations, E947D and K1873R, for strain SDLY107. Conclusion SDLY107 belonged to C4 subtype, amino acid mutations E947D and K1873R of which may be relevant to the pathogenicity of EV71.     

Molecular epidemiology of enterovirus 71 in Taiwan

Summary.  Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10⁻³vs. 1.4 × 1010⁻³) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance. Received June 13, 2000 Accepted July 29, 2000     

Identification of genetic diversity of porcine Norovirus and Sapovirus in Korea

It is well known that Norovirus (NoV) and Sapovirus (SaV) identified in humans and pigs have heterogeneous genome sequences. In this study, a total of three strains of NoV and 37 strains of SaV were detected in 567 porcine fecal samples by RT-PCR, corresponding detection rates of 0.5 and 6.5%, respectively. Phylogenetic analyses were conducted using amino acid sequences of the partial RNA-dependent RNA polymerase (RdRp) and complete capsid proteins of both viruses to determine their genogroups. Analysis with the RdRp sequences indicated that all three NoV strains HW41, DG32, and DO35 detected in this study were classified into genogroup II (GII). A further analysis with the complete capsid sequence demonstrated that the DO35 strain belonged to subgenotype b in GII-21 (GII-21b) along with the SW918 strain. A total of 26 strains out of 27 strains that were selected from the 37 porcine SaVs were classified into genogroup III when they were analyzed with the RdRp sequences. The remaining strain (DO19) was not clustered with any of the previously classified SaV strains, thereby suggesting the advent of a new genogroup virus. Additional analyses with the amino acid sequence of the capsid and the nucleotide sequence of the RdRp and capsid junction region supported the notion that the DO19 strain belonged to a novel genogroup of SaV. To the best of our knowledge, this is the first report to describe a novel porcine SaV belonging to an unknown genogroup in Korea.     

2008年广东省肠道病毒71型分离株全基因组核苷酸序列分析

目的 了解2008年广东省流行的肠道病毒(EV)71型基因特征.方法 选择2008年广东省分离的1株EV71毒株(GDFS-3),进行全基因组序列测定和基因进化特性的分析.结果 GDFS-3株与其他EV71型毒株相比,在编码区没有核苷酸的缺失和插入,其5'UTR和3'UTR的长度和序列有一定的差异.核苷酸同源性比较结果 表明,GDFS-3株与中国台湾流行株(TW984)的同源性最高(为96.0%),与新加坡流行株SIN5865及标准株MS、BrCr的同源性则在81.0%左右.氨基酸同源性比较结果 表明,GDFS-3株与TW984同源件最高(99.0%).根据VP1基因序列构建亲缘性关系树,GDFS-3株与C4亚型(subgenogroup)聚为一簇,与C4亚型代表株的核苷酸序列同源性为91.0%～95.0%.结论 遗传进化分析表明,GDFS-3株和中国台湾2004年流行的EV71毒株的亲缘关系最为密切,属于C4亚型,而与标准株BrCr和MS的亲缘关系较远.5'UTR的突变对于EV71毒力增强可能有重要作用.上述结果 有助于EV71型的基础研究和中国对于EV71所致疾病的预防.     

2008年深圳地区手足口病病原学调查

目的 研究2008年深圳地区手足口病病原体EV71和CoxA16感染情况,为手足口病的防治提供依据.方法 应用RT-PCR技术检测307例手足口病患儿标本病原体EV71、CoxA16基因,将PCR产物测序,并应用ChstaIW2在线分析软件进行序列分析、进化树分析.结果 不同标本中EV71的阳性率分别为:大便标本24.4%(75/307)、咽拭子为7.8%(24/307)、外周血12.5%(1/8);CoxA16的阳性率分别为,大便13.8%(28/203),咽拭子11.0%(20/181),其中EV71和CoxA16同时为阳性的有3份(0.98%);脑脊液标本未检测到EV71和CoxA16.与标准株BrCr序列比对分析,测序的14例标本中,8例的EV71新出现多个位点变异,其中2例标本的A2528G变异导致氨基酸改变,即N595D,1例标本的A2714G变异引起氨基酸改变,即1658V.进化树分析显示,有2例标本的病毒株与anhui毒株亲缘关系较近,与标准株BrCr及深圳株SHH02-6、SHZH02-40、SHZH03-58等亲缘关系较远;而其余12株与上述毒株亲缘关系较远.根据文献报道的EV71基因分型方法分析,测序的14株EV71基因型为C型.结论 在引起2008年深圳手足口病病原体中相当的比例是EV71,且可能部分是anhui病毒株的入侵.     

Evaluating the prevalence and molecular epidemiology of echovirus 11 isolated from sewage in Shandong Province, China in 2010

Echovirus 11 (E11) is an important human pathogen, but its genetic information in China is in scarce. In this study, 12 sewage samples from Jinan city and 18 from Linyi city were collected in Shandong Province, China in 2010, and E11 was the predominant serotype with 54 isolates from 16 samples. Numbers of E11 isolates reached peaks in August in both Jinan and Linyi city, while another peak occurred in December in Linyi. The complete VP1 genes of all these isolates were sequenced and phylogenetically compared with clinical isolates from Shandong in 1994-2010 (n?=?29) and global E11. Shandong isolates segregated into five clusters, four in genogroup A and one in genogroup C. Environmental isolates belonged to two clusters of genogroup A, with high inter-cluster genetic divergence (18.5-20.9%). No local clinical E11 was isolated in the two cities in 2010, revealing the value of environmental surveillance in investigating circulating viruses. These findings underscored the significance of environmental VP1 sequence divergences in comprehending the local enterovirus circulation, and updated the global molecular epidemiology of E11.     

Combined 5′ UTR RFLP analysis and VP1 sequencing for epidemic investigation of enteroviruses

Enteroviruses, the main cause of aseptic meningitis, consist of 100 serotypes, and many of them have been associated with large outbreaks. In the present study, a comparison of RFLP analysis of the 5′ untranslated region (5′UTR) and sequencing of both the 5′UTR and VP1 regions was conducted for epidemiological linkage of 27 clinical enterovirus strains. The clinical enterovirus strains were clustered into five restriction profile groups. Even though the restriction profile clusters of clinical isolates were not related to those of the respective prototype strains, epidemiological relationships between the members of each cluster were observed. The restriction profile clusters in the 5′UTR corresponded to the phylogenetic clusters in the VP1 genomic region. The incongruence between the topology of Gior strain in 5′UTR and VP1 phylogenetic trees indicates a recombination event. The proposed RFLP assay in combination with VP1 sequencing can offer crucial epidemiological information about the circulating enteroviruses.     

A large Finnish echovirus 30 outbreak was preceded by silent circulation of the same genotype

An outbreak of echovirus 30 (E-30) in 2009 was confirmed by both frequent isolation of the virus from sewage as well as from patient samples in Finland. Over the last 10 years E-30 had only been isolated sporadically in Finland. We here study the phylogenetic relationships of the strains from the outbreak in the context of E-30 circulation over the last 20 years. The analyzed region comprised 276 nucleotides in the 5′ end of VP1 (nucleotides 132–407 in the VP1 of the E-30 Bastianni strain). The Finnish strains were clustered into at least four distinct genogroups, with seven clusters exceeding the genotype demarcation of 12% and the 2009 epidemic strains forming the largest genogroup VII. Moreover, we detected largely divergent genotypes in 2007 and 2009. Interestingly, close genetic relatives of the epidemic strains had already been isolated a few years before the outbreak. Phylodynamic analysis estimated 8.9 years (95% highest posterior density intervals 7.0–11.0) as the age of genogroup VII, indicating a probable origin and evolutionary history prior to its introduction and epidemic expansion in Finland. Finally, the most recent common ancestor for the current E-30 diversity dates back to 1939 (95% highest posterior density intervals 1913–1956).     

Novel recombinant norovirus causing outbreaks of gastroenteritis in Santiago, Chile

Capsid and polymerase (RdRp) genes of 13 norovirus outbreak strains from Chile were compared. The genes sequences were discordant for five strains, and recombination was confirmed for two of them by amplification of a 1,360-bp gene segment containing a fragment of both genes. These strains belonged to a novel genogroup by RdRp sequence and to genogroup GII/3 by capsid sequence. Determining the clinical and epidemiological impact of human calicivirus recombination will require future studies.     

Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71

In Taiwan, enterovirus 71 (EV71) has played an important role in severe enterovirus-related cases every year since the devastating outbreak in 1998. Three genogroups A, B, C occur worldwide; with the B and C genogroups being subdivided into B1-B4 and C1-C4 subgenogroups respectively. To understand the mutation of the EV71 genogroup in Taiwan before and after 1998, a total of 54 worldwide strains were studied including 41 Taiwanese strains obtained in 1986 and 1998-2004. A fragment of 207 bp of the VP4 region was amplified and sequenced. Genetic analysis was performed using MEGA software (version 3.0) for the nucleotide sequence alignment and phylogenetic analysis. In Taiwan, the subgenogroup B1 was predominant before 1998 while subgenogroup C2 was the major etiologic group in 1998 outbreak. A minor etiologic group outbreak in 1998, subgenogroup B4, became predominant during the period from 1999 to 2003. In this study, subgenogroup C4 emerged and became predominant in 2004 in Taiwan. The nucleotide differences between B1 and C2, C2 and B4, B4 and C4 were 20%-26%, 19%-27%, 18%-22%, respectively. Nucleotide sequence alignment revealed 67 substitutions. Most of the substitutions (62/67) were silent mutations. This is the first report about the emergence of EV71 subgenogroup C4 in Taiwan.     

Genetic diversity of bovine <Emphasis Type="Italic">Picobirnavirus</Emphasis>, Brazil

Picobirnaviruses (PBVs) are emerging and opportunistic viruses with possible zoonotic potential. In this study, we present the detection, molecular characterization, and genotypic differentiation of PBVs from genogroup I in bovine stool samples from different Brazilian regions. A high proportion of PCR-positive samples (23.4%) was detected in a total of 77 analyzed. Nucleotide identity, alignment, and phylogenetic analyses revealed high diversity among the studied sequences. The results obtained indicate, for the first time, the circulation of bovine PBVs belonging to genogroup I in different Brazilian states, with heterogeneous phylogenetic-clustering profiles.     

Molecular characterization of enteric viral agents from children in northern region of Ghana

Viral gastrointestinal infections are among the most important causes of childhood morbidity and mortality, especially in non-industrialized countries. The objective of this study was the molecular characterization of rotaviruses, noroviruses, adenoviruses, astroviruses, and enteroviruses obtained from 367 children in the Northern Region of Ghana. One hundred and forty-two rotavirus-positive stool samples were examined. The most frequent type identified was G1P[8] occurring in 80% of the cases. Of 27 norovirus positive samples, 5 isolates belonged to genogroup I and 22 to genogroup II. Adenoviruses were detected in 73 samples; 23.3% of these belonged to genogroup F, 31.5% to D, 17.8% to A, 15.1% to C, and 12.3% to B. Astrovirus typing of 12 positive samples displayed a distribution into four different genotypes: five sequences clustered with AstV-8, four with AstV-2, two with AstV-5, and one with AstV-6. Twenty-three different enterovirus types were identified in 45 positive samples, coxsackievirus A24 being the most frequent pathogen (18%). This first, comprehensive molecular characterization of enteric viruses in northern Ghana provides baseline data for the molecular epidemiology of these pathogens and immunisation strategies. The available rotavirus vaccines cover the predominant G1P[8] type and would reduce substantially disease burden in that area.