辽宁省首次检出G9型A组轮状病毒

目的了解辽宁省A组轮状病毒的感染情况,为轮状病毒的预防控制提供科学依据。方法采集辽宁省沈阳、大连、丹东、阜新4个市门诊及住院疑似病毒性腹泻患者粪便标本135份,采用酶联免疫吸附试验(ELISA)检测轮状病毒抗原,阳性标本用逆转录-聚合酶链反应(RT-PCR)扩增A组轮状病毒VP7基因和VP4基因,RT-PCR产物进行核苷酸碱基序列的测定和比对,并构建VP7基因遗传进化树。结果 135份粪便标本中,共检测出A组轮状病毒阳性标本21份;A组轮状病毒G基因分型:G9型15株,G3型2株,G2型1株,G1型1株,未分型2株;P基因型分型:P[8]型20株,P[4]型1株;G/P基因型组合以G9P[8]为主共15株,G3P[8]2株,G1P[8]1株,G2P[4]1株,G/P[8]2株。结论辽宁省首次检出G9型A组轮状病毒,主要流行G/P基因型组合为G9P[8]。     

北京地区2007-2008年G9型轮状病毒VP4、VP6、NSP4和NSP5基因分析

本研究室在2007-2008年收集的临床标本中再次发现北京地区儿童中有G9型轮状病毒的感染,并已对其进行了VP7基因分析和VP4基因巢式PCR分型~([1,2]).为进一步了解北京G9型轮状病毒的重要蛋白基因的进化,本研究室对2例从社区感染患儿中发现的北京G9型VP4、VP6、NSP4和NSP5基因进行测序及进化分析,结果简报如下.     

中国1998～2004年G9型轮状病毒分子流行病学研究

目的研究中国流行的轮状病毒(Rotavirus,RV)G9型毒株的分子流行病学特征。方法在中国9个地区收集5岁以下腹泻患儿粪便标本,应用酶联免疫吸附试验(ELISA)方法检测RV,对阳性标本用逆转录-聚合酶链反应(RT-PCR)分型,选择G9型毒株进行VP7基因全长克隆测序和分子流行病学分析。结果1998~2004年共检测出RV1 268份,其中45份为G9型(3.5%),昆明最多(34/45),其次是兰州(8/45)、长春(2/45)、卢龙(1/45),北京、郑州、杭州、福州、广州未检测到G9型毒株。对35份G9型标本进行P分型鉴定:15份为P[8]型,12份为P[6]型,5份为P[4]型,1份为P[8 4]混合型,2份未能分型。中国1998~2000年以P[8]G9毒株流行为主,而2001年后以P[6]G9毒株为主。22株G9型病毒VP7基因序列比对结果表明,中国流行的G9型毒株彼此同源性高,同属G9第3亚型。结论中国流行的RV G9型株与世界各地的流行株同源性较高,国内传播范围扩大的趋势值得关注。     

武汉市儿童医院婴幼儿腹泻轮状病毒的VP4型别分析

目的研究武汉市儿童医院腹泻门诊A组轮状病毒VP4基因的分子流行病学特征. 方法利用聚丙烯酰胺凝胶电泳,将检测出的A组轮状病毒阳性样利用多重RT-PCR技术对VP4基因进行分型研究. 结果武汉地区793份腹泻患儿粪便样本经检测轮状病毒阳性257例,阳性率为32.4%.其中P [8]型232例(90.3%),P [4]型3例(1.2%),P [8]与P [4]混合感染15例(5.8%),尚有7例(2.7%)未能分出型别.对检测结果按采样时间、年龄和性别分布分别进行了分析. 结论武汉地区A组轮状病毒以P[8]型为主要流行基因型,患儿以6月至1岁为主,男女性别差异不大,武汉地区婴幼儿腹泻A组轮状病毒VP4基因分型研究将为轮状病毒疫苗的研制提供基础.     

福州地区2009-2014年腹泻儿童轮状病毒特征

目的 了解轮状病毒在福州地区腹泻儿童中的流行情况.方法 收集2009-2014年5岁以下腹泻住院儿童粪便标本,用ELISA法检测轮状病毒抗原,RT-PCR法确定基因型别.对G9轮状病毒阳性标本的VP7基因全长测序及进化分析.结果 福州地区腹泻儿童轮状病毒高峰期在10～12月,呈单峰流行态势.G9轮状病毒在2011年后成为福州地区优势流行型别,毒株VP7基因核苷酸序列相似性92.5％～100％,毒株间有较高的同源性,进化分析都属于G9第3亚型.结论 G9轮状病毒在福州地区流行强度逐渐加强,目前已成为优势流行型别.毒株同源性高,属G9型3亚型.     

青海省67份轮状病毒结构蛋白VP7基因分型和分布情况

目的 研究2016-2017年青海地区腹泻患者轮状病毒基因分型及流行病学分布。方法 收集门诊及住院部腹泻的粪便标本238份,采用实时荧光PCR法对轮状病毒A组进行检测,阳性标本进行VP7基因扩增和测序。结果 238份粪便标本中,通过实时荧光PCR 检测到轮状病毒A组阳性67份,阳性率为28.15%（67/238）;对67份轮状病毒A阳性标本进行VP7蛋白检测和测序,测序后得到29份核苷酸序列,用Clustral X Bootstap NJ Tree软件构件进化树,分析发现2016年3月-2017年12月青海轮状病毒以G9P8型为主,共26株,占89.66% (26/29),G2P4型2株(2/28),G3P8型1株(1/28), 轮状病毒腹泻发病高发季节为9-12月,其中以12月份检出最高,占总数的61.19%（41/67）。病人以成人为主,成人和5岁以下儿童比例为1.73[DK]∶1。结论 2016-2017年青海地区轮状病毒以流行病毒株G9P8型为主。     

酒泉地区婴幼儿腹泻的A组轮状病毒分子基因特征研究

目的采用基因分型方法调查研究酒泉地区婴幼儿A组轮状病毒（rotavirus，RV）腹泻的基因分型特点。方法对2001年11月至2002年12月酒泉地区收集的腹泻婴幼儿324份粪便标本，电脑随机抽样选取204份采用酶联免疫吸附试验（enzyme—linked immunosorbant assay，ELISA）和逆转录聚合酶链反应（RT—PCR），进行轮状病毒病原检测，扩增A组轮状病毒VP4和VP7基因，并对阳性标本进行基因分型。结果酶联免疫吸附试验法测得204份粪便标本的A组轮状病毒阳性率为54．9％（112／204）。对酶联免疫吸附试验呈阳性的标本进行逆转录-聚合酶链反应法检测A组轮状病毒G，P基因型，结果表明，酒泉地区流行的A组轮状病毒主要基因型为G3P[8]型（50．0％），其余依次为G1P[4]（15．8％）、G3P[4]（15．8％）、G1P[8]（10．6％）、G2P[4]（2．6％）和G1G3P[4]（2．6％）、G1G3P[8]（2．6％）。结论A组轮状病毒是酒泉地区婴幼儿非细菌性腹泻的主要病原，其中G3P[8]型是主要基因型。     

A组轮状病毒G3型哈尔滨地方株VP7基因序列分析

目的:了解我国A组轮状病毒G3型哈尔滨地方株与标准株及国内外部分地区G3型地方株VP7蛋白基因序列的差异,为轮状病毒疫苗在我国东北地区的研发与应用提供资料。方法:通过一步法RT-PCR获得了6株轮状病毒哈尔滨地方株VP7蛋白的cDNA片段,,对其进行扩增、克隆、测序,用DNASTAR生物软件进行序列分析。结果:6株哈尔滨地方株VP7蛋白推导氨基酸序列同源性99.4%～100%;与G3标准株Crw-8的同源性为94.2%;与G1、G2、G4标准株比对变异较大(75.2%～82.2%);哈尔滨地方株氨基酸序列在aa108、aa266、aa268、aa278位点的变异完全相同。结论:6株轮状病毒G3型哈尔滨地方株来源于相同的一支G3型轮状病毒毒株。     

呼和浩特地区2008-2009年婴幼儿轮状病毒性腹泻流行株分型研究

目的研究呼和浩特地区2008年6月～2009年5月婴幼儿腹泻轮状病毒（rotavirus,RV）的基因型。方法采集内蒙古妇幼保健院2008年6月～2009年5月所有≤59月龄的住院腹泻患儿粪便标本313份。采用酶联免疫吸附试验（ELISA）检测RV;对RV抗原阳性的标本用巢式逆转录聚合酶链式反应（nested RT-PCR）对6种流行的VP7基因型（G1、G2、G3、G4、G8、G9）和5种流行的VP4基因型（P[8]、P[4]、P[6]、P[9]、P[10]）进行分型;最后整理数据,应用SPSS 13.0统计软件包进行统计分析。结果 313份粪便标本中,125份为RV抗原阳性（39.94%）。对阳性标本进行VP7基因分型,结果为G1型64.8%（81/125）、G2型2.4%（3/125）、G3型14.4%（18/125）、G9型0.8%（1/125）、G1+G3混合型16.8%（21/125）、G2+G3混合型0.8%（1/125）;进行VP4基因分型,结果为P[8]型84.0%（105/125）、P[4]型4.0%（5/125）、P[8]+P[4]型1.6%（2/125）,13份...     

呼和浩特地区2008-2009年婴幼儿轮状病毒性腹泻流行株分型研究

目的研究呼和浩特地区2008年6月～2009年5月婴幼儿腹泻轮状病毒(rotavirus,RV)的基因型。方法采集内蒙古妇幼保健院2008年6月～2009年5月所有≤59月龄的住院腹泻患儿粪便标本313份。采用酶联免疫吸附试验(ELISA)检测RV;对RV抗原阳性的标本用巢式逆转录聚合酶链式反应(nested RT-PCR)对6种流行的VP7基因型(G1、G2、G3、G4、G8、G9)和5种流行的VP4基因型(P[8]、P[4]、P[6]、P[9]、P[10])进行分型;最后整理数据,应用SPSS 13.0统计软件包进行统计分析。结果 313份粪便标本中,125份为RV抗原阳性(39.94%)。对阳性标本进行VP7基因分型,结果为G1型64.8%(81/125)、G2型2.4%(3/125)、G3型14.4%(18/125)、G9型0.8%(1/125)、G1+G3混合型16.8%(21/125)、G2+G3混合型0.8%(1/125);进行VP4基因分型,结果为P[8]型84.0%(105/125)、P[4]型4.0%(5/125)、P[8]+P[4]型1.6%(2/125),13份未分出型别。结论 RV是...     

Molecular characterization of rotavirus strains detected during a clinical trial of a human rotavirus vaccine in Blantyre, Malawi

The human, G1P[8] rotavirus vaccine (Rotarix?) significantly reduced severe rotavirus gastroenteritis episodes in a clinical trial in South Africa and Malawi, but vaccine efficacy was lower in Malawi (49.5%) than reported in South Africa (76.9%) and elsewhere. The aim of this study was to examine the molecular relationships of circulating wild-type rotaviruses detected during the clinical trial in Malawi to RIX4414 (the strain contained in Rotarix?) and to common human rotavirus strains. Of 88 rotavirus-positive, diarrhoeal stool specimens, 43 rotaviruses exhibited identifiable RNA migration patterns when examined by polyacrylamide gel electrophoresis. The genes encoding VP7, VP4, VP6 and NSP4 of 5 representative strains possessing genotypes G12P[6], G1P[8], G9P[8], and G8P[4] were sequenced. While their VP7 (G) and VP4 (P) genotype designations were confirmed, the VP6 (I) and NSP4 (E) genotypes were either I1E1 or I2E2, indicating that they were of human rotavirus origin. RNA-RNA hybridization using 21 culture-adapted strains showed that Malawian rotaviruses had a genomic RNA constellation common to either the Wa-like or the DS-1 like human rotaviruses. Overall, the Malawi strains appear similar in their genetic make-up to rotaviruses described in countries where vaccine efficacy is greater, suggesting that the lower efficacy in Malawi is unlikely to be explained by the diversity of circulating strains.     

Genotypic linkages of VP4, VP6, VP7, NSP4, NSP5 genes of rotaviruses circulating among children with acute gastroenteritis in Thailand

Rotavirus is the main cause of acute viral gastroenteritis in infants and young children worldwide. Surveillance of group A rotavirus has been conducted in Chiang Mai, Thailand since 1987 up to 2004 and those studies revealed that group A rotavirus was responsible for about 20-61% of diarrheal diseases in hospitalized cases. In this study, we reported the continuing surveillance of group A rotavirus in 2005 and found that group A rotavirus was detected in 43 out of 147 (29.3%) stool samples. Five different G and P genotype combinations were detected, G1P[8] (27 strains), G2P[4] (12 strains), G9P[8] (2 strains), G3P[8] (1 strain), and G3P[10] (1 strain). In addition, analysis of their genotypic linkages of G (VP7), P (VP4), I (VP6), E (NSP4), and H (NSP5) genotypes demonstrated that the rotaviruses circulating in Chiang Mai, Thailand carried 3 unique linkage patterns. The G1P[8], G3P[8], and G9P[8] strains carried their VP6, NSP4, NSP5 genotypes of I1, E1, H1, respectively. The G2P[4] strains were linked with I2, E2, H2 genotypes, while an uncommon G3P[10] genotype carried unique genotypes of I8, E3 and H6. These findings provide the overall picture of genotypic linkage data of rotavirus strains circulating in Chiang Mai, Thailand.     

Molecular characterization of group A rotaviruses detected in children with gastroenteritis in Ireland in 2006-2009

Community and hospital-acquired cases of human rotavirus are responsible for millions of gastroenteritis cases in children worldwide, chiefly in developing countries, and vaccines are now available. During surveillance activity for human rotavirus infections in Ireland, between 2006 and 2009, a total of 420 rotavirus strains were collected and analysed. Upon either PCR genotyping and sequence analysis, a variety of VP7 (G1-G4 and G9) and VP4 (P[4], P[6], P[8] and P[9]) genotypes were detected. Strains G1P[8] were found to be predominant throughout the period 2006-2008, with slight fluctuations seen in the very limited samples available in 2008-2009. Upon either PCR genotyping and sequence analysis of selected strains, the G1, G3 and G9 viruses were found to contain E1 (Wa-like) NSP4 and I1 VP6 genotypes, while the analysed G2 strains possessed E2 NSP4 and I2 VP6 genotypes, a genetic make-up which is highly conserved in the major human rotavirus genogroups Wa- and Kun-like, respectively. Upon sequence analysis of the most common VP4 genotype, P[8], at least two distinct lineages were identified, both unrelated to P[8] Irish rotaviruses circulating in previous years, and more closely related to recent European humans rotaviruses. Moreover, sequence analysis of the VP7 of G1 rotaviruses revealed the onset of a G1 variant, previously unseen in the Irish population.     

Genetic diversity of species A rotaviruses detected in clinical and environmental samples,including porcine-like rotaviruses from hospitalized children in the Philippines

Rotaviruses are the major cause of severe acute diarrhea in infants and young children. Rotaviruses exhibit zoonosis and thereby infect both humans and animals. Viruses detected in urban rivers possibly reflect the presence of circulating viruses in the catchment. The present study investigates the genetic diversity of species A rotaviruses detected from river water and stool of hospitalized children with acute diarrhea in Tacloban City, the Philippines. Species A rotaviruses were detected by real-time RT-PCR and their genotypes were identified by multiplex PCR and sequencing of partial regions of VP7 and VP4. Rotaviruses were detected in 85.7% (30/35) of the river water samples and 62.7% (151/241) of the clinical samples. Genotypes of VP7 in the river water samples were G1, G2, G3, G4, G5, and G9, and those of VP4 were P[3], P[4], P[6], P[8], and P[13]. Genotypes of viruses from the clinical samples were G2P[4], G1P[8], G3P[8], G4P[6], G5P[6], and G9P[8]. Among those, G2P[4] in clinical samples (77.9%, 81/104) and P[4] of VP4 in river water samples (67.5%, 56/83)) were the most frequently detected rotavirus genotypes. However, G5 was the more frequently detected than G2 in the river water samples (42% vs. 13%) which may be originated from porcine rotavirus. Sequence analyses of eleven gene segments revealed one G5P[6] and two G4P[6] rotaviruses in the clinical samples, wherein, several gene segments were closely related to porcine rotaviruses. The constellation of these rotavirus genes suggests the emergence of reassortment between human and porcine rotavirus due to interspecies transmission.Although two commercial rotavirus vaccines are available now, these vaccines are designed to confer immunity against the major human rotaviruses. Constant monitoring of viral variety in populated areas where humans and domestic animals live in close proximity provides vital information related to the diversity of rotaviruses in a human population.     

Identification of a multi-reassortant G12P[9] rotavirus with novel VP1, VP2, VP3 and NSP2 genotypes in a child with acute gastroenteritis

The G12 rotavirus genotype is globally emerging to cause severe gastroenteritis in children. Common G12 rotaviruses have either a Wa-like or DS-1-like genome constellation, while some G12 strains may have unusual genome composition. In this study, we determined the full-genome sequence of a G12P[9] strain (ME848/12) detected in a child hospitalized with acute gastroenteritis in Italy in 2012. Strain ME848/12 showed a complex genetic constellation (G12-P[9]-I17-R12-C12-M11-A12-N12-T7-E6-H2), likely derived from multiple reassortment events, with the VP1, VP2, VP3 and NSP2 genes being established as novel genotypes R12, C12, M11 and N12, respectively. Gathering sequence data on human and animal rotaviruses is important to trace the complex evolutionary history of atypical RVAs.     

Whole genome sequencing reveals genetic heterogeneity of G3P[8] rotaviruses circulating in Italy

After a sporadic detection in 1990s, G3P[8] rotaviruses emerged as a predominant genotype during recent years in many areas worldwide, including parts of Italy. The present study describes the molecular epidemiology and evolution of G3P[8] rotaviruses detected in Italian children with gastroenteritis during two survey periods (2004–2005 and 2008–2013). Whole genome of selected G3P[8] strains was determined and antigenic differences between these strains and rotavirus vaccine strains were analyzed. Among 819 (271 in 2004–2005 and 548 in 2008–2013) rotaviruses genotyped during the survey periods, the number of G3P[8] rotavirus markedly varied over the years (0/83 in 2004, 30/188 in 2005 and 0/96 in 2008, 6/88 in 2009, 4/97 in 2010, 0/83 in 2011, 9/82 in 2012, 56/102 cases in 2013). The genotypes of the 11 gene segments of 15 selected strains were assigned to G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1; thus all strains belonged to the Wa genogroup. Phylogenetic analysis of the Italian G3P[8] strains showed a peculiar picture of segregation with a 2012 lineage for VP1-VP3, NSP1, NSP2, NSP4 and NSP5 genes and a 2013 lineage for VP6, NSP1 and NSP3 genes, with a 1.3–20.2% nucleotide difference from the oldest Italian G3P[8] strains. The genetic variability of the Italian G3P[8] observed in comparison with sequences of rotaviruses available in GenBank suggested a process of selection acting on a global scale, rather than the emergence of local strains, as several lineages were already circulating globally. Compared with the vaccine strains, the Italian G3P[8] rotaviruses segregated in different lineages (5–5.3% and 7.2–11.4% nucleotide differences in the VP7 and VP4, respectively) with some mismatches in the putative neutralizing epitopes of VP7 and VP4 antigens. The accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses might generate, over the years, vaccine-resistant variants.     

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.     

Evidence of discordant genetic linkage in the VP4, VP6, VP7 and NSP4 encoding genes of rotavirus strains from adolescent and adult patients with acute gastroenteritis

The vast diversity within rotavirus strains circulating in the developing countries continues to be a major challenge for the efficacy of currently used preset rotavirus vaccines. The sequence analysis and phylogeny of multiple genes of rotavirus strains enable identification of reassortant strains and their human or animal origin. The objective of this study was to monitor the genetic linkage between the rotavirus VP4(P), VP6(I), VP7(G) and NSP4(E) encoding genes. The G, P, I and E genotypes of a total of 80 rotavirus strains isolated from adolescent and adult cases of acute gastroenteritis at the two time points [1993–1996 (n = 67) and 2004–2007 (n = 13)] were determined by nucleotide sequencing and phylogenetic analysis. The rotavirus strains from the 1990s and 2000s revealed common combinations of genotypes (G1–P[8]–I1–E1, G2–P[4]–I2–E2, G3–P[8]–I1–E1 and G4–P[8]–I1–E1) in 47.8% and 30.8%, unusual combinations of the same genotypes (G2–P[8]–I2–E2, G9–P[6]–I1–E1, G9–P[6]–I1–E2, G9–P[6]–I2–E1 and G4–P[4]–I1–E2, G1–P[4]–I2–E1, G9–P[4]–I1–E1) in 7.5% and 23% and mixed infections of different G and P genotypes in 31.3% and 46.2%, respectively. Discordance in the association of I with E, G with I and E and P with I and E genotypes was found to be contributed respectively by 23.8–38.5%, 40.3–69.8% and 49.3–61.5% of the rotavirus strains at the two time points.The data suggest relatively high occurrence of intergenogroup reassortment in circulating rotavirus strains emphasizing the need for continuous surveillance and whole genome sequence based characterization of rotavirus strains for better understanding of their evolution and ecology.     

Sequence and phylogenetic analyses of human rotavirus strains: Comparison of VP7 and VP81 antigenic epitopes between Tunisian and vaccine strains before national rotavirus vaccine introduction

Group A rotaviruses (RVA) are the leading cause of severe gastroenteritis in infants and young children worldwide. Due to their epidemiological complexity, it is important to compare the genetic characteristics of vaccine strains with the RVA strains circulating before the introduction of the vaccine in the Tunisian immunization program. In the present study, the nucleotide sequences of VP7 and VP8¹ (n = 31), the main targets for neutralizing antibodies, were determined. Comparison of antigenic epitopes of 11 G1P[8], 12 G2P[4], 4 G3P[8], 2 G4P[8], 1 G6P[9] and 1 G12P[8] RVA strains circulating in Tunisia from 2006 to 2011 with the RVA strains present in licensed vaccines showed that multiple amino acid differences existed in or near putative neutralizing domains of VP7 and VP8¹. The Tunisian G3 RVA strains were found to possess a potential extra N-linked glycosylation site. The Tunisian G4 RVA were closely related to the G4 vaccine strain in RotaTeq, belonging to the same lineage, but the alignment of their VP7 amino acids revealed an insertion of an asparagine residue at position 76 which is close to a glycosylation site (aa 69–71). Despite several differences detected between Tunisian and vaccine strains, which may affect binding of neutralizing antibodies, both vaccines are known to protect against the vast majority of the circulating genotypes, providing an indication of the high vaccine efficiency that can be expected in a future rotavirus immunization program.