婴幼儿腹泻A群轮状病毒G和P的基因分型研究

目的研究浙江萧山医院婴幼儿童腹泻标本中人轮状病毒（Human Rotavims）毒株的感染情况及G和P基因型流行特点。方法收集该院2009年8月至2010年8月腹泻儿童15233份粪便标本采用酶联免疫吸附试验、逆转录-巢式聚合酶链反应进行轮状病毒病原检测,将128份阳性标本进行VP7和VP4基本分型。结果15233份婴幼儿腹泻标本中有2706份标本为轮状病毒阳性,阳性率17．8％;男孩和女孩检出率差异无统计学意义,以6-12月龄段检出率最高;对128份阳性标本进行G血清分型和P基因分型,G1型53份（41．4％）、G3型38份（29．7％）、G1G3型17份（13．3％）、G未分型20份（15．6％）;P[8]型72份（56．3％）、P[4]型16份（12．5％）、P[8]P[4]型3份（2．3％）、P未分型37份（28．9％）,G血清型和P基因型的组合以G1P[8]为主,占29．7％（38／128）。结论浙江萧山医院A群轮状病毒G血清以G1型为主,其次为G3型,P基因型以P[8]型为主。     

2009～2010年北京儿童腹泻中轮状病毒新VP4基因亚型的研究

P[8]b基因亚型是国内外新近发现的A组人轮状病毒(HRV)VP4基因的一种新亚型,本研究旨在建立有效鉴别HRV P[8]a和P[8]b基因亚型及P[4]和P[6]基因型的VP4基因打点杂交分型方法,并运用此方法对2009～2010年首都儿科研究所附属儿童医院门诊及住院腹泻患儿中P[8]b基因亚型HRV的流行情况及其G/P基因组合情况进行研究。通过对GenBank序列数据库可检索到的国内外HRV各种P基因型及亚型的VP4基因序列应用相关软件进行基因分析,在不同基因型别间核苷酸变异密集而相同P基因型内核苷酸高度保守的的位置设计各型别探针,并分别以本实验室上传GenBank的北京HRV地方株P[4]和P[6]基因型及P[8]a和P[8]b亚型的VP4基因作为相应型别探针的合成引物的设计模板及探针经PCR合成的合成模板,合成地高辛素标记的DNA探针。经测序验证所建立的VP4基因杂交分型方法结果可靠。对门诊88例(55%,88/160)及住院79例(70.5%,79/112)HRV腹泻患儿的P分型结果显示P[8]a亚型仍为主要型别,前者为96.6%(85/88),而后者为62.0%(49/79);P[8]b亚型在住院HRV感染腹泻患儿中占较高比例(27.9%,22/79),虽然其在门诊HRV感染患儿中也存在,但仅占2.3%(2/88);另外单纯P[4]基因型HRV感染仅在住院腹泻患儿中检测到1例(1.3%,1/79),而P[6]基因型在门诊及住院HRV感染腹泻患儿中均未检测到;本组标本中HRV P[8]b亚型主要与G9基因型组合。本研究表明G9P[8]b型HRV在北京腹泻儿童中有流行。     

胶体金法与RT-PCR法测定A群轮状病毒及G血清型分型

目的了解婴幼儿A群轮状病毒腹泻的流行病学特征。方法收集浙江省苍南县人民医院2009年1-12月份的婴幼儿腹泻粪便标本,采用胶体金免疫层析法和逆转录-聚合酶链反应法进行轮状病毒抗原检测和血清型分型,分析G血清型分型。结果 828份婴幼儿腹泻粪便轮状病毒阳性率为33.45%。在轮状病毒腹泻患儿中,1岁以内占49.82%,2岁以内占89.89%。本地区轮状病毒腹泻呈现季节高峰,11月至次年1月为轮状病毒腹泻流行期。毒株分型显示G3型为流行优势株（51.6%）,其次是G1型（26.6%）;另有13.7%为混合感染,包括G1＋G3型（10.5%）、G2＋G3型（2.4%）和G1＋G9型（0.8%）。结论 2009年浙江省苍南县婴幼儿轮状病毒腹泻的主要血清型是G3和G1,G3为优势毒株。 更多还原     

苏浙闽地区婴幼儿急性腹泻患者病毒检测及 临床特征分析

目的分析苏浙闽地区婴幼儿急性腹泻患者常见病毒及患儿临床特征,为婴幼儿急性腹泻的流行病学研究和临床治疗提供参考。方法选取2010至2014年苏浙闽地区多家医院门诊就诊的急性腹泻患儿1 452例,采用ELISA法检测A组轮状病毒(HRV),多重PCR法检测诺如病毒(NoV)、肠道腺病毒(EAdV)和星状病毒(HAstV),并用RT-PCR法对A组轮状病毒阳性的样本进行G和P分型。结果 1 452例患儿中由常见病毒引起的急性腹泻患儿有707例,占总调查患者的48.69%。急性腹泻患儿常见病毒感染中单一病毒感染占90.10%(637/707),其感染病毒的构成以轮状病毒最多(47.52%,336/707),其次为诺如病毒(36.07%,255/707)以及星状病毒(2.40%,17/707)和肠道腺病毒(4.10%,29/707)。病毒感染腹泻患儿中多重感染者共70例,占9.90%(70/707),以A组轮状病毒合并诺如病毒感染最为常见,占多重感染者的68.57%(48/70)。226份A组轮状病毒阳性标本中,G分型以G1为优势分型,占33.19%;P分型中则以P [11]比例最高,占38.50%;G、P型组合以P [11]G11为主(38.50%)。结论 A组轮状病毒是引发婴幼儿急性腹泻的主要病毒,诺如病毒次之,且婴幼儿病毒性腹泻患者可合并多种病毒感染。不同地区腹泻患儿病毒感染阳性率有差异,轮状病毒基因型表现则具有多样性。     

昆明婴幼儿腹泻的轮状病毒分子基因特征研究

目的了解昆明婴幼儿腹泻中轮状病毒感染情况。方法收集昆明医学院第一附属医院儿科2004年10-12月住院的腹泻的患儿粪便标本60份,采用聚丙烯酰胺凝胶电泳（PAGE）法和RT—PCR法进行轮状病毒分型。结果60份粪便标本中,29份检测到A组轮状病毒RNA基因,阳性检出率为48.3％,未发现B组及C组轮状病毒。其中RNA长型有29份（100％）,未发现短型和混合型。对29份有明确编号的轮状病毒阳性标本来源的患儿分析显示,平均发病年龄10.5个月。29份标本中,有20份可用RT—PCR分型,均为G3型,未发现其他型。结论A组轮状病毒是昆明地区5岁以下婴幼儿腹泻病的主要病原,3月龄-2岁婴幼儿是轮状病毒的易感人群,以冬季10月份至12月份为流行高峰,基因组以长型为主,血清型为G3型。     

2018-2019年福州腹泻住院儿童A组轮状病毒全基因组分析

了解2018-2019年福州市五岁以下腹泻住院儿童标本中A组轮状病毒(RVA)基因组特征.通过反转录-聚合酶链式反应(RT-PCR)对49份RVA阳性标本进行核酸扩增,对扩增到的40份标本进行全基因组二代测序,获得G1P[8]型RVA毒株7株,G9P[8]型RVA毒株33株.根据VP7节段分型,40株RVA毒株中有30株G9-Ⅵ亚型、3株G9-Ⅲ亚型和7株G1-Ⅰ亚型;根据VP4节段分型,40株RVA毒株均属于P[8]-3亚型.全基因组核苷酸序列分析表明,除了VP7节段外,序列差异比较大的有NSP4和NSP1两个节段.本研究获得11株G9P[8]型Waa-ike株(G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1),22株NSP4节段重配的G9P[8]-E2株(G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1),在测序的33株G9P[8]型RVA毒株中G9P[8]-E2重配株占66.7％.7株G1P[8]型RVA毒株均为Wa-like株.研究结果表明,2018-2019年福州地区流行的RVA毒株中G9P[8]-E2重配株已经成为优势株,为了更全面了解福州地区RVA毒株的遗传变异情况,后续还需加大标本量持续进行监测.     

2010-2013年上海长宁地区成人轮状病毒急性胃肠炎的分子特征

为研究上海长宁地区成人轮状病毒急性胃肠炎的分子特征,收集2010年6月-2013年12月长宁区定点医院成人急性胃肠炎患者的粪便标本1 554份,采用酶联免疫吸附试验(enzyme-linked immunosorbentassay,ELISA)进行A组轮状病毒检测,多重聚合酶链反应(polymerase chain reaction,PCR)进行B组和C组轮状病毒检测.结果显示,1 554例患者的平均年龄为(46.19±15.59)岁,其中男性691例、女性863例.共检出轮状病毒200株,检出率为12.87％.18～39岁组的轮状病毒检出率为9.15％,40～59岁组为15.58％,60～79岁组为15.16％,≥80岁组为18.57％.A组轮状病毒基因型检测结果显示,G型以G9亚型最多(29.82％),P型以P[8]亚型最多(77.19％),GP组合以G9P[8]最多(25.73％).结果提示,2010-2013年上海长宁地区成人轮状病毒急性胃肠炎在就诊患者中以中老年多见,基因型以G9P[8]最多.     

2018–2020年人轮状病毒锦州地方株VP4及VP7基因特征

【背景】人A组轮状病毒(Rotavirus Group A,RVA)是婴幼儿胃肠炎的主要病原体及发展中国家婴幼儿死亡的重要原因,目前无特效药物治疗,疫苗预防是唯一可行的预防感染方法。外衣壳蛋白VP7和VP4是疫苗设计的主要靶点,针对该基因加强RVA地方株分子流行病学监测十分必要。【目的】对锦州地方流行RVA株VP7和VP4基因进行型别鉴定和序列特征分析。【方法】收集锦州地区2018-2020年RVA感染腹泻患儿的粪便标本,提取病毒RNA,通过RT-PCR扩增VP7、VP4基因片段并测序,得到7株RVA VP7和VP4序列。使用在线基因分型工具Rota C V2.0对测序结果进行分型分析。应用BLAST、DNAStar、MEGA X、Bio Edit等生物软件与临床流行株及疫苗株进行系统发育分析及氨基酸序列比对分析。【结果】分型结果表明7株锦州地方株均为G9P[8]型,系统发育分析证实其VP7和VP4基因分别属于G9-Ⅵ和P[8]-3谱系,核苷酸序列相似性分别为99.32%-100%与99.41%-100%。JZ株VP7与疫苗株Rotavac和Rotasiil相比,在抗原表位区7-1a、7-1b、7-2中分别存在4个和3个氨基酸替换。JZ株VP4与疫苗株Rotarix和Rota Teq VP4氨基酸序列相比,发现7个和4个氨基酸替换,位于抗原表位区8-1和8-3。【结论】2018-2020年在辽宁锦州地区检测到7株G9P[8]型RVA株,VP7和VP4序列相似性高于99%,G9P[8]型可能是辽宁省锦州地区2018-2020年婴幼儿轮状病毒腹泻的主要流行基因型之一。与同基因型疫苗株比较,位于JZ株VP7和VP4抗原表位区的氨基酸位点差异对于野毒株免疫逃逸机制的研究具有意义。     

南京地区2012～2013年G9型轮状病毒VP7基因特征的分析

A组轮状病毒是引起成人和婴幼儿急性腹泻的主要病原。了解轮状病毒流行株的型别,对主要中和抗原VP7的编码基因进行遗传变异分析,可为当地轮状病毒疫苗的应用和开发提供指导。我们对2012年10月至2013年12月南京地区908例腹泻门诊患者的粪便标本进行轮状病毒检测,采用RT-PCR方法对随机抽取的50份阳性标本进行G分型,并对其中19份G9型轮状病毒的VP7基因序列测序分析。结果发现轮状病毒阳性率11.34%(103/908),其中以G9型为主,占78.0%(39/50),其次是G2、G1和G3型。对G9型轮状病毒VP7基因核苷酸序列进行分析,显示主要分为G9-VI亚型和G9-III亚型,以G9-VI亚型为主,且属于中国和日本G9型轮状病毒亚簇,部分毒株在A、B、C、F四个中和抗原表位中有变异,这可能有助于G9型轮状病毒的流行,值得引起注意。     

中国河北省卢龙县儿童轮状病毒腹泻研究

轮状病毒是我国儿童重症腹泻的主要病原。按照WHO轮状病毒监测方案,于1999年7月至2003年6月,在河北省卢龙县开展了医院和社区为基础<5岁儿童轮状病毒腹泻的监测。结果表明:卢龙县<5岁儿童腹泻的发病率为1 3次/人/年。4年中全县共有2350名<5岁急性腹泻患儿住院,占所有住院儿童的38%(2350/6213)。住院的腹泻患儿每年有两个高峰,一个是夏季(6～8月),占全年腹泻住院病例的22%;另一个在冬季(12月至次年2月),占58%。住院的轮状病毒腹泻只有一个高峰,是在冬季(12月至次年2月),高峰期的轮状病毒腹泻住院患儿数占全年轮状病毒腹泻患儿住院数的86%。按全年统计,轮状病毒腹泻占住院腹泻患儿的46%,轮状病毒腹泻的住院率为11/1000儿童/年。在门诊腹泻患儿中轮状病毒腹泻患儿占28%,在社区腹泻患儿中轮状病毒腹泻占10%。轮状病毒毒株的分布,G3型(45%)最常见,其次为G1型(35%)、G2型(8%)、G4型(3%)、G9型(0 6%),混合感染较少(1%)。还有8%的毒株未能分出型别。在轮状病毒腹泻患者中,9～11月龄的儿童检出率最高(53%),其次是12～17月龄(51%)、18～23月龄(36%)和6～8月龄(30%)。在4年研究期间共有5～10名1～59月龄儿童可能因为轮状病毒腹泻死亡,其中有1例确诊为P[8]G1型毒株感染。初步估计,该县1～59月龄儿童轮状病毒腹泻的死     

Distribution of human rotaviruses, especially G9 strains, in Japan from 1996 to 2000

A 4-year (1996-2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998-1999, and in Saga (78.4%) in 1999-2000. G9 strains isolated from 1998-1999 belonged to the P[8]-NSP4-Wa-group with long RNA pattern, while, G9 strains isolated from 1999-2000 belonged to three groups, the P[8]-NSP4-Wa-group with long RNA pattern, the P[4]-NSP4-KUN-group with short RNA pattern and a mixed-type group (P[4]/P[8]-NSP4-KUN/Wa-group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999-2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998-1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998-1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999-2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P-genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619-628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00-Ad2863VP7 (AB091746), 00-OS2986VP7 (AB091747), 00-SG2509VP7 (AB091748), 00-SG2518VP7 (AB091749), 00-SG2541 (AB091750), 00-SG2864 (AB091751), 00-SP2737VP7 (AB091752), 99-SP1542VP7 (AB091753), 99-SP1904VP7 (AB091754), 99-TK2082VP7 (AB091755) and 99-TK2091VP7 (AB091756).     

G and P genotypes of rotavirus circulating among children with diarrhea in the Colombian northern coast.

A study on the prevalence of rotavirus G and P genotypes was carried out based on 253 stool specimens obtained from children living in the Colombia northern coast region who were less than 3-years-old and who suffered from acute diarrhea. A previous study had detected the presence of rotavirus A in 90 (36.5%) of the 246 samples tested by enzyme immunoassay (EIA), and these strains were investigated in the present study. Of these, 50 strains yielded an RNA electropherotype, most of which (80.0%) had long profiles and 20.0% of which had short profiles. Genotyping of 84 positive samples indicated that 67.9% of the strains could be typed. G1 (57.9%), was the most predominant VP7 genotype, followed by G3 (21.1%), G9 (15.8%) and G2 (5.3%). Among the VP4 genotypes, P[4] (49.1%) was the most prevalent, followed by P[6] 36.4% and P[8] (14.5%). Neither G4 nor G8 nor P[9] types were detected. The most common G-P combinations were G3 P[4] (8.8%) and G9 P[6] (7.0%), followed by G1 P[4] and G1 P[8] (5.3% each). All G1 P[8] strains showed long RNA profiles, whereas G3 P[4] and G9 P[6] displayed both long and short patterns. Mixed infections involved 21.0% of strains. There was a marked diversity among strains collected, and novel strains, including G9, as well as other atypical combinations of G and P genotypes, such as G9 P[6] and G3 P[4], were found.     

Human rotavirus K8 strain represents a new VP4 serotype.

The complete VP4 gene of the human rotavirus (HRV) K8 strain (G1 serotype) was cloned and inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. A K8VP4 recombinant baculovirus was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with transfer vector DNA containing the K8VP4 gene and wild-type baculovirus DNA. Infection of Sf9 cells with this VP4 recombinant baculovirus resulted in the production of a protein that is similar in size and antigenic activity to the authentic VP4 of the K8 strain. Guinea pigs immunized with the expressed VP4 developed antibodies that neutralized the infectivity of the K8 strain. This antiserum neutralized HRV strains belonging to VP4 serotypes 1A, 1B, and 2 with efficiency eightfold or lower than that of the homologous virus, indicating that the human rotavirus K8 strain represents a distinct VP4 serotype (P3). In addition, low levels of cross-immunoprecipitation of the K8VP4 and its VP5 and VP8 subunits with hyperimmune antisera to HRV strains representing different VP4 serotype specificities also suggested that the K8 strain possesses a unique VP4 with few epitopes in common with other P-serotype strains.     

Genetic variation in the VP4 and NSP4 genes of human rotavirus serotype 3 (G3 type) isolated in China and Japan

Sequence analyses of the VP4 and NSP4 genes were performed on twenty human isolates of serotype G3 rotavirus obtained from China and Japan. One isolate from China, CHW17, possessed P[4] genotype VP4 and KUN group NSP4 genes which are associated with G2. One isolate (02/92) from Japan, which was shown to have a wider spacing between RNA segments 10 and 11 by RNA polyacrylamide gel electrophoretic analysis like AU-1, possessed P[9] genotype VP4 and AU-1 group NSP4 genes. The other isolates had P[8] genotype VP4 and Wa group NSP4 genes. While the nucleotide sequence conservation among the G3 VP7 genes was more than 79% (Wen et al, Arch. Virol., 1997, 142: 1481-1489), the conservation of VP4 and NSP4 genes in the same genotypes or groups was more than 85%.