抗扎伊尔型埃博拉病毒单克隆抗体的制备和鉴定

目的 采用扎伊尔型埃博拉病毒核蛋白(EBOV NP)的合成多肽为免疫原制备鼠源单克隆抗体,并用4种埃博拉流行株核蛋白基因的真核表达蛋白对制备的单克隆抗体进行特异性分析。方法 用人工合成的扎伊尔型埃博拉病毒核蛋白多肽免疫动物,进行细胞融合后筛选阳性杂交瘤细胞。构建埃博拉病毒4种亚型的真核表达载体转染HEK293T细胞以表达目的蛋白,再以真核表达蛋白为抗原,用免疫印迹方法分析扎伊尔型埃博拉病毒单克隆抗体的特异性。结果 完成了抗扎伊尔型单克隆抗体的制备,共得到能分泌抗扎伊尔型埃博拉病毒单克隆抗体的杂交瘤细胞12株,小鼠腹水抗体效价介于1∶10⁴~1∶10⁵,其中3株杂交瘤细胞株分泌的抗扎伊尔型埃博拉病毒单克隆抗体与其他3种亚型无交叉反应,抗体效价高、特异性好。结论 成功制备抗扎伊尔型埃博拉病毒单克隆抗体,为建立快速检测埃博拉病毒的方法奠定基础。     

新型疫苗佐剂Ov-ASP-1佐剂活性功能区的设计、表达及生物学特性研究

目的 获得保留Ov-ASP-1佐剂活性的功能区。方法 用前期制备的Ov-ASP-1单克隆抗体对Ov-ASP-1的9个肽进行特异性结合,根据肽的结合情况设计功能区ASPPRM,并进行密码子优化、构建原核表达载体后在大肠杆菌中表达。表达产物经 Western印迹鉴定后的用镍柱纯化蛋白,复性完全后对获得的ASPPRM活性蛋白进行生物学特性分析。结果 ASPPRM以包涵体形式表达,分子量为17 kD。将其和OVA共同免疫小鼠后ASPPRM组抗OVA的IgG抗体显著高于OVA组。结论 本研究获得了保留佐剂活性的ASPPRM蛋白,为后续ASPPRM体内的免疫增效作用及Ov-ASP-1佐剂活性功能域的探索奠定基础。     

一种不依赖于活病毒的中东呼吸综合征冠状病毒受体结合区特异性中和抗体检测方法的建立

目的 拟建立一种不依赖于活病毒和高等级生物安全条件,快速简便检测中东呼吸综合征冠状病毒(MERS-CoV)受体结合区(RBD)特异性的中和抗体。方法 利用哺乳动物表达系统表达重组rRBD-Fc蛋白;应用流式细胞术检测rRBD-Fc蛋白与Huh-7细胞结合的最佳浓度,建立通过流式细胞术检测rRBD-Fc与Huh-7结合活性的方法,并利用无关蛋白验证结合的特异性;在此基础上,利用RBD特异性中和抗体、RBD特异性非中和抗体、MERS-CoV RBD免疫小鼠血清和无关抗体建立能够检测中和抗体阻断RBD与Huh-7结合作用的RBD特异性中和抗体检测技术方法,并与实毒中和试验进行比较,评价方法的一致性;应用建立的中和抗体检测方法进行血清学检测。结果成功表达了rRBD-Fc蛋白,rRBD-Fc蛋白能够与Huh-7细胞特异性结合,并确定了rRBD-Fc与Huh-7细胞结合的最佳剂量;在此基础上建立了通过流式细胞术检测抗体阻断RBD与Huh-7结合作用的RBD特异性中和抗体检测技术方法,结果表明,检测方法能够区分具有中和活性的RBD特异性中和抗体、rRBD-Fc免疫小鼠血清和其他抗体,具有特异性,且能够反映中和活性的强弱。该方法在检测12株中和抗体时,与传统的实毒中和试验检测结果具有很好的一致性,能够有效应用于血清学检测。结论 成功建立了一种不依赖活病毒和高等级生物安全条件,即可快速检测MERS-CoV RBD特异性中和抗体的方法,为针对MERS-CoV疫苗设计和抗体效果快速评价、血清学调查和免疫保护机制研究提供了一种更加简便、安全的技术手段。     

河南省2008-2014年5岁以下腹泻儿童A组轮状病毒病原学及流行病学特征研究

目的 分析2008-2014年河南省5岁以下腹泻儿童A组轮状病毒的感染状况及流行病学特征。方法 采集河南省两个监测哨点医院5岁以下儿童腹泻病例的粪便样本2 098份,双抗体夹心法ELISA检测A组轮状病毒,阳性样本抽提病毒RNA,两步巢式多重RT-PCR进行G-P基因分型,同时收集病例临床与流行病学信息进行分析。结果 2 098份腹泻样本共检出A组轮状病毒688份,总阳性率32.8%。年检出率最高46.4%(2013年),最低26.7%(2009年)。轮状病毒检出率的季节性特征显著,存在秋季和春季两个高峰。A组轮状病毒型别组合以G9P^[8]、G2P^[4]、G3P^[8]、G1P^[8]为主,还存在部分混合感染型别。感染病例集中于4个月到1岁,以无临床症状或轻症为主。结论 河南省5岁以下腹泻患儿中存在较高的A组轮状病毒感染率,病原体可分为多种基因型别,感染人群具有显著的流行病学特征。     

寨卡病毒NS1蛋白的原核表达和单克隆抗体的制备

目的 高效表达和纯化重组的寨卡病毒NS1蛋白,制备抗NS1蛋白的单克隆抗体。方法 构建含有寨卡病毒NS1基因的原核表达质粒,利用大肠杆菌大量表达重组NS1蛋白,纯化蛋白后免疫小鼠并进行细胞融合,筛选制备高纯度的单克隆抗体。结果 在大肠杆菌中高效表达了重组NS1蛋白,重组NS1蛋白具有良好的免疫原性,筛选出3株分泌针对寨卡病毒NS1蛋白单克隆抗体的杂交瘤细胞株,分别命名为6B8、7D11和3E2,纯化的单克隆抗体与重组NS1蛋白有良好的特异性反应。结论 利用重组NS1蛋白免疫制备了抗NS1的单克隆抗体,为后续建立针对NS1蛋白的ELISA检测方法及相关研究提供了基础。     

2型猪链球菌细胞分裂蛋白GpsB的原核表达及其鉴定

目的 利用原核表达系统诱导表达2型猪链球菌(Streptococcus suis 2,S.suis 2)分裂相关因子GpsB重组蛋白,为后续研究奠定基础。方法 以S. suis 2菌株05ZYH33全基因组DNA为模板,经PCR扩增得到目的基因片段。目的基因经双酶切后连接至表达载体pET32a,转化大肠杆菌(Escherichia coli, E. coli)DH5α感受态细胞。重组质粒经测序鉴定正确后转化E. coli BL21感受态细胞。获得的重组表达菌经IPTG诱导表达目的蛋白。利用Ni离子亲和层析柱纯化重组蛋白,并进行SDS-PAGE和Western blot 鉴定。利用重组蛋白免疫小鼠制备多克隆抗体。结果 成功构建出重组表达载体pET32a^gpsB,并经IPTG诱导表达出目的蛋白。重组蛋白主要存在于表达菌裂解液上清中,分子质量约30 kD,与预期大小一致。Western blot 检测发现,该蛋白能被His-Tag 单克隆抗体特异性识别。制备的多克隆抗体能特异性识别重组GpsB蛋白(rGpsB)。结论 成功表达和纯化了rGpsB并获得了该蛋白的多克隆抗体,为进一步研究该蛋白在S.suis 2 分裂过程中的作用鉴定了基础。     

结核分枝杆菌Rv3621c原核表达及免疫功能研究

目的 以结核分枝杆菌(Mycobacterium tuberculosis,M.tb)H37Rv基因组为模板,构建、纯化及鉴定原核表达质粒pPROEX-Rv3621c,通过人群、小鼠试验进行免疫原性评价。方法 构建重组质粒pPROEX-Rv3621c,并以全血干扰素释放分析技术(Whole-blood IFN-γ release assay,WBIA)检测其能否被安徽省淮南市M.tb感染者T细胞特异性识别。rRv3621c混合佐剂MTM[母牛分枝杆菌(M.vaccae),人工合成海藻糖-6'6,二分枝菌酸(TDB),单磷酰脂质A(MPLA)]免疫小鼠后,检测血清中特异性抗体分泌水平、脾细胞中抗原特异性Th1型细胞因子分泌水平及肺脏细胞因子mRNA表达水平。结果 成功构建重组质粒pPROEX-Rv3621c,并使之诱导表达、纯化和鉴定。在rRv3621c蛋白诱导下,活动性结核(Active tuberculosis, ATB)患者外周血淋巴细胞释放的IFN-γ水平明显较高(t=4.813, P<0.01),且ATB患者产生的IFN-γ水平高于潜伏性结核(Latent tuberculosis infection, LTBI)人群(t=4.442, P<0.01)。BCG+Rv3621c/MTM组小鼠产生的特异性抗体滴度水平明显高于Rv3621c/MTM组(P<0.01)和BCG组(P<0.01),Rv3621c/MTM组和BCG+Rv3621c/MTM组小鼠的IgG2a/IgG1比值大于1,明显高于MTM组和BCG组。BCG+Rv3621c/MTM组小鼠均分泌高水平IFN-γ、TNF-α和IL-2。Rv3621c/MTM组小鼠肺脏组织中IFN-γ、TNF-α及iNOS表达水平较高。结论 M.tb感染者外周血T细胞可特异性识别rRv3621c蛋白,rRv3621c混合佐剂MTM可以诱导较强烈的抗原特异性Th1型免疫应答。     

寨卡病毒E蛋白及第三结构域的原核表达和多克隆抗体制备

目的 在大肠杆菌中克隆表达和纯化寨卡病毒(Zika Virus,ZIKV)包膜糖蛋白(Envelope Protein,E)及第三结构域(Envelope DomainⅢ,EDⅢ),并制备两种免疫原的鼠多克隆抗体。方法 通过Vero-E6细胞培养扩增ZIKV,提取病毒总RNA并反转录为cDNA,利用E和EDⅢ基因的cDNA序列构建原核表达载体pET32a/E和pET28a/EDⅢ,转入E.coli BL21(DE3),IPTG诱导表达,采用Ni⁺柱亲和层析法纯化蛋白。以纯化的重组蛋白为抗原免疫BALB/c小鼠,采集抗血清,间接ELISA法测定效价,Western Blot检测特异性。结果 成功表达并纯化重组蛋白E和EDⅢ,获得的多克隆抗体效价均达到1:409 600,Western Blot检测多克隆抗体可特异性识别重组E蛋白和EDⅢ以及天然E蛋白。结论 成功制备出特异性抗寨卡病毒E蛋白和EDⅢ的鼠源多克隆抗体,为深入探索寨卡病毒致病机制、检测方法和免疫策略奠定了研究基础。     

登革热病毒(DENV)包膜蛋白特异性人源抗体筛选与鉴定

目的 建立自外周血快速筛选识别登革热病毒衣壳的人源抗体的方法,从病毒感染者外周血记忆B细胞中筛选出特异性抗体并进行性质分析。方法 通过流式细胞术分选得到外周血中登革热病毒包膜蛋白特异性的单个记忆B细胞,结合高效单细胞PCR技术获得抗体基因序列,对重组表达的单克隆抗体进行结合活性及中和活性检测。结果 成功筛选到识别DENV-1包膜蛋白的6株人源单克隆抗体,其中4株抗体均具有很强的结合活性以及中和活性。结论 成功的通过流式细胞术对DENV特异性记忆B细胞进行分选,并结合单细胞PCR技术,实现了对DENV特异性人源单克隆抗体的高效筛选。通过该平台有希望筛选得到DENV的广谱中和抗体,用于登革热的预防和治疗。     

结核分枝杆菌EsxV亚单位疫苗黏膜免疫诱导的免疫应答

目的 研究结核分枝杆菌抗原Rv3619c(EsxV)黏膜免疫小鼠诱导的免疫应答水平。方法 PCR法扩增esxV基因克隆入原核表达载体pET28a(+),获得的重组E.coli诱导表达目的蛋白,SDS-PAGE电泳和Western Blot鉴定蛋白的表达,亲和层析法纯化EsxV蛋白。以EsxV和/或联合环二腺苷酸(c-di-AMP)经鼻黏膜免疫BALB/c小鼠,ELISA法检测小鼠特异性抗体水平及亚类,MTS法检测脾细胞增殖,qRT-PCR检测脾和肺脏细胞因子表达水平,ELISA法检测脾细胞因子分泌水平。结果 成功构建EsxV的原核表达载体pET28a(+)-esxV并诱导表达目的蛋白,亲和层析法获得了纯化的重组EsxV蛋白。EsxV经黏膜免疫可诱导显著的体液免疫应答,但诱导的细胞免疫应答水平不高。EsxV与c-di-AMP经黏膜接种可诱导特异性IgG水平增加,EsxV蛋白特异的脾细胞增殖,脾和肺细胞的IFN-γ转录增加。c-di-AMP显著提高EsxV特异的IFN-γ、IL-2、IL-10和IL-17细胞因子分泌,而不诱导炎症因子TNF-α和IL-6表达。结论 EsxV与c-di-AMP佐剂构建的亚单位疫苗可诱导特异性体液和细胞免疫应答,可进一步用于新型结核病亚单位疫苗的研制。     

Structural basis of P[II] rotavirus evolution and host ranges under selection of histo-blood group antigens

Group A rotaviruses cause severe gastroenteritis in infants and young children worldwide, with P[II] genogroup rotaviruses (RVs) responsible for >90% of global cases. RVs have diverse host ranges in different human and animal populations determined by host histo-blood group antigen (HBGA) receptor polymorphism, but details governing diversity, host ranges, and species barriers remain elusive. In this study, crystal structures of complexes of the major P[II] genogroup P[4] and P[8] genotype RV VP8* receptor–binding domains together with Lewis epitope–containing LNDFH I glycans in combination with VP8* receptor-glycan ligand affinity measurements based on NMR titration experiments revealed the structural basis for RV genotype-specific switching between ββ and βα HBGA receptor–binding sites that determine RV host ranges. The data support the hypothesis that P[II] RV evolution progressed from animals to humans under the selection of type 1 HBGAs guided by stepwise host synthesis of type 1 ABH and Lewis HBGAs. The results help explain disease burden, species barriers, epidemiology, and limited efficacy of current RV vaccines in developing countries. The structural data has the potential to impact the design of future vaccine strategies against RV gastroenteritis.

The major human rotaviruses (RVs), the P[8], P[4], and P[6] genotypes in the P[II] genogroup, are responsible for over 90% of human infections worldwide (1–3). Despite successes of the RotaTeq and Rotarix RV vaccines in many developed countries, their efficacy remains disappointingly poor in developing countries (4–6). Low efficacies of both vaccines in developing countries can be attributed to a lack of cross protection between P[8], which is more common in developed countries, and other P-type RVs, such as P[6] and P[11], that are less common in developed countries but more common in developing countries (7–13).Significant advances have been made in understanding RV evolution under the selection of stepwise synthesis of histo-blood group antigens (HBGAs) in humans. For example, P[II] RVs that mainly infect humans are thought to have originated from P[I] RVs with an animal host origin and evolved the ability to infect humans under selective pressure to bind polymorphic human HBGAs. This deduction is in agreement with a complete VP4 sequence phylogeny analysis that revealed that P[10]/P[12] in P[I] were genetically closer to P[19], P[6], and P[4]/P[8] in P[II] than other genotypes from other genogroups (14, 15). These observations led to the hypothesis that host ranges of P[II] genotypes for certain animal species and different human populations are dictated by the evolutionary stages of their HBGA receptors.P[19] appears to represent an early evolutionary branch of the P[II] genogroup since it recognizes type 1 precursor HBGAs and therefore commonly infects animals (porcine) but rarely humans. On the other hand, P[4] and P[8] appear to be more evolutionarily advanced since they have developed the ability to recognize more mature HBGA products that dominate in humans. P[6] appears to represent an intermediate stage of evolution close to P[19] that commonly infects both animals (porcine) and humans, likely because of its evolutionary status that allows it to recognize less mature type 1 HBGA precursor glycans shared between humans and animals (porcine). The deduced evolutionary path that enabled the transition from animal to human host, which is correlated with the emergence of the P[II] branch from the P[I] branch, may apply to other genotypes and genogroups and may be important for RV classification and epidemiology (16, 17).Evidence for HBGA-controlled RV host ranges and evolution is also available from structural analyses of genotype-specific interactions of RV VP8* domains with their glycan receptor ligands. For example, early structures showed that VP8* domains from animal and human RVs adopted similar galectin-like folds, and they recognize distinct HBGAs either through a ββ or βα site. However, our recent NMR spectroscopy–based docking and crystallographic studies showed that P[4], P[6], P[8], and P[19] VP8*s of P[II] interacted with H type 1 HBGA precursor using a common βα site (17–21), while P[8] VP8* bound Le^b tetra-saccharide and Lewis epitope–containing hexa-saccharide (LNDFH I) in the ββ site (22).To elucidate the molecular basis for receptor-binding bias between βα- and ββ-binding sites, we characterized relative binding affinities of major P[II] RV VP8* domains for glycans representing different HBGA synthetic stages, including the Lewis epitope–containing LNDFH I, using NMR heteronuclear single quantum coherence spectroscopy (HSQC)-monitored titrations. The structural basis for the bias of ββ sites for Lewis epitope HBGAs and βα sites for HBGAs lacking the Lewis epitope was elucidated from crystal structures of P[4] and P[8] bound to LNDFH I and P[6] bound to Lacto-N-tetraose (LNT). Sequence- and structure-based analyses of differences in P[II] VP8* receptor–binding interfaces revealed molecular details responsible for receptor switching between genotype-specific ββ and βα HBGA–binding sites. Overall, the results provide strong evidence for HBGA-controlled P[II] RV evolution from an animal host origin that resulted in diverse genotypes infecting children in different populations and which may impact future strategies for RV disease control and prevention.     

A Nanoparticle-Based Trivalent Vaccine Targeting the Glycan Binding VP8* Domains of Rotaviruses

Rotavirus causes severe gastroenteritis in children. Although vaccines are implemented, rotavirus-related diarrhea still claims ~200,000 lives annually worldwide, mainly in low-income settings, pointing to a need for improved vaccine tactics. To meet such a public health need, a P₂₄-VP8* nanoparticle displaying the glycan-binding VP8* domains, the major neutralizing antigens of rotavirus, was generated as a new type of rotavirus vaccine. We reported here our development of a P₂₄-VP8* nanoparticle-based trivalent vaccine. First, we established a method to produce tag-free P₂₄-VP8* nanoparticles presenting the VP8*s of P[8], P[4], and P[6] rotaviruses, respectively, which are the three predominantly circulating rotavirus P types globally. This approach consists of a chemical-based protein precipitation and an ion exchange purification, which may be scaled up for large vaccine production. All three P₂₄-VP8* nanoparticle types self-assembled efficiently with authentic VP8*-glycan receptor binding function. After they were mixed as a trivalent vaccine, we showed that intramuscular immunization of the vaccine elicited high IgG titers specific to the three homologous VP8* types in mice. The resulted mouse sera strongly neutralized replication of all three rotavirus P types in cell culture. Thus, the trivalent P₂₄-VP8* nanoparticles are a promising vaccine candidate for parenteral use against multiple P types of predominant rotaviruses.     

Continuing rotavirus circulation in children and adults despite high coverage rotavirus vaccination in Finland

ObjectivesTo determine occurrence of residual rotavirus (RV) disease in different age groups in Finland after five to nine years of high coverage (≥90%) mass-vaccination with RotaTeq^Ⓡ vaccine, and to examine the vaccine effect on circulating genotypes.MethodsSince 2013 all clinical laboratories in the country were obliged to send RV positive stool samples for typing. RVs were genotyped by RT-PCR for VP7 and VP4 proteins, sequenced and compared to reference strains.ResultsRV continued to circulate throughout the study period at low level with a small increase in 2017–2018. There were three age-related clusters: young children representing primary or secondary vaccine failures, school-age children who may not have been vaccinated, and the elderly. Genotype distribution differed from the pre-vaccination period with a steady decline of G1P[8], emergence of G9P[8] and especially more recently G12P[8]. In the elderly, G2P[4] was predominant but was also replaced by G12P[8] in 2017–18.ConclusionsRV vaccination with a high coverage keeps RV disease at low level but does not prevent RV circulation. New RV genotypes have emerged replacing largely the previously predominant G1P[8]. Increase of overall RV activity with emergence of G12P[8] in the latest follow-up season 2017–18 might be a potential alarm sign.     

Phylogenetic analysis of human rotavirus in Myanmar: detection of Indian-Bangladeshi G1/G2 lineages, Chinese G3 lineage and OP354-like P[8] lineage (P[8]b subtype)

As a first phylogenetic study of human rotavirus in Myanmar, VP7 and VP8* gene sequences of 5 group A human rotaviruses detected in children in Yangon City were determined and analyzed for their relatedness to rotavirus strains reported in other countries. VP7 genes of the two G1P[8] strains and the two G2P[4] strains clustered phylogenetically with those of Indian-Bangladeshi lineages with extremely high sequence identities. In contrast, a G3P[8] strain exhibited a close relatedness of VP7 gene to G3 rotaviruses currently prevailing in China, which had been referred to as a new variant G3 rotavirus. While VP8* genes of P[4] and P[8] strains clustered with those of Indian and Bangladeshi strains, only the G1 strain was grouped into a rare P[8] subtype, ie, P[8]b (OP354-like P[8]) with close relatedness to the P[8]b strains in eastern India and Thailand. The coexistence in Myanmar of G1/G2 and G3 rotaviruses, which are virtually identical to those predominating in India/Bangladesh and China, respectively, suggests the spread of these predominant rotaviruses from the two regions into Myanmar.     

Sequence analysis of VP4 genes of wild type and culture adapted human rotavirus G1P[8] strains

ObjectiveTo conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361, 0613158, 061060 and 0715880) and cell culture adapted (06361-CA, 0613158-CA, 061060-CA and 0715880-CA) G1P[8] rotavirus strains.MethodsFull-length VP4 genes of each of the four wild type G1P[8] rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected to RT-PCR amplification and nucleotide sequencing.ResultsAll four cell culture adapted G1P[8] rotavirus strains showed nucleotide and amino acid substitutions in the VP4 gene as compared to their wild type strains. The number of substitutions however, varied from 1-64 and 1-13 respectively. The substitutions were distributed in both VP5* and VP8* subunits of VP4 gene respectively of permeabilization and hemagglutinating activity. The presence of unique amino acid substitutions was identified in two of the four wild type (V377G, S387N in 061060 and I644L in 0715880) and all four cell culture adapted (A46V in 0613158-CA, T60R in 06361-CA, L237V, G389V and Q480H in 061060-CA and S615G and T625P in 0715880-CA) strains for the first time in the VP4 gene of P[8] specificity. Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains.ConclusionsAmino acid substitutions detected in the VP4 genes of G1P[8] rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation, immunogenicity and conformation is needed for the development of newer rotavirus vaccines.     

Rotavirus vaccines: a long and difficult journey

Diarrhea is the second cause of death worldwide. The main causative agent in infants and children less than 5 years is rotavirus. Consequently, for the World Health Organization and the Global Alliance for Vaccines and Immunization (GAVI), rotavirus vaccination is an urgent priority. The global distribution of rotavirus serotypes varies from country to country, but in 80-90% of cases is caused by five types: G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. Currently, two rotavirus vaccines are available, an attenuated monovalent G1P[8] vaccine, and a pentavalent human-bovine recombinant vaccine containing five strains of bovine WC3 rotavirus, four with a gene codifying the VP7 protein of human rotaviruses G1, G2, G3 and G4, and a fifth expressing the VP4 P[8] genotype.     

南京地区2011-2012年度门诊婴幼儿轮状病毒腹泻的分子流行病学研究

目的 了解南京地区门诊婴幼儿轮状病毒腹泻的分子流行病学特征及轮状病毒的G/P分型状况。方法 2011年7月至2012年6月对门诊腹泻患儿标本进行轮状病毒胶体金检测,阳性标本通过巢式反转录聚合酶连反应进行G/P分型,同时测序得出亚型。 结果 在2 081例腹泻标本中共检出胶体金阳性标本165例,阳性率为7.9%。轮状病毒感染的高峰季节在10月到次年2月之间, 94%的感染患儿都在2岁以下,其中13～24月龄为高发年龄段。轮状病毒分型结果表明G1, G3, G9为主要G分型,而P\[8\]则是最常见的P分型。常见的G／P组合为：G1P\[8\](16.4%), G3P\[8\](13.9%), G9P\[8\](24.8%)。进一步VP7和VP4基因测序比对后发现G1和G3毒株各分属于一个亚型G1 Ic,G3 Ia,而G9毒株大部分属于G9 IV亚型,同时也有一小部分毒株属于G9 III亚型,P\[8\]型毒株主要为P\[8\] II亚型,少数为P\[8\] IV亚型。 结论 研究结果表明G9P\[8\]是最主要的流行型别,同时G1P\[8\]和G3P\[8\]也占较大比例。     

2019-2020年湖北部分地区G9P[8]型人轮状病毒基因特征北大核心CSCD

目的对人A组轮状病毒进行检测及分离鉴定,并研究其各基因片段的遗传进化关系。方法2019-2020年对湖北武汉市和襄阳市临床腹泻病人的粪便样品进行采集,共319份。设计特异性轮状病毒VP6基因引物,RT-PCR检测轮状病毒的感染情况。将阳性样品接种于MA104细胞进行轮状病毒的分离。RT-PCR特异性扩增VP6基因和特异性间接免疫荧光对其进行病毒鉴定及病毒增殖检测;并进一步通过RT-PCR扩增轮状病毒的11个基因片段,在线工具Rota C V2.0对测序结果进行分型分析。Mega软件对其全基因组序列进行遗传进化分析。结果轮状病毒感染阳性标本共69份,阳性率为21.63%。成功分离获得11株人轮状病毒,主要衣壳蛋白VP7和VP4基因型均为G9P[8]型。其中3株轮状病毒归属于类Wa株毒株,基因型图谱为G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1。8株毒株在Wa-like的基因型中具有DS-1-like的NSP4为E2基因型特征。基因型图谱为G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1。结论G9P[8]型人轮状病毒毒株在2019-2020年湖北部分地区占主导趋势,且其NSP4基因以E2基因型为主要流行形式。     

Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus nonstructural protein 4 and viral protein (VP) 4, VP6, and VP7 in infants who received selected live oral rotavirus vaccines

Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus enterotoxin nonstructural protein (NSP)-4, independent neutralization antigens viral protein (VP)-4 and VP7, and group A rotavirus common antigen VP6 were analyzed by an immunocytochemistry assay in infants who received 1 of several live oral rotavirus vaccines. Significant serum immunoglobulin (Ig) A and IgG antibody responses to homotypic and/or heterotypic NSP4s of genotype [A], [B], or [C] were detected after vaccination. The magnitude of antibody responses to homotypic and heterotypic NSP4s was not significantly different, irrespective of the NSP4 genotype of the administered vaccine strain. In addition, there were no significant differences between IgA antibody responses to homotypic and heterotypic VP7s. In contrast, IgA antibody responses to VP4 were predominantly homotypic. IgA antibody responses to VP7 were lower in magnitude than those to VP4 but were comparable to those to NSP4. Antibody titers to homotypic and/or heterotypic NSP4s were positively correlated with those to VP6 before and after vaccination.     

Distribution of human rotavirus G and P genotypes in a hospital setting from Northern India

Rotavirus gastroenteritis is a major cause of severe dehydrating diarrhea in children worldwide. Rotavirus G and P genotyping is essential for epidemiological surveillance and for better formulation of candidate rotavirus vaccines. Out of 862 diarrheal stool samples collected from hospitalized children aged < 2 years during February 2005 - March 2007, 318 (36.9%) were positive for rotavirus by ELISA. G and P genotyping was performed on 100 randomly selected positive samples using a seminested multiplex RT-PCR assay. The result of G genotyping indicates G1 (60%) was the most predominant VP7 type, followed by G2 (16%), G9 (8%) and G3 (3%). Two cases of G12 genotype were also observed. P genotypes identified were P[8] (40%) followed by P[4] (26%) and P[6] (17%). The most common G-P combinations were G1P[8] (26%), followed by G1P[4] and G1P[6]. Mixed infection involved 28% of strains. In this study the G1 and P[8] genotypes were the leading G and P types. Two cases with G12 genotype were also observed during the study.