两株Wa株人源轮状病毒NSP4蛋白的表达及其致ICR小鼠腹泻的毒力比较

目的研究两株Wa株人源轮状病毒NSP4蛋白139位氨基酸位点的变异对毒力的影响。方法从2002～2004年昆明地区婴幼儿轮状病毒感染者大便样品中,通过RT-PCR,PCR扩增了22株轮状病毒株的NSP4全长基因、cDNA序列,并对cDNA序列测序,发现这些病毒株的NSP4氨基酸序列高度保守,仅在79位、139位氨基酸位点存在差异。选取139位氨基酸不同(V/I)的两株病毒株,在E.coli BL21中,用pGEX5X-1载体融合表达了两株病毒的86～175位氨基酸(GST-NSP4_(86-175);并酶切融合头得到两种NSP4_(86-175)蛋白,在ICR乳鼠中比较这4种不同蛋白致小鼠腹泻的差异。结果发现GST-NSP4_(86-175)蛋白毒性比NSP4_(86-175)蛋白强,而139位氨基酸不同(V/I)的两株病毒株的NSP4_(86-175)蛋白毒性没有明显的差异。结论139位氨基酸位点的变异并不影响NSP4蛋白的毒力改变。     

新的成人腹泻轮状病毒J19株NSP4和NSP5基因克隆和序列分析

目的克隆新的成人腹泻轮状病毒J19株NSP4和NSP5基因,并分析其基因序列.方法利用一种改进的非依赖核酸序列的单引物扩增方法扩增J19株NSP4和NSP5基因,克隆到pMD18-T载体中并进行测序.在此基础上,将J19株NSP4和NSP5的蛋白序列与其他轮状病毒蛋白序列进行比较分析和种系进化分析.结果J19株NSP4和NSP5基因为基因10和11,全长为739 bp和649 bp,它们分别编码213个和176个氨基酸.与J19株NSP4和NSP5蛋白序列一致性较高的分别是B组成人腹泻轮状病毒Bang373株（20.3%）和B组猪轮状病毒db101株（29.5%）.对J19株的NSP4和NSP5的遗传进化分析表明,J19株在进化树上的位置都靠近A、B和C组轮状病毒分支的根部,而且它比较偏向B组轮状病毒的分支.结论J19株的NSP4和NSP5与其他轮状病毒的相应蛋白序列存在显著差异.J19株NSP4和NSP5的蛋白序列比较和遗传进化分析表明新的成人腹泻轮状病毒与成人腹泻轮状病毒可能有共同起源;但是新的成人腹泻轮状病毒与成人腹泻轮状病毒存在显著差异.     

新成人腹泻轮状病毒J19株NSP1、NSP2和NSP3基因序列分析

目的 克隆新成人腹泻轮状病毒J19株三个非结构蛋白NSP1、NSP2和NSP3基因，并分析其基因序列。方法 利用一种改进的非依赖核酸序列的单引物扩增方法扩增J19株三个基因，克隆到pMD18-T载体中并进行测序。在此基础上，将J19株的NSP1、NSP2和NSP3的蛋白序列与其他轮状病毒蛋白序列进行比较分析和种系进化分析。结果J19株的NSP1、NSP2和NSP3基因为基因5、7和8，它们的全长1307个、1004个和932个核苷酸，编码395个、297个和262个氨基酸。与J19株的NSP1、NSP2和NSP3蛋白序列一致性较高的分别是B组轮状病毒KB63株（26．3％）、WH1株（46．6％）和IDIR株（29．6％）。对J19株的NSP1、NSP2和NSP3的遗传进化分析表明，J19株在进化树上的位置都靠近A、B和C组轮状病毒分支的根部，而且它比较偏向于B组轮状病毒的分支。结论 J19株的NSPI、NSP2和NSP3与其他轮状病毒的相应蛋白序列存在显著差异。J19株NSP1、NSP2、NSP3的蛋白序列比较和遗传进化分析表明新成人腹泻轮状病毒与成人腹泻轮状病毒可能有共同起源；但是新成人腹泻轮状病毒与成人腹泻轮状病毒存在显著差异。     

轮状病毒非结构蛋白4的克隆、表达及其对胞内钙离子的干扰

目的构建轮状病毒（rotavirus, RV）非结构蛋白4（nonstructural protein 4, NSP4）和其第86～175位氨基酸肽段（86 to 175 amino acid peptide of nonstructural protein 4, NSP486-175）基因的原核表达系统,初步证实重组表达产物的生物学活性。方法胶体金法筛选A组轮状病毒抗体阳性粪便,提取病毒RNA并逆转录为cDNA,PCR扩增出NSP4和NSP486-175基因,插入到表达载体pET-28a （＋）。构建pET-28a（＋）-NSP4和pET-28a（＋）-NSP486-175原核表达载体,转化E．coli BL21（DE3）,IPTG诱导表达,Ni-NTA亲和层纯化及Western blot 鉴定。 Fluo-3 AM标记Caco-2细胞内游离的Ca2＋（ in-trancellular calcium,[Ca2＋]i）,激光共聚焦显微镜检测NSP486-175蛋白对[Ca2＋]i的效应。结果 NSP4和NSP486-175 DNA测序结果与 NCBI 中相关基因一致。完整的 NSP4在大肠埃希菌中难以表达。NSP486-175蛋白主要以可溶性方式存在,相对分子质量为10000。外源性添加NSP486-175蛋白至Caco-2细胞荧光强度和分布改变。结论构建了pET-28a（＋）-NSP4和pET-28a（＋）-NSP486-175表达载体,目的蛋白能够导致细胞内游离的Ca2＋失衡,初步证实具有生物学活性,可进一步用于轮状病毒致病机制及疫苗的相关研究。     

中国轮状病毒非结构蛋白NSP4基因变异特征的分析

目的　研究我国轮状病毒流行株NSP4基因的变异特点。方法　对近年来从我国不同地区获得的 2 7份人轮状病毒流行株的NSP4基因用RT PCR进行扩增 ,克隆后进行全长cDNA序列分析 ,并利用Clustal× 1.8,TreeView3 2及DNAStar软件与参比株Wa、KUN、AU 1、EW及来自GenBank的OSU、SA11、Hochi、US2 44、Bristol株的NSP4序列进行分析比较。采用PCR分型方法对VP7血清型进行鉴定 ,确定轮状病毒G型与NSP4基因型的关系。结果　氨基酸同源性比较表明 ,我国轮状病毒不同流行株NSP4之间同源性为 81.7%～ 99.4% ,据此可将 2 7株RVNSP4分为 2组 ,分别以Wa株和KUN株为代表 ,其中以Wa组为主。组内同源性分别为 92 .0 %～ 99.4%和 92 .0 %～ 98.9% ,组内变异率分别为 0～ 8 5 %及 1 2 %～ 8 5 %。两组间变异率达 16 6%～ 2 1 0 %。氨基酸进化树提示在Wa组内包括 3个亚组。轮状病毒G血清型与NSP4基因型之间的联系不确定。结论　我国流行株NSP4基因主要可分为Wa组和KUN组 ,在Wa组内可形成三个亚组 ,并且在高变区有特征性的氨基酸位点。NSP4的变异与年份有关而与地域关系不密切     

B组轮状病毒WH-1株 NSP2基因序列和蛋白质结构分析

目的 克隆成人腹泻标本WH-1中B组轮状病毒组(group Brotavirus，GBRV)非结构蛋白NSP2基因，分析其核苷酸序列，比较与其它GBRV基因同源性，预测其mRNA二级结构、蛋白质二级结构。方法利用逆转录-聚合酶链反应(RT-PCR)技术，从成人腹泻标本WH-1中扩增GBRV NSP2基因，克隆载体pUCmT，对NSP2基因进行核苷酸序列分析。利用GeneBee软件比较与其它GBRV毒株NSP2基因同源性，Rnaviz2．0软件绘制NSP2基因的二级结构，PredictProtein软件分析NSP2蛋白结构。结果成人腹泻标本WH-1中GBRV非结构蛋白NSP2基因全长1007bD，与ADRV核苷酸序列的同源性达98％，与印度加尔各达分离株CAI-1达82％，与IDIR(鼠)同源性仅为79％。氨基酸序列与ADRV的同源性达98．4％，与CAL-1达97．7％，而与IDIR仅为89．0％。其mRNA折叠形成多达26个发卡环状结构。NSP2蛋白是由301个氨基酸残基组成的多肽，含有2个潜在的N-糖基化位点和多个磷酰化位点。结论成人腹泻标本WH-1中GBRV非结构蛋白NSP2基因和氨基酸序列与人GBRV有较高的同源性，而与动物中GBRV同源性相对较低，其中与ADRV的同源性最高，推测GBRV WH-1株与ADRV具有相同起源。     

我国各血清型轮状病毒非结构蛋白NSP4基因特征的初步分析

目的 对我国血清学调查中发现的四型（G型）轮状病毒NSP4序列进行测定和分析,了解NSP4的基因类型和变异状况。方法 利用银染测离方法对G1、G2、G3、G9型毒株NSP4 cDNA序列进行测定。结果 四个血清型毒株NSP4的核苷酸和氨基酸序列的同源性分别为78.8%,93.5%;83.0%,95.5%。G1、G3、G9型标本多属Wa组;G2型标本多属KUN组。NSP4的变异主要位于VP4结合域内的135－145位氨基酸。结论 Wa组和KUN组是我国主要的两种NSP4基因型。     

轮状病毒NSP486-175蛋白对大鼠心肌细胞损伤作用及相关机制研究

目的研究轮状病毒非结构蛋白4的86～175位氨基酸肽段(NSP486-175)对大鼠心肌细胞损伤作用及其相关机制。方法利用前期制备好的活性NSP486-175蛋白处理大鼠H9C2心肌细胞, 观察处理后的细胞生长形态变化, 检测细胞培养液中LDH活性;Fluo-3AM标记细胞内游离的钙离子, 蛋白质处理细胞后, 用激光共聚焦显微镜检测大鼠心肌细胞内钙离子浓度变化。流式细胞术分析蛋白质对细胞凋亡的影响, RT-PCR检测Bax、Bcl-2、caspase-3、78 kDa葡萄糖调控蛋白(GRP78)、C/EBP同源蛋白(CHOP)及caspase-12 mRNA转录水平;Western blot检测caspase-3、caspase-8、caspase-9、GRP78、CHOP和caspase-12蛋白表达水平。结果正常生长的心肌细胞呈典型的成肌样细胞形态, 细胞呈梭形, 边界清晰。纯化的NSP486-175蛋白刺激后出现明显的细胞病变效应, 细胞空泡变性, 皱缩变圆, 细胞碎裂;蛋白质处理组细胞培养液中的LDH活性均增高;与对照组相比, NSP486-175处理组细胞的钙离子荧光增强, ...     

轮状病毒非结构蛋白NSP4研究进展

轮状病毒（rotavirus,RV)是病毒性腹泻的主要病因，NSP4（nonstructural protein4)作为轮状病毒的一种非结构蛋白，在病毒形态发生和致病性中发挥重要作用，近年来日益受到重视，本文就NSP4的研究进展作一综述。     

轮状病毒非结构蛋白NSP4研究进展

轮状病毒(rotavirus,RV)是病毒性腹泻的主要病因.NSP4(nonstructural protein 4)作为轮状病毒的一种非结构蛋白,在病毒形态发生和致病性中发挥重要作用,近年来日益受到重视.本文就NSP4的研究进展作一综述.     

Diarrhea induction by rotavirus NSP4 in the homologous mouse model system.

Comparison of the NSP4 amino acid sequences from 31 strains of mammalian rotaviruses revealed the presence of four distinct NSP4 alleles; i.e., the Wa, KUN, AU-1, and EW alleles. The EW allele consists only of NSP4s from murine rotavirus strains and is divergent from other NSP4 alleles from the evolutionary perspective. There have been conflicting reports regarding the enterotoxigenic activity of NSP4 in the mouse model system; heterologous simian and porcine rotavirus NSP4s function as an enterotoxin in mice, while a homologous EC NSP4 does not play a dominant role as an enterotoxin in the cystic fibrosis conductance regulator knockout mice. To further examine the enterotoxigenic activity of NSP4, we expressed in Escherichia coli a recombinant protein consisting of glutathione S-transferase and amino acid residues 86-175 of the EW NSP4. We found that this fusion protein caused diarrhea in the majority (8/14) of 5- to 6-day-old CD1 mice. This study confirmed and extended that group A rotavirus NSP4s were able to induce diarrhea in neonatal mice and had an enterotoxigenic activity.     

G3P2 rotaviruses causing diarrhoeal disease in neonates differ in VP4, VP7 and NSP4 sequence from G3P2 strains causing asymptomatic neonatal infection

Summary During longitudinal epidemiological studies of rotavirus infections in children in Melbourne, Australia human G3P2 rotavirus strains causing asymptomatic or symptomatic infections have been identified. Eleven strains (AS strains) associated with asymptomatic infection of newborn babies from 1974–1984, and five strains (S strains) associated with symptomatic infection of newborn babies (4) or a 22 week old infant (1) during 1980–1986 were studied. The entire nucleotide sequences of genes coding for VP4, VP7, NSP4 and VP6 were derived for representative AS and S strains. The nucleotide sequences of neutralization epitope regions present on the outer capsid proteins VP4 and VP7 (regions C and F) showed extensive conservation of nucleotide and deduced amino acid sequence in all strains. Minor variations were observed over the 12 year period in VP7 epitope regions A and B in some strains. Specific conserved amino acids differences between the asymptomatic and symptomatic strains were observed in the genes encoding VP4 at aa133 and 303 (asparagine or threonine) and 380 (serine or isoleucine), VP7 at aa27 (threonine or isoleucine), aa29 (isoleucine or threonine), aa42 (valine or alanine) and aa238 (asparagine or aspartic acid/serine) and NSP4 at aa135 (isoleucine or valine). No amino acid changes were identified in gene 6. The observed amino acid differences occurred in proteins that have been implicated in virulence, and correlate with differences in clinical symptoms of infants infected with these strains. These results permit speculation about the genetic basis for virulence of human strains.The sequence data reported in this paper have been deposited in GenBank nucleotide sequence database under numbers U16299 and U42628.     

Species-specific but not genotype-specific primary and secondary isotype-specific NSP4 antibody responses in gnotobiotic calves and piglets infected with homologous host bovine (NSP4[A]) or porcine (NSP4[B]) rotavirus

Using recombinant baculoviruses expressing rotavirus NSP4 [A], [B], [C], and [D] genotypes of bovine, porcine, human, simian, or murine origin, we analyzed serum antibody responses to NSP4s in gnotobiotic calves and piglets infected by the oral/alimentary or intraamniotic route with bovine (NSP4[A]) (Wyatt, R.G., Mebus, C.A., Yolken, R.H., Kalica, A.R., James, H.D., Jr., Kapikian, A.Z., Chanock, R.M., 1979. Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus. Science 203(4380), 548-550) or porcine (NSP4[B]) (Hoshino, Y., Saif, L.J., Sereno, M.M., Chanock, R.M., Kapikian, A.Z., 1988. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 62(3), 744-748) rotaviruses. Following primary infection and challenge with virulent rotaviruses, the animals developed higher or significantly higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous host heterotypic NSP4s, indicating that antibody responses were species specific rather than genotype specific. Antibody responses to NSP4s corresponded closely with the phylogenetic relationships of NSP4s within a species-specific region of amino acids (aa) 131-141. In contrast, NSP4 genotypes determined by amino acid full-length sequence identity predicted poorly their "serotypes". In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, in an approach to immunization with a live oral rotavirus vaccine, the NSP4 protein does not appear to play an important role in protection against rotavirus disease and infection.     

A Human Vaccine Strain of Lamb Rotavirus (Chinese) NSP4 Gene: Complete Nucleotide Sequence and Phylogenetic Analyses

A lamb strain of rotavirus has recently been licensed for use in China as a live vaccine to prevent rotavirus diarrhea in children. As rotavirus NSP4, especially the cytotoxic domain alone is considered to be associated with diarrhea, we sequenced gene segment 10, which encodes NSP4, of lamb rotavirus. Comparative analyses was performed to identify differences from human rotavirus strains, that might be associated with attenuation, and to ascertain whether the lamb rotavirus gene fits among the NSP4 of other sequenced rotavirus strains. Our comparative nucleotide sequence analysis suggests its close identity (91.17% homology) with that of group-A equine rotavirus (strain HI23). Multiple alignment of the deduced amino acid sequence of lamb NSP4 with that of other group A rotaviruses demonstrated homology ranging from 63.42% with that of porcine YM strain to 93.71% with equine HI23 strain of rotavirus. A group A-specific NSP4 monoclonal antibody recognized the glycosylated and unglycosylated forms of the protein from virus-infected lysates, suggesting a well-conserved group-specificity of the lamb NSP4. Phylogenetic analysis of the lamb rotavirus gene, with 60 other NSP4 gene sequences of human and animal rotavirus strains, demonstrated that the lamb rotavirus strain belongs to genotype A. Comparative analysis also revealed that although it is a vaccine strain, the NSP4 cytotoxic domain of lamb strain demonstrated an overall amino acid conservation similar to that of other strains, whose NSP4 alone causes diarrhea in animal models. These results taken together with our previous observations clearly reaffirm the idea that the attenuation phenotype of rotaviruses does not involve NSP4 cytotoxic domain, perhaps due to the suppression of NSP4 cytotoxic activity by other rotaviral proteins.     

Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4

Long electropherotype with Subgroup I specificity is a common feature of animal rotaviruses. In an epidemic of infantile gastroenteritis in Manipur, India, long but SG I strains predominated in the outbreak in the year 1987-88. One such strain isolated from that region, following the outbreak had G9P [19] specificity. As this is a rare combination, the gene sequences encoding VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 of this strain were analyzed. All these genes except VP7 were closely related to porcine rotaviruses (95-99% identity at amino acid level) and clustered with the porcine strains in phylogenetic analysis. In addition, it had subgroup I nature and belonged to NSP4 genotype B which is characteristic of animal rotaviruses. This is the first report of a rotavirus with VP6 and NSP4, two crucial proteins thought to be involved in host range restriction and pathogenicity, were of porcine origin and caused diarrhoea in a human host. Among the genes of this strain sequenced so far, only VP7 had highest identity to human strains at amino acid level. This study suggests reassortment may be occurring between human and other animal strains and some of the reassortant viruses may be virulent to humans.     

Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997.

Serotyping of human rotavirus was conducted in 396 Japanese and 100 Thai rotavirus-positive fecal specimens collected from 1995 to 1997. Serotype G9 was found to be the third most common serotype with frequency of 16.2% in Thailand from 1996 to 1997. It was also detected in Japan with a low frequency (0.7%) in this year. The genetic analyses of VP4 and NSP4 genes of these G9 strains showed that 1 strain from Japan possessed P[8] genotype and NSP4 Wa-group with long electropherotype (e-type). In contrast, 5 strains from Thailand belonged to P[6] and 1 strain belonged to P[4]. All of the Thai strains were in the NSP4 KUN-group with a short e-type. Sequence analysis of their VP7 gene revealed that there was the highest homology among fecal G9 strains (> 96.3%, amino acid identity) and a relatively high degree of homology to standard viruses, F45 from Japan (95.4-96.3%, amino acid identity) and 116E from India (92-92.3%, amino acid identity). However, immunological analysis using G9 specific monoclonal antibodies (Mabs) against VP7 protein showed that the G9 strains isolated from the two countries had different antigenic specificity. It was confirmed further by intraserotypical phylogenetic analysis of VP7 amino acid. These results indicated that the prevalence of G9 rotavirus in 1996-1997 in Thailand was relative to the continuing recent emergence of it on a worldwide basis, while the Japanese G9 strain isolated in this survey was identified to have progenitors common to the F45 strain that was prevalent in 1985 in Japan. Phylogenetic analysis of VP7 amino acid of G1-14 prototype rotavirus showed that the G9 strains were most closely related to the equine G14 rotavirus FI23 strain but G3 strains, interserotypically. These findings suggest that G9 rotaviruses might be divided into two or more subtypes.     

Molecular characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for multiple human-animal interspecies transmissions

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian-like VP7 and caprine-like VP4 genes, was isolated from a 2-year-old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000-2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4 gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4 and VP7 genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4 genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses

Summary.  Previous sequence analyses of the rotavirus nonstructural NSP4 from human and some animal rotavirus strains revealed the presence of three distinct NSP4 alleles or genetic groups. To examine the species of origin relatedness and diversity of NSP4, the nucleotide and deduced amino acid sequences of the gene encoding the NSP4 from 15 animal rotavirus strains of porcine, equine, bovine, lapine and canine origin were determined and compared to human and other animal strains sequenced previously. Lapine and equine strains were shown to belong to the NSP4 genotype A. Murine NSP4 sequences formed a previously unrecognized fourth distinct NSP4 genotype (genotype D) that was more divergent compared to NSP4 genotype A, B, and C than the latter three are among each other. Within NSP4 genotypes, strains isolated from rabbits, horses, cows (genotype A) and pigs (genotype B) clustered according to species of origin, suggesting a conserved pattern of evolution within species. NSP4 sequence comparison among one wildtype and two tissue culture-adapted lapine strains, known to cause disease in neonatal rabbits, failed to identify amino acid changes within the variable region spanning amino acids 130 to 141, suggesting that disease in rabbits is the result of the lapine virus infection and replication, including production of the NSP4 enterotoxin. Accepted September 3, 1999/Received July 19, 1999