The molecular biology of rotaviruses. VII. Detailed structural analysis of gene 10 of bovine rotavirus

cDNA cloning and nucleotide sequence analysis have allowed detailed structural studies on RNA segment 10 of the U.K. bovine rotavirus to be undertaken. The complete sequence of 751 nucleotides was determined and found to contain only a single long open reading frame capable of coding for a protein of 175 amino acids. The gene has an unusually long 3' untranslated region of 184 nucleotides or some 24.5% of the total sequence, whose start was confirmed by analysing the carboxyterminal amino acid of the gene 10 product, the glycoprotein VP10c. Analysis of purified virions radio-labelled with [3H]glucosamine and [3H]mannose showed that VP10c is not a detectable component of the virus particle. The two potential glycosylation sites were found to be very close to the amino terminus of the putative translation product, strongly suggesting that the glycoprotein VP10c does not contain a cleavable signal sequence.     

Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo-Uruguay from 1996-1999

The gene encoding the protein VP7 that induces the major neutralizing response has been sequenced from 34 human rotaviruses isolated from children with acute diarrhea in Montevideo (Uruguay) over a 4-year period (1996-1999). These sequences were analyzed and compared to representative corresponding sequences available on databases. In most years, serotype G1 was present as the single serotype, except in 1999 when serotypes G1 and G4 were present simultaneously. Two G1 VP7 lineages were identified. Serotype G2 was present in 1997. The G4 isolates are grouped with Argentine strains and emerged during 1998 in a recently defined sublineage. Neither serotype G3 nor the emerging serotype G9 were isolated during the study. Antigenic domains of isolates and of representative reference strains of each serotype were compared. Sequences of strains isolated during the same year, showed a high degree of homology among strains belonging to the same serotype.     

轮状病毒抗原蛋白G3VP7基因在花生中遗传转化的研究

为提高外源蛋白的表达量和简化分离纯化过程,使用植物油体表达载体,以花生子叶节为转化受体,通过农杆菌介导将轮状病毒抗原蛋白G3VP7基因开展遗传转化的研究。从转化植株中随机选取11株表现Kan抗性植株进行PCR检测,结果有6株能扩增出特异性条带,阳性率为54.5%。对转基因植株进一步进行PCR-Southern杂交分析,发现转基因植株中有6株PCR反应呈阳性,其中有3株PCR-Southern杂交有特异性目标带出现。结果表明,外源基因已经整合到了花生基因组上。该研究为以植物为载体生产廉价、高效的植物口服疫苗奠定了基础。     

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.     

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains

Of five globally important VP7 (G) serotypes (G1-4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.     

Molecular characterization of VP4, VP6, VP7, NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain

An unusual strain of human rotavirus G3P[10] (CMH079/05) was detected in a stool sample of a 2‐year‐old child admitted to the hospital with severe diarrhea in Chiang Mai, Thailand. Analysis of the VP7 gene sequence revealed highest identities with unusual human rotavirus G3 strain CMH222 at 98.7% on the nucleotide and 99.6% on the amino acid levels. Phylogenetic analysis of the VP7 sequence confirmed that the CMH079/05 strain formed a cluster with G3 rotavirus reference strains and showed the closest lineage with the CMH222 strain. Analysis of partial VP4 gene of CMH079/05 revealed highest degree of sequence identities with P[10] rotavirus prototype strain 69M at nucleotide and amino acid levels of 92.9% and 94.6%, respectively. Phylogenetic analysis of the VP4 sequence revealed that CMH079/05 and 69M clustered closely together in a monophyletic branch separated from other rotavirus genotypes. To our knowledge, this is a novel G–P combination of G3 and P[10] genotypes. In addition, analyses of VP6, NSP4, and NSP5/6 genes revealed these uncommon genetic characteristics: (i) the VP6 gene differed from the four other known subgroups; (ii) the NSP4 gene was identified as NSP4 genetic group C, an uncommon group in humans; and (iii) the NSP5/6 gene was most closely related with T152, a G12P[9] rotavirus previously isolated in Thailand. The finding of uncommon G3P[10] rotavirus in this pediatric patient provided additional evidence of the genetic diversity of human group A rotaviruses in Chiang Mai, Thailand. J. Med. Virol. 81:176–182, 2009. © 2008 Wiley‐Liss, Inc.     

轮状病毒VP7基因的克隆与表达

目的构建轮状病毒VP7的重组表达质粒，在大肠杆菌中对其进行表达。方法克隆编码轮状病毒VP7的基因，并在原核表达系统中表达了带有6xHis标签的融合蛋白。结果经双酶切鉴定和基因测序证明成功重组了轮状病毒VP7基因的pQE-30重组质粒；蛋白质PAGE电泳表明，获得了分子量约为48000Mr的蛋白质。结论本研究获得了重组的轮状病毒VP7蛋白。     

Analysis of the VP6 gene of human and porcine group A rotavirus strains with unusual subgroup specificities

Full‐length VP6 amino acid sequences of human and porcine rotaviruses with subgroup (SG) (I + II) and SG non‐(I + II) were analyzed in comparison with those of SG I and SG II. In human rotaviruses, the strains in the same SG shared a very high degree of amino acid identity, ranging from 97.4% to 99.4% for SG I, 95.9% to 100% for SG II, and 99.4% to100% for SG non‐(I + II), while viruses in different SGs shared somewhat lower sequence identity at 90.4–93.1%. Conserved amino acids that distinguished the strains of SG I from SG II were observed at 21 positions. The viruses with SG non‐(I + II) shared sequence identity with SG II as high as 97.2–99.7%, suggesting that they belonged to genogroup II. Similarly, porcine rotaviruses in the same SG shared 96.4–99.7% for SG I, 98.2–100% for SG II, 97.4–100% for SG (I + II), and 96.2–99.7% for SG non‐(I + II), while strains in different SGs shared sequence identity ranging from 91.9% to 94.4%. Interestingly, the strains with SG (I + II) and SG non‐(I + II) shared a high degree of sequence identity with SG I, at 96.4–100% and 94.7–99.7% respectively, suggesting that they are related to porcine SG I strains. The conserved amino acids which distinguished SG I from SG II were observed at 13 positions. The strains with SG I, SG (I + II), and SG non‐(I + II) showed identical amino acid residues at these positions. Phylogenetic analysis strongly supported the findings of the sequence analysis. J. Med. Virol. 81:183–191, 2009. © 2008 Wiley‐Liss, Inc.     

Diversity in the VP7 encoding genes of rotavirus strains isolated from adolescent and adult cases of acute gastroenteritis

A study was conducted to examine the diversity in the VP7 genes of rotavirus strains circulating in adolescent and adult cases of acute gastroenteritis during two different time periods, 1993-1996 and 2004-2007. The multiplex RT-PCR carried out on 131 rotavirus positive fecal specimens detected 65 (49.6%) single and 48 (36.6%) mixed infections of VP7 genotypes that included 43G1 (38.1%), 37G2 (32.7%), 8G3 (7.1%), 15G4 (13.3%), and 10G9 (8.8%) specificities. Sequencing and phylogenetic analysis of the VP7 gene amplicons revealed the presence of G1-IA (4.7%), G1-IB (69.8%), and G1-IC (25.5%) lineages within the G1 strains, G2-IIb1 (70.3%) and G2-IIb2 (29.7%) lineages within G2 strains, G3-3S1 (12.5%) and G3-3S4 (87.5%) lineages within G3 strains, G4-Ia (6.7%) and G4-Ib (93.3%) lineages within G4 strains, and G9-III lineage within G9 strains. The variability within VP7 genotypes was evident by 1.4-8.0% and 1.3-3.9% amino acid divergence respectively from the prototype strains and between the groups of strains at the two time points. This is the first report describing the phylogenetic analysis of VP7 genes of rotaviruses from adolescent and adult cases of acute gastroenteritis in India. Since adults infected with rotavirus could act as a source of infection and affect the epidemiology of rotaviruses in children, genetic analysis of the rotavirus strains circulating in adults is required. The intragenotypic diversity within VP7 genes demonstrated by the present study highlights the need for constant surveillance of rotavirus infections to understand better the evolution and transmission of group A rotaviruses in the community.     

Sequence Analysis of the VP4, VP6, VP7, and NSP4 Gene Products ofthe Bovine Rotavirus WC3

The bovine rotavirus (BRV) WC3 serves as the background strain in the development of a multivalent reassortant vaccine against rotavirus gastroenteritis in infants. The genes encoding the outer capsid spike protein VP4, the inner capsid protein VP6, the outer capsid glycoprotein VP7, and the viral enterotoxin NSP4 of BRV WC3 were sequenced. Comparative analysis of the deduced amino acids of the sequenced genes indicated that the BRV WC3 strain shares a high degree of amino acid identity with serotype P7[5] VP4 (93–96%), serotype G6 VP7 (91–97%), subgroup (SG) I VP6 (96–99%), and NSP4 genogroup A (96–98%) BRV strains. Our results confirm and extend previous studies which suggested that the VP4 of BRV WC3 was closely related to that of the P7[5] prototype, BRV UK. In addition, the VP6 and VP7 of BRV WC3 were very similar to the VP6 and VP7 of both SG I and G6 BRV NCDV and UK strains. However, the NSP4 of BRV WC3 was more closely related to that BRV NCDV, the P6[1] prototype, than to that of BRV UK.     

Bovine Rotavirus 993/83 Shows a Third Subtype of Avian VP7 Protein

VP7 genes of rotavirus (RV) 993/83 isolated from a German calf with diarrhea and of RV PO-13 isolated from a Japanese pigeon were sequenced. Alignment of the deduced VP7 amino acid sequence showed 98.8% sequence identity, while only 70% and 84% identity was seen with VP7 from chicken RV Ch-2 and turkey RV Ty-1, respectively. Over the antigenic regions A, B, and C mammalian RV 993/83 showed more aa identity with mammalian G3 RVs than with chicken RV Ch-2, which could explain the strong one-way cross-neutralization observed between RV 993/83 and G3 RVs. Despite marked VP7 sequence diversity avian RVs could not be differentiated into distinct G types. This revised version was published online in July 2006 with corrections to the Cover Date.     

VP7 and VP4 genotypes of rotaviruses cocirculating in Iran, 2015 to 2017: Comparison with cogent sequences of Rotarix and RotaTeq vaccine strains before their use for universal mass vaccination

The present study was conducted to analyze the genotypic diversity of circulating species A rotavirus (RVA) strains in Iran and also to investigate comparative analysis between the genotypes of VP4 and VP7 of cocirculating RVA and vaccine strains before the vaccine is introduced in the national immunization program. The G3-lineage I was found in this study as the most common G genotype which was followed by G9-lineage III, G1-lineages I, II, G12-lineage III, G2-lineage IV, and G4-lineage I. Also, P[8]-lineages III, IV was found as the predominant P genotype which was followed by P[4]-lineage V, and P[6]-lineage I. Overally, G3P[8] was determined as the most common combination. Moreover, the analysis of the VP7 antigenic epitopes showed that several amino acid differences existed between circulating Iranian and the vaccine strains. The comparison of genotype G1 of Iranian and vaccine strains (RotaTeq and Rotarix), and genotypes G2, G3, and G4 of Iranian and RotaTeq vaccine strains revealed three to five amino acids differences on the VP7 antigenic epitopes. Furthermore, analyzing of the VP8* epitopes of Iranian P[8] strains indicated that they contained up to 11 and 14 amino acid differences with Rotarix and RotaTeq, respectively. Based on different patterns of amino acid substitutions in circulating and vaccine strains, the emergence of antibody escaping mutants and potentially the decrease of immune protection might ensue in vaccinated children. However, considering the broad cross-protective activity of RVA vaccines, their efficacy should be monitored after the introduction in Iran.     

Surveillance for rotavirus in Argentina

Group A rotaviruses are the major cause of severe gastroenteritis in young children worldwide. Because rotavirus vaccination appeared imminent, a nationwide surveillance program was organized between October 1996 and October 1998 in the largest Argentine cities. Surveillance for disease burden, rotavirus detection, and rotavirus typing was undertaken at nine locations. Results showed rotavirus to be associated with 42% of diarrhea admissions. Although the prevalent G types changed from year to year, common G types were found in 96% of the cases and were usually associated with common P types. Uncommon G types, G9 and G5, were found at low prevalence and uncommon G/P combinations occurred at almost every study site. These data suggest that a rotavirus vaccine could substantially decrease the rotavirus disease burden in Argentina, but that introduction of a vaccine should be accompanied by a concurrent surveillance system.     

轮状病毒结构蛋白VP7 HLA-A2.1限制性CTL表位的初步鉴定

目的 预测并初步鉴定轮状病毒Wa株结构蛋白VP7 HLA—A2．1限制性CTL表位。方法 用BIMAS软件预测VP7 HLA—A2．1限制性CTL表位;分子模拟技术对分值最高的4条肽进行分子模建;最后测定候选肽与HLA-A2．1分子的亲和力及结合稳定性。结果 结合BIMAS及分子模拟的预测结果,选择4条肽QLYCDYNLV（132—140）、LLNYILKSV（18—26）、VLMKYDQSL（140—148）及VNWKKWWQV（287—295）作为候选表位肽;4条肽与HLA—A2．1结合的荧光系数（FI）值分别为2．58、3．83、3．19及0．82,肽-HLA-A2．1复合物半数解离时间（DC50）分别为2-4、6-8、8及2h。结论 QLYCDYNLV（132—140）、LLNYILKSV（18—26）及VLMKYDQSL（140—148）为潜在的HLA-A2．1限制性CTL表位。     

Cross-reactivity between the major Parietaria allergen and rotavirus VP4 protein

BACKGROUND: The present study investigates immunological cross-reactivity between Par o 1, the major pollen allergen of Parietaria, and the VP4 protein of rotavirus, a microorganism that is world-wide the main etiological agent of gastroenteritis in children. METHODS: IgG and IgE cross-reactivity was assessed by direct binding and competitive inhibition assays (ELISA and DARIA), using recombinant VP4 from rhesus infectious rotavirus (RR), synthetic peptides and Par o 1-specific antibodies affinity purified from pooled and individual human sera. RESULTS: Antibodies specifically binding Par o 1, affinity purified from the sera of 35 individuals with skin test positivity to Parietaria and from 14 pools, were extensively cross-reactive with RRVP4. Cross-reactive binding was specifically inhibited by synthetic peptides derived from the C-terminal sequences of the VP4 proteins from human and rhesus infectious rotavirus. CONCLUSIONS: This study reports the first evidence of cross-reactivity between an allergen and a viral antigen.     

A long‐term survey on the distribution of the human rotavirus G type in Thailand

The distribution of the G type of human rotavirus was surveyed in Thailand between July 1993 and June 2007. A significant yearly change in the distribution of the G type distribution was found. From 1993–1994 to 1998–1999, the G1 type was the most dominant. In 1999–2000, G9 began to appear at a high frequency. In 2000–2001, 2001–2002, and 2002–2003, G9 was very common. In 2003–2004, G1 became the most prevalent type again, and since then it has been detected at the highest frequency. G12 strains, which were first detected in 1998–1999, were also found in 2004–2005 and 2006–2007. The G4 and G3 types were moderately prevalent in 2001–2002 and 2004–2005, respectively. Nucleotide sequence analysis of the VP7 genes of the G9 and G12 strains which reemerged in Thailand showed that they were each similar to the contemporary strains in other countries. J. Med. Virol. 82:157–163, 2010. © 2009 Wiley‐Liss, Inc.