Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.     

Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.     

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.     

Diagnosis of rotavirus infection with cloned cDNA copies of viral genome segments.

The diagnostic potential of cloned cDNA copies of human rotavirus (strain WA) genome segments for the detection of rotavirus in clinical specimens has been determined. A hybridization assay in which a mixture of 32P-labeled cDNAs representing the 11 rotavirus segments was used as a probe compared favorably with three frequently used diagnostic tests for rotavirus in terms of both specificity and sensitivity. Significantly, clinical isolates could be readily distinguished when cloned cDNA copies of individual genome segments were used independently as a probe. In assays in which genome RNA from rotaviruses of known subgroups and serotypes were tested, cloned probes that encode nonstructural viral proteins hybridized efficiently to genome RNAs of all strains, whereas cloned probes corresponding to genome segments 6 and 9 exhibited the potential for differentiating strains of different subgroups and serotypes. Cloned cDNA copies of rotavirus genome segments therefore offer considerable potential for improved general diagnosis of rotavirus in clinical specimens, as well as for epidemiological studies in which virus isolates can be distinguished on the basis of nucleotide sequence homology of individual genome segments.     

A candidate packaging signal of human rotavirus differentiating Wa‐like and DS‐1‐like genomic constellations

Rotavirus A (RVA) possesses a genome of 11 segmented RNAs. In human RVA, two major genomic constellations are represented by prototype strains Wa and DS‐1. Here packaging signals differentiating Wa‐like and DS‐1‐like genomic constellations were searched for by analyzing genomic sequences of Wa‐like and DS‐1‐like strains. One pair of 11 nucleotide sites in the coding regions of viral structural protein (VP) 2 and VP6 was found to be complementary specifically among Wa‐like strains. These sites tended to be free from base‐pairing in secondary structures of genomic segments, suggesting that they may serve as a packaging signal in Wa‐like strains.     

Rotavirus VP2 core shell regions critical for viral polymerase activation

The innermost VP2 core shell of the triple-layered, icosahedral rotavirus particle surrounds the viral genome and RNA processing enzymes, including the RNA-dependent RNA polymerase (VP1). In addition to anchoring VP1 within the core, VP2 is also an essential cofactor that triggers the polymerase to initiate double-stranded RNA (dsRNA) synthesis using packaged plus-strand RNA templates. The VP2 requirement effectively couples packaging with genome replication and ensures that VP1 makes dsRNA only within an assembling previrion particle. However, the mechanism by which the rotavirus core shell protein activates the viral polymerase remains very poorly understood. In the current study, we sought to elucidate VP2 regions critical for VP1-mediated in vitro dsRNA synthesis. By comparing the functions of proteins from several different rotaviruses, we found that polymerase activation by the core shell protein is specific. Through truncation and chimera mutagenesis, we demonstrate that the VP2 amino terminus, which forms a decameric, internal hub underneath each 5-fold axis, plays an important but nonspecific role in VP1 activation. Our results indicate that the VP2 residues correlating with polymerase activation specificity are located on the inner face of the core shell, distinct from the amino terminus. Based on these findings, we predict that several regions of VP2 engage the polymerase during the concerted processes of rotavirus core assembly and genome replication.     

新成人腹泻轮状病毒J19株的全基因组克隆和VP4、VP6和VP7基因序列分析

为了进一步了解新成人腹泻轮状病毒J19株的基因和蛋白特征,利用一种改良的非依赖核酸序列的单引物扩增方法扩增J19株的11个基因,克隆到pMD18-T载体中并进行测序。在此基础上,将主要抗原蛋白VP4、VP6和VP7的蛋白序列与其它轮状病毒的相关蛋白序列进行比较分析并对VP6蛋白序列做遗传进化分析。结果获得J19株11个基因的全长基因序列。基因序列分析表明J19株的第3、6和第9基因分别长2 512bp、1 287bp和820bp,它们分别预测编码抗原蛋白VP4(823aa)、VP6(396aa)和VP7(258aa)。组成J19株的VP4、VP6和VP7蛋白序列对B组轮状病毒的CAL株、IDIR株以及ADRV株的相关蛋白序列的一致性分别是27.6%、38.5%和22.3%。对分组抗原蛋白VP6的遗传进化分析表明,J19株在进化树上的位置靠近外群蛋白分支以及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。J19株的VP4、VP6和VP7蛋白序列与其它轮状病毒的相应蛋白序列存在显著差异。VP6蛋白序列的遗传进化分析表明J19株可能是一个新组轮状病毒的代表性毒株;同时,它也可能是一个与B组轮状病毒的起源和进化密切相关的毒株之一。关于新成人腹泻轮状病毒J19株11个基因的克隆及VP4、VP6和VP7基因的序列分析,这是第一次报道。     

Sequence analysis of gene 11 equivalents from "short" and "super short" strains of rotavirus

The molecular basis for the aberrant migration pattern of the gene 11 equivalent in rotaviruses with "short" (human DS-1) and "super short" (human 69M and bovine VMRI) electropherotypes was investigated. The mRNAs of these viruses were synthesized in vitro, and the entire gene 11 equivalent of each of these viruses was sequenced with specific synthetic oligonucleotide primers. These sequences were compared with previously published sequences of "long" pattern rotavirus gene 11 segments. The increased lengths of the gene 11 equivalents of DS-1, 69M, and VMRI are due to a prolonged, 3' untranslated region in this gene segment. The 3' untranslated region of the VMRI gene 11 equivalent contains a clear duplication of a portion of its coding sequence. A stretch of 18 consecutive nucleotides within the 330-nucleotide, 3' untranslated region of 69M is identical to a section of UK coding sequence. The DS-1 and the remainder of the 69M 3'-end additional sequences are similar to each other, but neither is similar to any other currently available rotavirus gene sequence. This finding suggests that a process other than homologous duplication is involved in the evolution of these sequences. The widespread occurrence of human and animal rotaviruses with short and super short electropherotypes provides evidence that intragenic and possibly intergenic recombinational events associated with an error-prone viral RNA polymerase may play a role in increasing the genetic repertoire of rotaviruses.     

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。     

Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability.

A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.     

Sequence of the fourth gene of human rotaviruses recovered from asymptomatic or symptomatic infections.

The complete nucleotide sequence of the fourth gene of symptomatic (Wa, DS-1, P, and VA70) and asymptomatic (M37, 1076, McN13, and ST3) rotaviruses of serotype 1, 2, 3, or 4 was determined by the dideoxy chain termination method. In each strain, the fourth gene, which encodes the outer capsid protein VP3, is 2,359 base pairs in length and has 5'- and 3'-noncoding regions of 9 and 25 nucleotides, respectively. The gene has a single long open reading frame of 2,325 base pairs that is capable of coding for a protein of 775 amino acids. A total of 14 N-terminal and 12 C-terminal amino acids are completely conserved or almost completely conserved, respectively, among nine human rotavirus VP3 genes that have been sequenced. In addition, there is conservation of arginine at the two trypsin cleavage sites as well as conservation of clusters of amino acids in different regions of the two VP3 cleavage products, VP8 and VP5. Three distinct forms of VP3 were identified among the nine human rotavirus strains analyzed. Three symptomatic rotaviruses (serotypes 1, 3, and 4) possess highly related VP3 genes (92.2 to 97% nucleotide identity). Two symptomatic serotype 2 rotaviruses possess VP3 genes which are even more closely related to each other (98.6% nucleotide identity) and only moderately related to the aforementioned VP3 genes of serotypes 1, 3, and 4 (87.4 to 88.2% nucleotide identity). The four asymptomatic rotaviruses, which constitute the third group, possess highly related VP3 genes (95.5 to 97.5% nucleotide identity) which are distinct from those of the virulent rotaviruses (73 to 74.8% nucleotide identity). At 91 positions in the protein sequence of VP3, an amino acid is conserved among the asymptomatic rotaviruses, while a different amino acid is conserved among the symptomatic rotaviruses. Notably, five regions are conserved among the symptomatic rotaviruses, while a different set of sequences are conserved among the asymptomatic rotaviruses. It is possible that some or all of these regions of sequence dimorphism may be responsible for the difference in virulence of these two groups of human rotaviruses. There are 13 regions in the VP3 protein sequence which exhibit the greatest variability; the majority of these variable regions are observed between amino acids 106 to 192. These regions may represent potential antigenic sites related to heterotypic rotavirus neutralization.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Molecular characterization of two strains of porcine group C rotavirus

Group C rotaviruses are an important cause of acute gastroenteritis in humans and animals. Fecal samples were collected from a porcine herd in July, 2009. Group C rotavirus RNA was detected using RT-PCR for the VP6 gene. The identified strain was further characterized by sequencing and phylogenetic analysis of the partial VP4, and complete VP6 and VP7 gene sequences. The partial VP4 and complete VP6 gene sequences of the CUK-5 strain were most closely related to those of the CUK-6 strain of group C rotaviruses. Phylogenetic analysis of the VP7 gene of the 2 strains (CUK-5 and CUK-6) and reference strains of group G rotavirus by the neighbor-joining method also confirmed that CUK-5 and CUK-6 belonged to type G5 and G1 strains, respectively. This study provides useful data for the prediction of newly appearing variants of porcine group C rotaviruses in neighboring countries through comparisons with GCRVs and fundamental research for vaccine development.     

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.     

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.