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。     

Whole-Genome Analysis of a Rare Human Korean G3P Rotavirus Strain Suggests a Complex Evolutionary Origin Potentially Involving Reassortment Events between Feline and Bovine Rotaviruses

A rare human rotavirus, G3P[9] strain RVA/Human-tc/KOR/CAU12-2-51/2013/G3P[9], was isolated from the stool of a 9-year-old female hospitalized with acute watery diarrhea in August 2012 in South Korea using a cell culture system, and its genome was analyzed. The complete genomic constellation of the CAU12-2-51 strain revealed a novel genotype constellation for human rotavirus, G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. Phylogenetic analysis revealed that the CAU12-2-51 strain originated from feline- and bovine-like reassortment strains. The genes encoding VP4, VP7, NSP1, NSP3, NSP4, and NSP5 were related to human/feline-like and feline rotavirus strains, whereas the remaining five genes encoding VP1, VP2, VP3, VP6, and NSP2 were related to the human/bovine-like and bovine rotavirus strains. This novel strain was identified for the first time, providing evidence of feline/bovine-to-human transmission of rotavirus. The data presented herein provide information regarding rotavirus diversity and evolution.     

Whole Genomic Analysis of an Unusual Human G6P[14] Rotavirus Strain Isolated from a Child with Diarrhea in Thailand: Evidence for Bovine-To-Human Interspecies Transmission and Reassortment Events

An unusual rotavirus strain, SKT-27, with the G6P[14] genotypes (RVA/Human-wt/THA/SKT-27/2012/G6P[14]), was identified in a stool specimen from a hospitalized child aged eight months with severe diarrhea. In this study, we sequenced and characterized the complete genome of strain SKT-27. On whole genomic analysis, strain SKT-27 was found to have a unique genotype constellation: G6-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The non-G/P genotype constellation of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) is commonly shared with rotavirus strains from artiodactyls such as cattle. Phylogenetic analysis indicated that nine of the 11 genes of strain SKT-27 (VP7, VP4, VP6, VP2-3, NSP1, NSP3-5) appeared to be of artiodactyl (likely bovine) origin, while the remaining VP1 and NSP2 genes were assumed to be of human origin. Thus, strain SKT-27 was found to have a bovine rotavirus genetic backbone, and thus is likely to be of bovine origin. Furthermore, strain SKT-27 appeared to be derived through interspecies transmission and reassortment events involving bovine and human rotavirus strains. Of note is that the VP7 gene of strain SKT-27 was located in G6 lineage-5 together with those of bovine rotavirus strains, away from the clusters comprising other G6P[14] strains in G6 lineages-2/6, suggesting the occurrence of independent bovine-to-human interspecies transmission events. To our knowledge, this is the first report on full genome-based characterization of human G6P[14] strains that have emerged in Southeast Asia. Our observations will provide important insights into the origin of G6P[14] strains, and into dynamic interactions between human and bovine rotavirus strains.     

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).     

Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Whole Genomic Analysis of Human G12P[6] and G12P[8] Rotavirus Strains that Have Emerged in Myanmar

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.     

Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Assessment of gastroenteric viruses frequency in a children's day care center in Rio De Janeiro, Brazil: a fifteen year study (1994-2008)

This 15-year study aimed to determine the role of the main viruses responsible for acute infantile gastroenteritis cases in a day care center in the city of Rio de Janeiro, Brazil. From 1994 to 2008, 539 fecal samples were obtained from 23 outbreaks as well as sporadic cases that occurred in this period. The detection of Rotavirus group A (RVA), norovirus (NoV) and astrovirus (AstV) was investigated both by classical and molecular methods of viral detection. RVA was detected by enzymatic immune assay and/or polyacrylamide gel electrophoresis and genotyped by using semi-nested multiplex PCR. NoV and AstV were subsequently tested by real time PCR in all RVA-negative samples and genotyped throughout genome sequencing. Three protocols for molecular characterization of NoV nucleotide sequencing were performed with the partial nucleotide sequencing of genomic regions known as region B (polymerase gen), C and D (capsid gen).Viruses were identified in 47.7% (257/539) of the cases, and the detection rates of RVA, NoV and AstV in16.1% (87/539), 33.4% (151/452), and 6.3% (19/301), respectively. Most gastroenteritis cases were reported in autumn and winter, although NoV presented a broader monthly distribution. Viruses' detection rates were significantly higher among children aged less than 24 months old, although NoV cases were detected in all age groups. RVA genotypes as G1P[8], G9P[8], G2P[4], G3P[8] and G1+G3P[8] and RVA was no longer detected after 2005. NoV characterization revealed genotypes variability circulating in the period as GI.2, GI.3, GI.8 GII.2, GII.3, GII.4, GII.4 variants 2001 and 2006b, GII.6, GII.7, GII.12 and GII.17. AstV genotypes 1, 2, 4 and 5 were also characterized. Those data demonstrate the impact of NoV infection in cases of infantile gastroenteritis, surpassing RVA infection responsible for high morbidity rate in children under five years old.     

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.     

Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.     

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.     

Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs

VP7 of group A rotavirus(RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector,three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains.Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6 F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.     

Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system

The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.