Characterization of neutralization specificities of outer capsid spike protein VP4 of selected murine,lapine, and human rotavirus strains

Neutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine x human, three lapine x human, and two human x human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study.     

Serological and genomic characterization of human rotaviruses detected in China

A total of 1,385 stool specimens were collected from children with diarrhea at two hospitals in Wuhan, Hubei Province, China, in 1994 and 1995, and screened for rotavirus by polyacrylamide gel electrophoresis of viral RNA. Group A rotavirus was detected with high frequency; 56.5% (87/154) and 40.8% (502/1,231) of the specimens collected in 1994 and 1995, respectively, were positive for rotavirus. Assignment of G serotype and P type (VP4 genotype) of group A rotavirus by ELISA with monoclonal antibodies and/or PCR, respectively, showed that strains of G2-P[4] and G1-P[8] specificity were predominant in 1994 and in 1995, respectively. In contrast, a single strain was found to have a P[9] type specificity, and no G4 strain was detected. Unusual combinations of RNA pattern-subgroup-G serotype-P type, such as long pattern-subgroup I-G1-P[8], short pattern-subgroup II-G3-P[4] and short pattern-subgroup I-G1-P[4], were detected in four specimens. Nucleotide sequences of the VP8* and/or NSP5 genes from two Chinese P[8] strains 470 and 582 and one Chinese P[9] strain 512 as well as five Japanese P[9] strains (K8, AU1, M318, O264, and O265) were determined and compared with the published sequences of the corresponding gene. In the phylogenetic tree of VP8* sequences of P[9] strains, which formed two clusters each having strain K8 or AU-1 as the representative strain, the Chinese P[9] strain was found in the cluster represented by AU-1, although it was most distantly related to other strains. While NSP5 sequences of human strains with P[9] specificity were related to simian and bovine strains, that of Chinese P[8] strains was most closely related to those of porcine strains. A single group C rotavirus (No. 208) was detected. Nucleotide sequences of its VP4, VP6, VP7, and NSP4 genes were very similar to those of group C human rotaviruses detected worldwide. J. Med. Virol. 55:168–176, 1998. © 1998 Wiley-Liss, Inc.     

A hospital based study on inter- and intragenotypic diversity of human rotavirus A VP4 and VP7 gene segments,Germany

BackgroundEfforts to reduce the impact of group A rotaviruses on human morbidity and mortality rely on oral immunisation with live attenuated or recombinant vaccines. A major challenge in immunisation is the vast inter- and intragenotypic diversity accomplished by circulating rotaviruses.ObjectivesTo monitor rotavirus inter- and intragenotypic diversity in hospitalised children.Study designFrom January 2008 to December 2009 stool samples from 1994 paediatric in-patients suffering from diarrhoea were screened for rotavirus. Rotavirus G- and P-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed.ResultsRotavirus A was detected in stool samples of 341 children, comprising G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], as well as uncommon G12P[6] genotypes and mixed infections. Predominant strains shifted from G1P[8] and G9P[8] genotypes in the first season to G3P[8] and G4P[8] genotypes in the second season. The highest intragenotypic diversity was detected in G1 strains and consisted of co-circulating G1-Ic, G1-Id, G1-Ie and G1-II rotaviruses. The G2 analysis revealed different intragenotypic lineages: G2-IIa, G2-IIb and G2-IIc. Interestingly, the circulating G4-Ib rotaviruses were characterised by insertions of 3 or 6 additional coding nucleotides within variable region 4 of VP7. Whereas different G9-III VP7 gene segments were detected G3-Ia sequences were highly homologous. In the VP4 analysis P[8]-III gene segment predominated over P[4]-Vb, P[8]-I, P[8]-IV and P[6]-I.ConclusionsA remarkable rotavirus heterogeneity was detected in the limited local setting and time span. Continued monitoring and nucleotide sequencing is necessary to document possible effects of rising immunisation levels on intragenotypic rotavirus diversity.     

Changing pattern of human group A rotaviruses: emergence of G12 as an important pathogen among children in eastern India.

BACKGROUND: Rotavirus genotypes, G1-G4 and G9 are associated with childhood diarrhoea throughout the world. In our previous study, we detected G1, G2, G4 and three G12 strains from Kolkata, India. OBJECTIVES: To study the prevalence of G- and P-genotypes of rotaviruses associated with dehydrating diarrhoea in children admitted to two leading hospitals in eastern India. STUDY DESIGN: An active surveillance was conducted for elucidation of rotavirus infection in two leading hospitals in Kolkata, West Bengal and Berhampur (GM), Orissa, India, separated by 603km from January 2003 to April 2005. The rotaviruses were detected by RNA electrophoresis in polyacrylamide gels. G- and P-typing of the positive samples were accomplished by amplifying VP7 and VP4 genes by RT-PCR and genotyped by seminested multiplex PCR methods. Sequencing, sequence analysis and phylogenetic analysis of VP7 genes of G12 strains were carried out to understand the variations between the strains isolated from different parts of the world. RESULTS: The genotypic distribution varied remarkably from our earlier study period (1998-2001) with G1 (53.8%) being the most predominant strain followed by G2 (22.5%), G12 (17.1%), G9 (2.1%) and not a single G3 or G4 isolate was detected separately. 35.2% samples exhibited mixed P-types followed by P[4] (31.7%), P[8] (21.8%) and P[6] (9.8%). The phylogenetic analysis of G12 strains revealed that the G12 strains detected from different parts of the world clustered into three different lineages. Though VP7 sequences of G12 strains isolated from Kolkata and Berhampur are conserved, their P-types were different. CONCLUSION: During this study period we reported emergence of G12 strains as an important pathogen among children in eastern India, thus necessitating its inclusion in future polyvalent vaccine to control rotavirus diarrhoea.     

Characterization of nontypeable rotavirus strains from the United States: identification of a new rotavirus reassortant (P2A[6],G12) and rare P3[9] strains related to bovine rotaviruses

Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% a.a. and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8-98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2-98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% a.a. homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains.     

Characterization of inter-genogroup reassortant rotavirus strains detected in hospitalized children in Italy

Analysis of archival stool collections provides an invaluable source of virus strains and genetic material that may be exploited for molecular, epidemiological, and biological studies. The aim of this study was the molecular characterization of unusual human rotavirus (HRV) strains displaying atypical combinations of electropherotype (e-type) and VP4 and/or VP7 genotypes. Analysis of a panel of archival stools collected in northern Italy revealed continual circulation of P[8]G1 HRVs during 1987-1990 and the onset of P[6] + P[8]G1 strains after 1989. Interestingly, nine G1 strains, associated with either P[8], P[4] + P[8], P[6] + P[8], or untypeable VP4 genes, and two P[4]G1 + G2 strains, displayed short RNA e-type. The genetic constellation of the unusual strains was investigated by analysis of the VP4, VP6, VP7, and NSP4 genes. All the G1 strains with short e-type were subgroup (SG)II or SGI + SGII, and possessed a NSP4 of genogroup B or A + B. Conversely, the P[4]G1 + G2 strains were SGI and possessed a genogroup A NSP4. Sequence analysis of the VP7 and VP4 genes revealed that the unusual P[8]G1 and P[4]G1 + G2 viruses emerged by reassortment of strains circulating locally, rather than by introduction of new strains.     

Detection of unusual rotavirus genotypes G8P[8] and G12P[6] in South Korea

Five hundred four fecal specimens, collected between 2004 and 2006 from young children with acute diarrhea, were screened for rotavirus by ELISA with VP6-specific antibody. Of these samples, 394 (78.2%) were confirmed as group A rotavirus and they underwent G- and P typing using a combination of ELISA, RT-PCR, and sequence analysis methods. The dominant circulating G serotype was G1 (35.6%) followed by G3 (26.4%), G4 (14.7%), and G2 (11.9%). There was a low prevalence of G9 (1.0%) and of unusual G type rotavirus, in particular, G12 (0.5%) and G8 (0.3%). Of the P genotype rotavirus in circulation, P[8] (53.0%) was most common followed by P[6] (15.5%), P[4] (15.2%), and P[9] (2.3%). Determination of G- and P type combinations revealed that G1P[8] strains were most prevalent (25.4%), amid G3P[8] (16.8%), G2P[4] (6.3%), and G4P[6] (6.1%) strains. Unusual or rare combinations such as G2P[6], G2P[8], G3P[4], G2P[9], G1P[9], G3P[9], G12P[6], G1P[4], G3P[6], and G8P[8] were also found. Owing to the recent emergence of G8 and G12 rotavirus, the findings from this study are important since they provide new information concerning the local and global spread of rotavirus genotypes.     

Characterisation of rotavirus G9 strains isolated in the UK between 1995 and 1998

G9P[6] and G9P[8] rotavirus strains were identified during 1995/96 through the molecular epidemiological surveillance of rotavirus strains circulating in the UK between 1995 and 1998. An increase in the incidence and spread of sporadic infections with rotavirus genotype G9P[8] across the UK was detected in the two following seasons. Partial sequencing of the VP7 gene showed that all the UK strains shared a high degree of homology and were related very closely to G9 strains from the US and from symptomatic infections in India (> or =96% homology). The UK strains were related more distantly to the apathogenic Indian strain 116E (85-87.8% homology). Phylogenetic analysis revealed clustering of the UK strains into 3 different lineages (I to III) and into two sub-lineages within lineage I. There were correlations between VP7 sequence clustering, the P type and the geographical origin of the G9 strains. Partial sequencing of the VP4 gene showed high degree of homology (>98%) among all the P[6] strains, and the sequences obtained from the P[8] strains clustered into 2 of the 3 global lineages described for P[8] strains associated with other G types. These data suggest that G9 strains may be a recent importation into the UK, and that G9P[8] strains may have emerged through reassortment in humans between G9P[6] strains introduced recently and the more prevalent cocirculating G1, G3 and G4 strains that normally carry VP4 genes of P[8] type.     

Genetic characterization of a novel,naturally occurring recombinant human G6P[6] rotavirus

A binary classification system has been established for group A rotaviruses, with the viral capsid protein VP7 defining G types and VP4 defining P types. At least 15 G types and 21 P types have been isolated globally with various G and P combinations. Most of the currently circulating human rotaviruses belong to G1P[8], G2P[4], G3P[8], and G4P[8]. We report a human rotavirus strain (B1711) with a novel genotypic VP7/VP4 combination of G6P[6]. This unique rotavirus was isolated from a 13-month-old human immunodeficiency virus (HIV)- negative child of an HIV-seropositive Malian mother that was hospitalized with severe diarrhea in Belgium after returning from a trip to Mali. The VP7 and VP4 genes of the rotavirus strain were sequenced, and phylogenetic trees were constructed. Nucleotide and amino acid sequence comparisons with 15 known G genotypes indicated that the VP7 sequence of strain B1711 was most closely related to an American (Se584) and an Italian (PA151) human G6 strain (95 to 96% nucleotide and 98% amino acid identity). Comparison of the VP4 sequence with 21 P types showed the closest similarity to P[6] genotypes, with greatest similarity to a G8P[6] Malawi strain (mw131) (97% nucleotide and 98% amino acid identity). The B1711 strain is the first reported rotavirus isolate with a G6P[6] genotypic combination. The discovery and surveillance of novel human and nonhuman rotavirus G or P types or of novel G/P combinations is essential for the design of future rotavirus vaccines and for our understanding of rotavirus diversity and evolution.     

Molecular epidemiology of group A rotavirus in outpatient diarrhea infants and children in Chongqing,China, 2011-2015

Human group A rotavirus (RVA) is the leading cause of acute viral gastroenteritis in children under 5 years old worldwide. The aim of this study was to investigate the genotype distribution of RVA in the Midwest of China. Sentinel-based surveillance of acute diarrhea was conducted at Children's Hospital of Chongqing Medical University from 2011 to 2015. RVA was tested by using enzyme-linked immunosorbent assays. The partial VP4 genes and VP7 genes of rotavirus were amplified and sequenced, and genotyping and phylogenetic analyses were performed. Among the 2236 stool specimens collected from children with acute gastroenteritis, 681 (30.46%) were positive for RVA. The majority of children (89.28%) who tested positive for RVA were children aged ≤2 years. The seasonal peak of RVA was in the winter. As for genotype, four strain combinations, G9P[8], G3P[8], G1P[8], and G2P[4] contributed to 75.62% (515/681) of the RVA-associated diarrhea cases. After a marked increase in G9P[8] (30.77%) in 2013, G9P[8] became the predominant genotype in 2014 and 2015, whilst the prevalence of G1P[8] was decreased to 2.72% in 2015. Unusual G-P combinations (eg, G1P[4], G9P[4], G4P[6], G3P[4], G2P[8]) were also detected sporadically over the study period. Phylogenetic tree analysis results showed that the VP7 sequences of G9 strains were clustered into two main lineages, and 77.34% of them were clustered into lineage VI, with the highest nucleotide similarity to the strain JS12-17(China). VP4 gene sequences of P[8] strains were almost P[8]-lineage 3. Substantial temporal variation in the circulation of various genotypes of rotavirus in Chongqing was observed during 2011-2015, and highlights the need for continuous surveillance of RVA infection for better understanding and control of RVA infection.     

Emergence of human G9 rotavirus with an exceptionally high frequency in children admitted to hospital with diarrhea in Chiang Mai, Thailand

Among 315 fecal specimens collected from children hospitalized with diarrhea in Chiang Mai, Thailand, in 2000-2001, group A rotavirus was detected in 107 (34.0%). Of these, 98 (91.6%) were G9, 6 (5.6%) were G3 and 3 (2.8%) were G2, respectively. Identification of their P-types demonstrated that 103 (96.3%) were P[8], 3 (2.8%) were P[4], and 1 (0.9%) was P[3] genotypes. Determination of G- and P-type combination revealed that all of G9 isolates were associated with P[8]. G9P[8] was the most predominant genotype and accounted for the majority (91.6%) of rotaviruses detected in this study. Molecular characterization of these G9 isolates demonstrated that all had long electropherotype, 96 of 98 (98.0%) belonged to subgroup II, one belonged to subgroup I and the other one was subgroup unidentifiable. All of G9 isolates possessed NSP4 genetic group B except for one isolate that showed dual genetic group specificities, B and C. The full-length VP7 gene nucleotide sequences among 15 representatives of these G9 strains were found to be highly homologous with percent identities of 99.3%-100%. Comparison with other G9 strains recently isolated showed that their nucleotide sequences were closely related to those of the US strain, US1205 (98.7%-99.0%) and Thai strain, 97CM108 (98.1%-99.0%). Interestingly, they were most closely related to the Japanese strain, 00-SG2509VP7, isolated in the same epidemic season, with percent nucleotide sequence identity of 99.4%-99.8%. The data imply that G9 strains isolated in this study and a G9 strain isolated in Japan in the year 2000 might have descended from the same ancestor.