Genetic characterization of a novel, naturally occurring recombinant human G6P rotavirus.
ABSTRACT: 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, G2P, G3P, and G4P. We report a human rotavirus strain (B1711) with a novel genotypic VP7/VP4 combination of G6P. 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 genotypes, with greatest similarity to a G8P Malawi strain (mw131) (97% nucleotide and 98% amino acid identity). The B1711 strain is the first reported rotavirus isolate with a G6P 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.
Project description:Rotaviruses with G6P specificity are mostly isolated in cattle and have been established as a rare cause of gastroenteritis in humans. This study reports the first detection of G6P rotavirus strain in Ghana from the stool of an infant during a hospital-based rotavirus surveillance study in 2010.Viral RNA was extracted and rotavirus VP7 and VP4 genes amplified by one step RT-PCR using gene-specific primers. The DNA was purified, sequenced and genotypes determined using BLAST and RotaC v2.0. Phylogenetic tree was constructed using maximum likelihood method in MEGA v6.06 software and statistically supported by bootstrapping with 1000 replicates. Phylogenetic distances were calculated using the Kimura-2 parameter model.The study strain, GHA-M0084/2010 was characterised as G6P. The VP7 gene of the Ghanaian strain clustered in G6 lineage-III together with artiodactyl and human rotavirus (HRV) strains. It exhibited the highest nucleotide (88.1 %) and amino acid (86.9 %) sequence identity with Belgian HRV strain, B10925. The VP8* fragment of the VP4 gene was closely related to HRV strains detected in France, Italy, Spain and Belgium. It exhibited the strongest nucleotide sequence identity (87.9 %) with HRV strains, PA169 and PR/1300 (Italy) and the strongest amino acid sequence identity (89.3 %) with HRV strain R2775/FRA/07 (France).The study reports the first detection of G6P HRV strain in an infant in Ghana. The detection of G6P, an unusual strain pre-vaccine introduction in Ghana, suggests a potential compromise of vaccine effectiveness and indicates the necessity for continuous surveillance in the post vaccine era.
Project description:An unusual rotavirus strain, SKT-27, with the G6P genotypes (RVA/Human-wt/THA/SKT-27/2012/G6P), 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-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 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 strains that have emerged in Southeast Asia. Our observations will provide important insights into the origin of G6P strains, and into dynamic interactions between human and bovine rotavirus strains.
Project description:We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.
Project description:The human, G1P rotavirus vaccine (Rotarix™) significantly reduced severe rotavirus gastroenteritis episodes in a clinical trial in South Africa and Malawi, but vaccine efficacy was lower in Malawi (49.5%) than reported in South Africa (76.9%) and elsewhere. The aim of this study was to examine the molecular relationships of circulating wild-type rotaviruses detected during the clinical trial in Malawi to RIX4414 (the strain contained in Rotarix™) and to common human rotavirus strains. Of 88 rotavirus-positive, diarrhoeal stool specimens, 43 rotaviruses exhibited identifiable RNA migration patterns when examined by polyacrylamide gel electrophoresis. The genes encoding VP7, VP4, VP6 and NSP4 of 5 representative strains possessing genotypes G12P, G1P, G9P, and G8P were sequenced. While their VP7 (G) and VP4 (P) genotype designations were confirmed, the VP6 (I) and NSP4 (E) genotypes were either I1E1 or I2E2, indicating that they were of human rotavirus origin. RNA-RNA hybridization using 21 culture-adapted strains showed that Malawian rotaviruses had a genomic RNA constellation common to either the Wa-like or the DS-1 like human rotaviruses. Overall, the Malawi strains appear similar in their genetic make-up to rotaviruses described in countries where vaccine efficacy is greater, suggesting that the lower efficacy in Malawi is unlikely to be explained by the diversity of circulating strains.
Project description:The genome of rotaviruses consists of 11 segments of double-stranded RNA, and each genome segment has multiple genotypes. Thus, the genotype constellation of an isolate is often indicative of its host species. Albeit rarely, interspecies transmission occurs either by virions with nonreassorted or reassorted genomic segments. A rotavirus with the G6P genotype, Ro8059, was isolated from the stool of a 1-year-old child during routine characterization of diarrheal specimens from a sentinel clinic in Israel in 1995. Since genotype G6P is generally associated with bovine rotaviruses, and the child developed diarrhea within days of his first contact with calves at an urban farm, the aim of this study was to characterize the whole genomic constellation of Ro8059 and four G6P bovine strains, BRV101, BRV105, BRV106, and CR231/39, by RNA-RNA hybridization and full genome sequencing to determine whether some or all of the segments were of bovine origin. The genome constellations of all four bovine G6P strains were G6-P-I2-R2-C2-M2-A3-N2-T6-E2-H3 for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Ro8059 shared the same genotype constellation with these bovine strains, with high nucleotide sequence identities (95.84 to 100%) for each of the 11 segments indicating that Ro8059 represented a direct interspecies whole-genome transmission of a nonreassorted rotavirus from a calf to a human infant. We conclude that this was the earliest example with a complete epidemiological link in which an entirely bovine rotavirus directly infected a human child and caused a symptomatic diarrheal illness. Thus, not all bovine rotaviruses are always naturally attenuated to the human host.
Project description:During an epidemiological study on rotaviruses among diarrheic children in the northeastern and middle belt regions of Nigeria, the distribution of G and P types was investigated in 127 stool specimens. By PCR G typing, the G type of rotaviruses in 97 samples was identified. Interestingly, an unusual G8 type, as well as common G1, G2, and G3 types, was detected more frequently (31 of 112; 27.7%). Eleven samples contained multiple G types, and a G9 strain (Bulumkutu) was identified for one of the probable mixed infections. In PCR P typing, P was detected most frequently, P being the second most common type, while the P type of 73 samples could not be identified. One rotavirus strain with a G8 type specificity could be cultivated in cell culture, and the P type of this strain was found to be P, which is usually carried by bovine strains. When the combinations of G and P types were examined, the unusual strains G2P and G8P were often identified. Sequence analysis was performed for the VP7 gene of the G9 strain Bulumkutu and the VP4 and VP7 genes of G8P strain HMG035. The VP7 sequence of the Nigerian serotype G9 was more closely related to that of a Brazilian strain than to those of other African strains. The VP7 and VP4 genes of G8P strain HMG035 were found to be very similar to that of a Thai bovine strain A5, suggesting that bovine strains may have been transmitted directly to humans. These results highlight an unexpected diversity among rotavirus strains in Nigeria and emphasize the need for further serological and genetic surveys on more rotavirus strains in African countries, including Nigeria.
Project description:Several G8P and G8P rotavirus strains were isolated from hospitalized patients in the Democratic Republic of Congo in 2003. To investigate their overall genomic relatedness and to determine to which genogroup they belonged, the complete genomes of strains DRC88 (G8P) and DRC86 (G8P) were determined. Genomic comparison of these two African G8 strains revealed that 10 out of their 11 gene segments, except for VP4, were nearly identical (>98.9% identical at the nucleotide level), suggesting that this rare G8P rotavirus strain originated recently from a reassortment between a common G8P strain and a strain with a P specificity. A very close evolutionary relationship between 9 out of the 11 gene segments of DRC88 and DRC86 and rotavirus strains belonging to the DS-1-like (G2P) "genogroup" was found, and several possible reassortment events preceding the occurrence of G8P and G8P human rotaviruses were hypothesized. Since the genes of G2P rotavirus strains are very well adapted to infect humans, the acquirement of a new VP7 (G8) gene, and especially the replacement of P (believed to be of animal origin) by P (most common in human rotaviruses), might make DRC88-like rotaviruses very well equipped to become a predominant human rotavirus strain and an important pathogen on the African continent and the rest of the world. These findings have important implications for rotavirus vaccine development and highlight that typing of new rotavirus strains by merely sequencing their VP7 and VP4 genes provides us with only the tip of the iceberg regarding rotavirus diversity.
Project description:In the present investigation we molecularly characterized nontypeable rotavirus strains previously identified during surveillance in New Delhi, India. The majority of strains were demonstrated to belong to genotype G1 (54.5%) or P (77.8%) on the basis of nucleotide sequencing of fragments from their VP7 and VP4 genes. The other genotypes detected included G2, G8, G9, G12, and P. A G8P strain, strain DS108, was detected for the first time in northern India. The VP7 gene of DS108 was most homologous with the VP7 gene of a bovine G8 strain, strain A5 (98.9%), indicating its bovine parentage. In contrast, the VP4 gene had a high degree of nucleotide sequence homology (92.9% to 99.1%) with the VP4 genes of human P strains. The VP6 gene and nonstructural genes (NSP1 to NSP3 and NSP5) were most homologous with the VP6 gene and nonstructural genes of human rotaviruses belonging to the DS1 genogroup. Interestingly, the NSP4 gene of DS108 clustered within genotype E6 that until now had only two representative strains, both with G12P specificity (strains RV176-00 and N26-02). Together, these results indicate that G8 strain DS108 belongs to the DS1 genogroup and could be the result of the acquisition of the VP7, VP4, and NSP4 genes by a human G2P strain from more than one donor, similar to the evolution of G12P strain RV176-00. The present study highlights the importance of characterizing multiple genes of nontypeable rotavirus strains to detect novel strains and get a more complete picture of rotavirus evolution.
Project description:In a 2-year study of viral gastroenteritis in children in Blantyre, Malawi, the diversity of rotavirus strains was investigated by using electropherotyping, reverse transcription-PCR amplification of the VP7 and VP4 genes (G and P genotyping), and nucleotide sequencing. Of 414 rotavirus strains characterized, the following strain types were identified: P, G1 (n = 111; 26.8%); P, G8 (n = 110; 26.6%); P, G3 (n = 93; 22.5%); P, G8 (n = 31; 7.5%); P, G4 (n = 21; 5.1%); P, G3 (n = 12; 2.9%); P, G1 (n = 7; 1.7%); P, G9 (n = 3; 0.7%); P, G4 (n = 3; 0.7%); P, G3 (n = 1; 0.2%); and mixed (n = 15; 3.6%). While all strains could be assigned a G type, seven strains (1.7%) remained P nontypeable. The majority of serotype G8 strains and all serotype G9 strains had short electropherotype profiles. All remaining typeable strains had long electropherotypes. Divergent serotype G1 rotaviruses, which contained multiple base substitutions in the 9T-1 primer binding site, were commonly identified in the second year of surveillance. Serotype G2 was not identified. Overall, G8 was the most frequently identified VP7 serotype (n = 144; 34.8%) and P was the most frequently detected VP4 genotype (n = 227; 54.8%). Partial sequence analysis of the VP4 gene of genotype P rotaviruses identified three distinct clusters, which predominantly (but not exclusively) comprised strains belonging to a distinct VP7 serotype (G1, G3, or G4). As a result of mutations in the 1T-1 primer binding site, strains belonging to each cluster required a separate primer for efficient typing. One cluster, represented by P, G4 strain OP354, was highly divergent from the established Wa and F45 VP4 P lineages. As is the case for some other countries, the diversity of rotaviruses in Malawi implies that rotavirus vaccines in development will need to protect against a wider panel of serotypes than originally envisioned.
Project description:Background:Rotaviruses are the major causes of pediatric gastroenteritis worldwide. The genotypic distribution of rotavirus strains shows temporal and geographical fluctuations, and knowledge of the molecular epidemiology of rotaviruses is important for the development of vaccines and diagnostic reagents. We investigated VP4 and VP7 capsid genotypes of rotaviruses isolated from 211 stool specimens collected from Korean neonates in a neonatal intensive care unit from September 2017 to March 2018. Results:Of 211 stool specimens, 15 specimens (7.1%) were rotavirus-positive. Eleven specimens (73.3%) were G8P type and 4 (26.7%) were G4P type. Sequence analysis revealed that all G8 sequences in this study showed the highest nucleotide identity to G8 sequences of G8P rotavirus strains isolated in Vietnam in 2014, and P gene sequences showed the highest nucleotide identity to P sequences of G4P strains detected in Korea in 2012. Only one amino acid difference in VP7 was found in 3 of the 11 G8P strains in this study, but multiple amino acid substitutions in VP7 were detected between these G8P strains and the commonly used vaccine strains. Conclusions:This study showed that rotavirus G8P strains were firstly detected at high frequency in Korean neonates from September 2017 to March 2018. These new rotavirus G8P strains were estimated to be derived from reassortment events between the G8 of G8P strains in Asian region and the P of G4 in Korea. Whether the emergence of this unusual G8P strain reflects continuous prevalence or transient occurrence will require continuous monitoring of rotavirus epidemiology.