Changing patterns of rotavirus genotypes in ghana: emergence of human rotavirus G9 as a major cause of diarrhea in children.
ABSTRACT: Genotyping of human rotaviruses was performed on 312 rotavirus-positive samples collected from 2,205 young children with diarrhea in the Upper East District of Ghana, a rural community. Of the 271 (86.9%) rotavirus strains that could be VP7 (G) or VP4 (P) characterized, 73 (26.9%) were of G9 specificity. The predominant G9 genotype was G9P, which constituted 79.5% of all G9 strains detected, followed by G9P (12.3%), G9P (2.7%), and G9P (1.3%). G9 strains with mixed P types constituted 2.7% of all G9 strains found in the study. All the G9P strains had a long RNA electrophoretic pattern with VP6 subgroup II specificity. Four G9 isolates, GH1319, GH1416, GH3550, and GH3574, which were selected based on the abundance of stool material and were representative of the three electropherotypes observed, were cloned and sequenced. The Ghanaian isolates shared more than 98% sequence nucleotide homology with other G9 strains from the United States (US1205), Malawi (MW69), Brazil (R160), Japan (95H115), and Nigeria (Bulumkutu). However, they showed only 95% nucleotide homology with the Thai G9 strain Mc345. Phylogenetic analysis of the nucleic acid sequence revealed the existence of at least three clusters, with Ghanaian strains forming one cluster, Nigerian and Brazilian strains forming a second cluster, and U.S., Malawian, and Japanese strains forming a third.
Project description:Molecular epidemiologic studies collecting information of the spatiotemporal distribution of rotavirus VP7 (G) and VP4 (P) genotypes have shown evidence for the increasing global importance of genotype G9 rotaviruses in humans and pigs. Sequence comparison of the VP7 gene of G9 strains identified different lineages to prevail in the respective host species although some of these lineages appear to be shared among heterologous hosts providing evidence of interspecies transmission events. The majority of these events indicates the pig-to-human spillover, although a reverse route of transmission cannot be excluded either. In this study, new variants of G9 rotaviruses were identified in two children with diarrhea and numerous pigs in Taiwan. Whole genome sequence and phylogenetic analyses of selected strains showed close genetic relationship among porcine and human strains suggesting zoonotic origin of Taiwanese human G9 strains detected in 2014-2015. Although the identified human G9P and G9P rotaviruses represented minority strains, the repeated detection of porcine-like rotavirus strains in Taiwanese children over time justifies the continuation of synchronized strain surveillance in humans and domestic animals.
Project description:In 2010, the rare OP354-like Pb rotavirus subtype was detected in children less than 2 years old in Ghana. In this follow-up study, to provide insight into the evolutionary history of the genome of Ghanaian Pb strains RVA/Human-wt/GHA/GHDC949/2010/G9P and RVA/Human-wt/GHA/GHM0094/2010/G9P detected in an infant and a 7-month old child hospitalised for acute gastroenteritis, we sequenced the complete genome using both Sanger sequencing and Illumina MiSeq technology followed by phylogenetic analysis of the near-full length sequences. Both strains possessed the Wa-like/genotype 1 constellation G9Pb-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence comparison and phylogenetic inference showed that both strains were identical at the lineage level throughout the 11 genome segments. Their VP7 sequences belonged to the major sub-lineage of the G9-lineage III whereas their VP4 sequences belonged to Pb cluster I. The VP7 and VP4 genes of the study strains were closely related to a Senegalese G9Pb strain detected in 2009. In the remaining nine genome segments, both strains consistently clustered together with Wa-like RVA strains possessing either Pa or Pb mostly of African RVA origin. The introduction of a Pb subtype VP4 gene into the stable Wa-like strain backbone may result in strains that might propagate easily in the human population, with a potential to become an important public health concern, especially because it is not certain if the monovalent rotavirus vaccine (Rotarix) used in Ghana will be efficacious against such strains. Our analysis of the full genomes of GHM0094 and GHDC949 adds to knowledge of the genetic make-up and evolutionary dynamics of Pb rotavirus strains.
Project description:The changing epidemiology of group A rotavirus (RV) strains in humans and swine, including emerging G9 strains, poses new challenges to current vaccines. In this study, we comparatively assessed the pathogenesis of porcine RV (PRV) G9P and evaluated the short-term cross-protection between this strain and human RV (HRV) Wa G1P in gnotobiotic pigs. Complete genome sequencing demonstrated that PRV G9P possessed a human-like G9 VP7 genotype but shared higher overall nucleotide identity with historic PRV strains. PRV G9P induced longer rectal virus shedding and RV RNAemia in pigs than HRV Wa G1P and generated complete short-term cross-protection in pigs challenged with HRV or PRV, whereas HRV Wa G1P induced only partial protection against PRV challenge. Moreover, PRV G9P replicated more extensively in porcine monocyte-derived dendritic cells (MoDCs) than did HRV Wa G1P. Cross-protection was likely not dependent on serum virus-neutralizing (VN) antibodies, as the heterologous VN antibody titers in the sera of G9P-inoculated pigs were low. Thus, our results suggest that heterologous protection by the current monovalent G1P HRV vaccine against emerging G9 strains should be evaluated in clinical and experimental studies to prevent further dissemination of G9 strains. Differences in the pathogenesis of these two strains may be partially attributable to their variable abilities to replicate and persist in porcine immune cells, including dendritic cells (DCs). Additional studies are needed to evaluate the emerging G9 strains as potential vaccine candidates and to test the susceptibility of various immune cells to infection by G9 and other common HRV/PRV genotypes.The changing epidemiology of porcine and human group A rotaviruses (RVs), including emerging G9 strains, may compromise the efficacy of current vaccines. An understanding of the pathogenesis and genetic, immunological, and biological features of the new emerging RV strains will contribute to the development of new surveillance and prevention tools. Additionally, studies of cross-protection between the newly identified emerging G9 porcine RV strains and a human G1 RV vaccine strain in a susceptible host (swine) will allow evaluation of G9 strains as potential novel vaccine candidates to be included in porcine or human vaccines.
Project description:Despite the development of vaccines in 2006, rotavirus is still a major cause of acute gastroenteritis worldwide. This study was performed to analyze the presence of circulating rotaviruses before and after the introduction of rotavirus vaccines to allow phylogenetic comparisons of vaccine strains in northern Taiwan.Rotavirus genotyping and sequencing of rotavirus VP7 and VP4 PCR products were performed by Reverse Transcriptase Polymerase Chain Reaction and DNA autosequencing. Phylogenies were constructed by the neighbor-joining and maximum-likelihood methods using CLUSTAL W software included in the MEGA software package (version 6.0).Between April 2004 and December 2012, a total of 101 rotavirus specimens from pediatric patients with acute gastroenteritis hospitalized in Chang Gung Children's Hospital were amplified, and their VP4 and VP7 sequences were determined. These 101 specimens consisted of 55 pre-vaccine strains (G1 [13, 23.6%], G2 [12, 21.8%], G3 [16, 29.1%], and G9 [14, 25.5%]) and 46 post-vaccine strains (G1 [25, 54.3%], G2 [12, 26.1%], G3 [5, 10.9%], and G9 [4, 8.7%]). The most common combination of the G and P types was G2P, accounting for 36% cases, followed by G9P (25%), G1P (20%), G3P (15%), G3P (10%), G1P (5%), and G2P (5%). Phylogenetic analysis showed that only the G1 and P genotypes clustered in the same lineages with the rotavirus vaccine strains.Based on our results, the inclusion of G9, modified G2 and G3 with target lineages, and the combination G2P and G9P in the rotavirus vaccines in Taiwan is warranted as a vaccination strategy.
Project description:The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394-4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P, G2P, G3P, and G4P, were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P and G2P. Rotavirus G9P and G9P strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.
Project description:BACKGROUND: We report the first multi-site rotavirus genotype analysis in Canada. Prior to this study, there was a dearth of rotavirus G and P genotyping data in Canada. Publically funded universal rotavirus vaccination in Canada started in 2011 and has been introduced by four provinces to date. Uptake of rotavirus vaccines in Canada prior to 2012 has been very limited. The aim of this study was to describe the genotypes of rotavirus strains circulating in Canada prior to widespread implementation of rotavirus vaccine by genotyping samples collected from selected paediatric hospitals. Secondly we identified rotavirus strains that differed genetically from those included in the vaccines and which could affect vaccine effectiveness. METHODS: Stool specimens were collected by opportunity sampling of children with gastroenteritis who presented to emergency departments. Samples were genotyped for G (VP7) genotypes and P (VP4) genotypes by hemi-nested multiplex PCR methods. Phylogenetic analysis was carried out on Canadian G9 strains to investigate their relationship to G9 strains that have circulated in other regions of the world. RESULTS: 348 samples were collected, of which 259 samples were rotavirus positive and genotyped. There were 34 rotavirus antigen immunoassay negative samples genotyped using PCR-based methods. Over the four rotavirus seasons, 174 samples were G1P, 45 were G3P, 22 were G2P, 13 were G9P, 3 were G4P and 2 were G9P. Sequence analysis showed that all Canadian G9 isolates are within lineage III. CONCLUSIONS: Although a limited number of samples were obtained from a median of 4 centres during the 4 years of the study, it appears that currently approved rotavirus vaccines are well matched to the rotavirus genotypes identified at these hospitals. Further surveillance to monitor the emergence of rotavirus genotypes in Canada is warranted.
Project description:Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and genotype G9P is one of the five most common genotypes detected in humans. In order to gain insight into the degree of genetic variability of G9P strains circulating in Cameroon, stool samples were collected during the 1999-2000 rotavirus season in two different geographic regions in Cameroon (Southwest and Western Regions). By RT-PCR, 15 G9P strains (15/89=16.8%) were identified whose genomic configurations was subsequently determined by complete or partial gene sequencing. In general, all Cameroonian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6-100% nucleotide identity amongst themselves and 81.2-99.5% nucleotide identity with global G9 strains. The full genome classification of all Cameroonian strains was G9-P-I1-R1-C1-M1-A1-N1-T1-E1-H1 but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinct lineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P, which shared the genomic constellation of other Cameroonian G9P strains, contained a novel G9 subtype which diverged significantly (18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide and amino acid alignments revealed that the 3' end of this gene is highly divergent from other G9 VP7 genes suggesting that it arose through extensive accumulation of point mutations. The results of this study demonstrate that diverse G9 strains circulated in Cameroon during 1999-2000.
Project description:Of 322 stool specimens collected from children with diarrhea from October 2005 through September 2006 in Wuhan, China, group A rotavirus was identified in 101 (31.4%). The most prevalent group A rotavirus genotype was G3P (62.6%), followed by G1P(17.6%), G1+G3P(8.8%), G3P(6.6%), G1P(2.2%), and G9P(2.2%). The G9 strains were first detected in Wuhan.
Project description:BACKGROUND:Rotavirus is a leading cause of severe diarrheal disease, and one of the common causes of death in children aged under five years old. The dominant epidemic strains may change in different years in the same area. In order to provide evidence for rotavirus epidemic control and inform vaccine development, we analyzed epidemiological patterns and genetic characteristics of rotavirus in Beijing during 2011-2016. METHODS:Stool specimens of outpatient children under five years old were collected from three children's hospitals on a weekly basis. Group A rotavirus antigens were detected using enzyme-linked immunosorbent assay (ELISA) kit. The partial VP4 genes and VP7 genes of rotavirus were both amplified and sequenced. Genotyping and phylogenetic analyses were performed. Logistic regression and Chi-square tests were performed to determine differences across age groups, districts and years in rotavirus prevalence and genotype distribution. RESULTS:A total of 3668 stool specimens from children with acute diarrhea identified through hospital-based surveillance were collected from 2011 to 2016 in Beijing. A total of 762 (20.8%) specimens tested positive for rotavirus. The rotavirus-positive rate was highest among the 1-2 years old age group (29.0%, 310/1070). November, December and January were the highest rotavirus-positive rate months each year. G9 was the most common G genotype (64.4%, 461/716), and P  was the most common P genotype (87.0%, 623/716) among the 716 rotavirus-positive specimens. G9P , G3P  and G2P  were the most common strains. The rotavirus-positive rates of samples in 2012 and 2013 were higher than that in 2011, and the dominant genotype changed from G3P  to G9P  in 2012 and 2013. VP7 gene sequences of G9 strains in this study clustered into two main lineages. Most of the G9 strains exhibited the highest nucleotide similarity (99.1%~?100.0%) to the strain found in Japan (MI1128). VP4 gene sequences of P  strains were almost Pb. CONCLUSIONS:Rotavirus accounted for more than one fifth of childhood diarrhea in Beijing during the study period. Targeted measures such as immunization with effective rotavirus vaccines should be carried out to reduce the morbidity and mortality due to rotavirus.
Project description:The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P specificity, of which 43 (89.6%) were characterized as Pa subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the Pa sequences placed them in subcluster IIIc within lineage III together with contemporary G1P, G3P, G8P, and G9P strains detected globally from 2006-2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian Pa strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P strains. In summary, the G1, G9, and Pa sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers.