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Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

ABSTRACT: Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.

SUBMITTER: Jere KC

PROVIDER: S-EPMC4181975 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

Jere Khuzwayo C KC O'Neill Hester G HG Potgieter A Christiaan AC van Dijk Alberdina A AA

PloS one 20140930 9

Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the c ...[more]

PMID: 25268783

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Project description:Group A rotaviruses (RVA) are among the main global causes of severe diarrhea in children under the age of 5years. Strain diversity, mixed infections and untypeable RVA strains are frequently reported in Africa. We analysed rotavirus-positive human stool samples (n=13) obtained from hospitalised children under the age of 5years who presented with acute gastroenteritis at sentinel hospital sites in six African countries, as well as bovine and porcine stool samples (n=1 each), to gain insights into rotavirus diversity and evolution. Polyacrylamide gel electrophoresis (PAGE) analysis and genotyping with G-(VP7) and P-specific (VP4) typing primers suggested that 13 of the 15 samples contained more than 11 segments and/or mixed G/P genotypes. Full-length amplicons for each segment were generated using RVA-specific primers and sequenced using the Ion Torrent and/or Illumina MiSeq next-generation sequencing platforms. Sequencing detected at least one segment in each sample for which duplicate sequences, often having distinct genotypes, existed. This supported and extended the PAGE and RT-PCR genotyping findings that suggested these samples were collected from individuals that had mixed rotavirus infections. The study reports the first porcine (MRC-DPRU1567) and bovine (MRC-DPRU3010) mixed infections. We also report a unique genome segment 9 (VP7), whose G9 genotype belongs to lineage VI and clusters with porcine reference strains. Previously, African G9 strains have all been in lineage III. Furthermore, additional RVA segments isolated from humans have a clear evolutionary relationship with porcine, bovine and ovine rotavirus sequences, indicating relatively recent interspecies transmission and reassortment. Thus, multiple RVA strains from sub-Saharan Africa are infecting mammalian hosts with unpredictable variations in their gene segment combinations. Whole-genome sequence analyses of mixed RVA strains underscore the considerable diversity of rotavirus sequences and genome segment combinations that result from a complex evolutionary history involving multiple host species.

Project description:BackgroundRotavirus is a leading cause of morbidity and mortality in young children worldwide. In China, the universal immunization of children with the rotavirus vaccine has not been introduced, and the two globally distributed vaccines (RotaTeq and Rotarix) are not licensed in the country. We aim to determine the prevalence and strain diversity of rotavirus in children with diarrhea aged ≤ five years across China.Materials and methodsSentinel-based surveillance of acute diarrhea was conducted at 213 participating hospitals in China from January 1, 2009, through December 31, 2015. Group A rotavirus (RVA) was tested by using enzyme-linked immunosorbent assays, and G- and P-genotype of RVA were tested by RT-PCR methods.ResultsOf 33,616 children with diarrhea, 10,089 (30%) were positive for RVA; RVA-associated diarrhea was identified in 2247 (39.5%, n = 2247/5685) inpatients and 7842 (28.1%, n = 7842/27931) outpatients. Children living in low-middle-income regions suffered from the highest burden of rotavirus, with 40.7% of diarrhea cases attributed to rotavirus infection, followed by 31.3% in upper-middle-income and 11.2% in high-income regions. The majority of children (88.9%, n = 8976/10089) who tested positive for RVA were children aged ≤ 2 years. The seasonal peak of RVA was in the winter. Among all 2533 RVA strains genotyped, five strain combinations, G9P[8], G3P[8], G1P[8], G2P[4] and G3P[4], contributed to 71.3% (1807/2533) of the RVA-associated diarrhea cases. The predominant strain of RVA has rapidly evolved from G3P[8] and G1P[8] to G9P[8] in the recent years, with the proportion of G9P[8] having increased remarkably from 3.4% in 2009 to 60.9% in 2015.ConclusionsThe burden of diarrhea attributed to rotavirus is high in China, highlighting the potential value of vaccination. The rapid shift of RVA strains highlights the importance of conducting rotavirus surveillance to ensure that currently marketed vaccines provide protective efficacy against the circulating strains.

Project description:BACKGROUND: Tanzania currently rolls out vaccination against rotavirus-diarrhea, a major cause of child illness and death. As the vaccine covers a limited number of rotavirus variants, this study describes the molecular epidemiology of rotavirus among children under two years in Dar es Salaam, Tanzania, prior to implementation of vaccination. METHODS: Stool specimens, demographic and clinical information, were collected from 690 children admitted to hospital due to diarrhea (cases) and 545 children without diarrhea (controls) during one year. Controls were inpatient or children attending child health clinics. Rotavirus antigen was detected using ELISA and positive samples were typed by multiplex semi-nested PCR and sequencing. RESULTS: The prevalence of rotavirus was higher in cases (32.5%) than in controls (7.7%, P<0.001). The most common G genotypes were G1 followed by G8, G12, and G4 in cases and G1, G12 and G8 in controls. The Tanzanian G1 variants displayed 94% similarity with the Rotarix vaccine G1 variant. The commonest P genotypes were P[8], P[4] and P[6], and the commonest G/P combination G1 P[8] (n?=?123), G8 P[4] and G12 P[6]. Overall, rotavirus prevalence was higher in cool (23.9%) than hot months (17.1%) of the year (P?=?0.012). We also observed significant seasonal variation of G genotypes. Rotavirus was most frequently found in the age group of four to six months. The prevalence of rotavirus in cases was lower in stunted children (28.9%) than in non-stunted children (40.1%, P?=?0.003) and lower in HIV-infected (15.4%, 4/26) than in HIV-uninfected children (55.3%, 42/76, P<0.001). CONCLUSION: This pre-vaccination study shows predominance of genotype G1 in Tanzania, which is phylogenetically distantly related to the vaccine strains. We confirm the emergence of genotype G8 and G12. Rotavirus infection and circulating genotypes showed seasonal variation. This study also suggests that rotavirus may not be an opportunistic pathogen in children infected with HIV.

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Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

Publications

Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

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