Next-generation sequencing of a human-animal reassortant G6P[14] rotavirus A strain from a child hospitalized with diarrhoea.

We previously reported the VP4 and the VP7 genotypes of the first G6P[14] rotavirus strain (RVA/Human-wt/GHA/M0084/2010/G6P[14]) from the stool of an infant with diarrhoea in Ghana. In the current study, we obtained the complete genome sequences using Illumina MiSeq next-generation sequencing to enable us to determine the host species origin of the genes by phylogenetic analysis. The genotype constellation was G6-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3. Phylogenetic analysis showed that M0084 was a reassortant strain from RVAs of both artiodactyl and human host species origin. The level of sequence identity of the individual genes of M0084 to other sequences in the GenBank ranged from 95.2 to 99.5%; however, there was no single strain from the GenBank database with a complete genome sequence that was highly similar to that of M0084. To help trace the source of such unique gene pools being introduced into human RVAs, it will be useful to examine RVA sequences from potential reservoirs such as sheep and goats, which are common domestic animals in this locality.

Whole Genome Sequences of the Wildtype AU-1 Rotavirus A Strain: The Prototype of the AU-1-like Genotype Constellation.

Most human rotaviruses belong to the Wa-like, DS-1-like, or AU-1-like genotype constellation. The AU-1-like constellation, albeit minor, captured attention because its prototype strain AU-1 originated from feline rotavirus, leading to the concept of interspecies transmission of rotavirus. The AU-1 genome sequence determined by various laboratories over the years has documented two conflicting VP7 sequences in the GenBank. As culture-adaptation may introduce changes in the viral genome, the original fecal (wild-type) and the seed stock of culture-adapted AU-1 genomes were sequenced using the Illumina's MiSeq platform to determine the authentic AU-1 sequence and to identify what mutational changes were selected during cell-culture adaptation. The wild-type and culture-adapted AU-1 genomes were identical except for one VP4-P475L substitution. Their VP7 gene was 99.9% identical to the previously reported AU-1 VP7 under accession number AB792641 but only 92.5% to that under accession number D86271. Thus, the wild-type sequences determined in this study (accession numbers OR727616-OR727626) should be used as the reference. The VP4-P475L mutation was more likely incidental than inevitable during cell-culture adaptation. This was the first study in which the whole genomes of both wild-type and cultured RVA strains were simultaneously determined by deep sequencing.

Emergence of Intergenogroup Reassortant G9P[4] Strains Following Rotavirus Vaccine Introduction in Ghana.

Rotavirus (RVA) is a leading cause of childhood gastroenteritis. RVA vaccines have reduced the global disease burden; however, the emergence of intergenogroup reassortant strains is a growing concern. During surveillance in Ghana, we observed the emergence of G9P[4] RVA strains in the fourth year after RVA vaccine introduction. To investigate whether Ghanaian G9P[4] strains also exhibited the DS-1-like backbone, as seen in reassortant G1/G3/G8/G9 strains found in other countries in recent years, this study determined the whole genome sequences of fifteen G9P[4] and two G2P[4] RVA strains detected during 2015-2016. The results reveal that the Ghanaian G9P[4] strains exhibited a double-reassortant genotype, with G9-VP7 and E6-NSP4 genes on a DS-1-like backbone (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). Although they shared a common ancestor with G9P[4] DS-1-like strains from other countries, further intra-reassortment events were observed among the original G9P[4] and co-circulating strains in Ghana. In the post-vaccine era, there were significant changes in the distribution of RVA genotype constellations, with unique strains emerging, indicating an impact beyond natural cyclical fluctuations. However, reassortant strains may exhibit instability and have a limited duration of appearance. Current vaccines have shown efficacy against DS-1-like strains; however, ongoing surveillance in fully vaccinated children is crucial for addressing concerns about long-term effectiveness.

Sub-genotype phylogeny of the non-G, non-P genes of genotype 2 Rotavirus A strains.

Recent increase in the detection of unusual G1P[8], G3P[8], G8P[8], and G9P[4] Rotavirus A (RVA) strains bearing the DS-1-like constellation of the non-G, non-P genes (hereafter referred to as the genotype 2 backbone) requires better understanding of their evolutionary relationship. However, within a genotype, there is lack of a consensus lineage designation framework and a set of common sequences that can serve as references. Phylogenetic analyses were carried out on over 8,500 RVA genotype 2 genes systematically retrieved from the rotavirus database within the NCBI Virus Variation Resource. In line with previous designations, using pairwise comparison of cogent nucleotide sequences and stringent bootstrap support, reference lineages were defined. This study proposes a lineage framework and provides a dataset ranging from 34 to 145 sequences for each genotype 2 gene for orderly lineage designation of global genotype 2 genes of RVAs detected in human and animals. The framework identified five to 31 lineages depending on the gene. The least number of lineages (five to seven) were observed in genotypes A2 (NSP1), T2 (NSP3) and H2 (NSP5) which are limited to human RVA whereas the most number of lineages (31) was observed in genotype E2 (NSP4). Sharing of the same lineage constellations of the genotype 2 backbone genes between recently-emerging, unusual G1P[8], G3P[8], G8P[8] and G9P[4] reassortants and many contemporary G2P[4] strains provided strong support to the hypothesis that unusual genotype 2 strains originated primarily from reassortment events in the recent past involving contemporary G2P[4] strains as one parent and ordinary genotype 1 strains or animal RVA strains as the other. The lineage framework with selected reference sequences will help researchers to identify the lineage to which a given genotype 2 strain belongs, and trace the evolutionary history of common and unusual genotype 2 strains in circulation.

Genetic analysis of Ghanaian G1P[8] and G9P[8] rotavirus A strains reveals the impact of P[8] VP4 gene polymorphism on P-genotyping.

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[8] 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[8] specificity, of which 43 (89.6%) were characterized as P[8]a 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 P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006-2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] 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 P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a 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.

Whole genome characterization and evolutionary analysis of OP354-like P[8] Rotavirus A strains isolated from Ghanaian children with diarrhoea.

In 2010, the rare OP354-like P[8]b 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 P[8]b strains RVA/Human-wt/GHA/GHDC949/2010/G9P[8] and RVA/Human-wt/GHA/GHM0094/2010/G9P[8] 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 G9P[8]b-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 P[8]b cluster I. The VP7 and VP4 genes of the study strains were closely related to a Senegalese G9P[8]b strain detected in 2009. In the remaining nine genome segments, both strains consistently clustered together with Wa-like RVA strains possessing either P[8]a or P[8]b mostly of African RVA origin. The introduction of a P[8]b 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 P[8]b rotavirus strains.

Epidemiological Survey of Rotaviruses Responsible for Infantile Diarrhea by the Immunomolecular Technique in Cotonou (Benin, West Africa).

Rotavirus remains the main causative agent of gastroenteritis in young children, in countries that have not yet introduced the vaccine. Benin, in order to implement the WHO recommendations, projects to introduce the rotavirus vaccine in 2018 as part of its Expanded Program on Immunization. But before the introduction of this vaccine, epidemiological data on rotavirus infections and rotavirus genotypes circulating in Benin should be available. The aim of this study is to generate epidemiological data on infantile rotavirus diarrhea in Benin. In order to determine the epidemiological characteristics and electrophoretypes of rotavirus responsible for gastroenteritis in diarrheic children aged 0 to 5 years, 186 stool samples were collected according to the WHO Rotavirus Laboratory Manual from March 2014 to February 2015 at Suru-Lere University Hospital Center. Detection of rotavirus antigen was performed by the ELISA test, followed by molecular characterization using polyacrylamide gel electrophoresis. 186 stool samples were analyzed for rotavirus, and seventy-three (39.2%) were found to be positive for rotavirus antigen by ELISA. Children aged 3 to 24 months were the most affected by rotavirus diarrhea in this study. Of the seventy-three children affected with rotavirus diarrhea, 27 (37%) had vomiting accompanied by dehydration and fever. Results based on electrophoresis showed that, among the 73 samples tested, 38 yielded typical rotavirus electrophoretic migration profiles.

Expression and molecular characterization of spherical particles derived from the genome of the hyperthermophilic euryarchaeote Pyrococcus furiosus.

Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.

Molecular epidemiology of group A human rotaviruses in North West region, Cameroon.

BACKGROUND: Rotavirus (RV) is the most common cause of severe diarrhea in children <5 years of age worldwide accounting for 527,000 deaths annually. Over 80% of these deaths occur in South Asia and sub-Saharan Africa. RV vaccines have significantly reduced RV-associated morbidity and mortalities in several countries like the United States and Mexico while vaccine trials have proved efficacious in Ghana and other developing countries. However, there is paucity of data on RV infection in Cameroon where diarrhea is a major childhood disease. METHODS: A total of 534 stool specimens collected between January 2003 and December 2004 from children with acute gastroenteritis in five health districts in the NWR of Cameroon were screened for group A human rotavirus antigen by ELISA and their electropherotypes determined by Polyacrylamide gel electrophoresis. RESULTS: RV was detected in 153 (28.7%) diarrheic specimens with infection occurring throughout the year, being more common in children under two years of age (P < 0.01) with the highest incidence in the 7-9 months age group (P <0.05). Sub clinical infections (9%) occurred mostly in children aged 0 - 6 months old (P<0.01). Source of drinking water was not associated with RV infection. Eleven electropherotype patterns were detected with predominance of long electropherotypes (92.8%) and mixed electropherotypes were seen only in hospitalized children. Some isolates showed overlapping or merged genome segments 7 and 8 or 9 and presenting with 10 segments of the RV genome. CONCLUSION: RV is a significant cause of pediatric diarrhea in the NWR affecting mostly children under 2 years of age. Continuous RV surveillance and nationwide surveys are recommended to improve the health of young children in Cameroon. More research is needed to fully characterize the isolated RV strains.