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.

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.

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.

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.

Rotavirus strain distribution in Ghana pre- and post- rotavirus vaccine introduction.

BACKGROUND: Ghana introduced the monovalent rotavirus vaccine (Rotarix) into its national paediatric vaccination programme in May2012. Vaccine introduction was initiated nationwide and achieved >85% coverage within a few months. Rotavirus strain distribution pre- and post-RV vaccine introduction is reported. METHODS: Stool samples were collected from diarrhoeic children <5 years of age hospitalized between 2009 and 2016 at sentinel sites across Ghana and analyzed for the presence of group A rotavirus by enzyme immunoassay. Rotavirus strains were characterized by RT-PCR and sequencing. RESULTS: A total of 1363 rotavirus EIA-positive samples were subjected to molecular characterization. These were made up of 823 (60.4%) and 540 (39.6%) samples from the pre- and post-vaccine periods respectively. Rotavirus VP7 genotypes G1, G2 and G3, and VP4 genotypes P[6] and P[8] constituted more than 65% of circulating G and P types in the pre-vaccine period. The common strains detected were G1P[8] (20%), G3P[6] (9.2%) and G2P[6] (4.9%). During the post-vaccine period, G12, G1 and G10 genotypes, constituted more than 65% of the VP7 genotypes whilst P[6] and P[8] made up more than 75% of the VP4 genotypes. The predominant circulating strains were G12P[8] (26%), G10P[6] (10%) G3P[6] (8.1%) and G1P[8] (8.0%). We also observed the emergence of the unusual rotavirus strain G9P[4] during this period. CONCLUSION: Rotavirus G1P[8], the major strain in circulation during the pre-vaccination era, was replaced by G12P[8] as the most predominant strain after vaccine introduction. This strain replacement could be temporary and unrelated to vaccine introduction since an increase in G12 was observed in countries yet to introduce the rotavirus vaccine in West Africa. A continuous surveillance programme in the post-vaccine era is necessary for the monitoring of circulating rotavirus strains and the detection of unusual/emerging genotypes.

Whole Genomic Analysis of Human G12P[6] and G12P[8] Rotavirus Strains that Have Emerged in Myanmar.

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.

Whole genomic analysis of porcine G10P[5] rotavirus strain P343 provides evidence for bovine-to-porcine interspecies transmission.

Porcine group A rotavirus (RVA) strain P343 (RVA/Pig-tc/THA/P343/1991/G10P[5]) was suggested to have VP7 and VP4 genes of bovine origin. In order to obtain precise information on the exact origin and evolution of this unusual porcine strain, the remaining nine genes (VP6, VP1-3, and NSP1-5) of strain P343 were sequenced and analyzed in the present study. On whole genomic analysis, strain P343 was found to have a bovine RVA-like genotype constellation (G10-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3) different from those of typical porcine RVA strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strain P343 appeared to be of bovine origin. Therefore, strain P343 was suggested to be a bovine RVA strain that was transmitted to pigs.