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

Molecular Epidemiology of Norovirus (NoV) Infection in Mie Prefecture: The Kinetics of Norovirus Antigenemia in Pediatric Patients

Few studies have shown the presence of norovirus (NoV) RNA in blood circulation but there is no data on norovirus antigenemia. We examined both antigenemia and RNAemia from the sera of children with NoV infections and studied whether norovirus antigenemia is correlated with the levels of norovirus-specific antibodies and clinical severity of gastroenteritis. Both stool and serum samples were collected from 63 children admitted to Mie National Hospital with acute NoV gastroenteritis. Norovirus antigen and RNA were detected in sera by ELISA and real-time RT-PCR, respectively. NoV antigenemia was found in 54.8% (34/62) and RNAemia in 14.3% (9/63) of sera samples. Antigenemia was more common in the younger age group (0–2 years) than in the older age groups, and most patients were male. There was no correlation between stool viral load and norovirus antigen (NoV-Ag) levels (rs = −0.063; Cl −0.3150 to 0.1967; p = 0.6251). Higher levels of acute norovirus-specific IgG serum antibodies resulted in a lower antigenemia OD value (n = 61; r = −0.4258; CI −0.62 to −0.19; p = 0.0006). Norovirus antigenemia occurred more commonly in children under 2 years of age with NoV-associated acute gastroenteritis. The occurrence of antigenemia was not correlated with stool viral load or disease severity

Understanding Pediatric Norovirus Epidemiology: A Decade of Study among Ghanaian Children

Understanding the epidemiology of human norovirus infection in children within Ghana and the entire sub-Saharan African region, where future norovirus vaccines would have the greatest impact, is essential. We analyzed 1337 diarrheic stool samples collected from children <5 years from January 2008 to December 2017 and found 485 (36.2%) shedding the virus. GII.4 (54.1%), GII.3 (7.7%), GII.6 (5.3%), GII.17 (4.7%), and GII.5 (4.7%) were the most common norovirus genotypes. Although norovirus GII.4 remained the predominant capsid genotype throughout the study period, an increase in GII.6 and GII.3 capsid genotypes was observed in 2013 and 2014, respectively. The severity of clinical illness in children infected with GII.4 norovirus strains was similar to illness caused by non-GII.4 strains. Since the epidemiology of norovirus changes rapidly, establishment of systematic surveillance within sentinel sites across the country would enhance the monitoring of circulating norovirus strains and allow continuous understanding of norovirus infection in Ghana

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

Additional file 1: Table S1. of Detection of the first G6P[14] human rotavirus strain in an infant with diarrhoea in Ghana

List of primers used in this study. (DOCX 16 kb

Identification of Novel Ghanaian G8P[6] Human-Bovine Reassortant Rotavirus Strain by Next Generation Sequencing

<div><p>Group A rotaviruses (RVAs) are the most important etiological agent of acute gastroenteritis in children <5 years of age worldwide. The monovalent rotavirus vaccine Rotarix was introduced into the national Expanded Programme on Immunization (EPI) in Ghana in May 2012. However, there is a paucity of genetic and phylogenetic data on the complete genomes of human RVAs in circulation pre-vaccine introduction. The common bovine rotavirus VP7 genotype G8 has been sporadically detected in Ghanaian children, usually in combination with the VP4 genotype P[6]. To investigate the genomic constellations and phylogeny of RVA strains in circulation prior to vaccine introduction, the full genomes of two unusual G8P[6] strains, GH018-08 and GH019-08, detected during burden of disease surveillance, were characterized by Illumina MiSeq sequencing. The Ghanaian isolates, GH018-08 and GH019-08, exhibited the unusual, previously unreported genotype constellation G8-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H3. Phylogenetic analyses confirmed that 10 out of the 11 genes of GH018-08 and GH019-08 were identical/nearly identical, with significant variation detected only in their VP1 genes, and clearly established the occurrence of multiple independent interspecies transmission and reassortment events between co-circulating bovine/ovine/caprine rotaviruses and human DS-1-like RVA strains. These findings highlight the contribution of reassortment and interspecies transmission events to the high rotavirus diversity in this region of Africa, and justify the need for simultaneous monitoring of animal and human rotavirus strains.</p></div

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

Genotype constellation of GH018-08 and GH019-08 with selected reference RVA strains of known genomic constellation.

a<p>RVA strain names according to proposed nomenclature of new classification system. <b>Boldface</b> font indicates RVA strains sequenced in this study. GenBank accession numbers for reference strains are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100699#pone.0100699.s003" target="_blank">Table S2</a>.</p>b<p>DS-1-like genotypes indicated by <b>boldface</b> font;</p>†<p>Typical bovine-like RVA genotypes.</p>‡<p>AU-1-like genotype.</p