Loan Phuong Do

Molecular evolution of the VP7 gene of Japanese G2 rotaviruses before vaccine introduction

Changes in the prevalence of G2 rotavirus after vaccine introduction are an important issue. However, such changes in a given country should be interpreted in the global context over time. We determined 35 Japanese G2 sequences and compared them with 508 globally collected G2 sequences. The D96N substitution, a substitution known to be associated with an abrupt increase in G2 strains and antigenic changes, emerged in those strains that formed a nascent lineage outside of the currently predominant lineage (sublineage IVa). Further studies are warranted to monitor the potential of their global spread, since they also appeared in Europe and Australia.

A rare G1P[6] super-short human rotavirus strain carrying an H2 genotype on the genetic background of a porcine rotavirus

Rotavirus strains with a rearranged 11th genome segment may show super-short RNA electropherotypes. Examples from human strains were limited to seven strains, 69M, 57M, B37, Mc345, AU19, B4106 and BE2001, which have a variety of G and P genotypes. AU19 is a rare G1P[6] human rotavirus strain detected in a Japanese infant with severe acute gastroenteritis. This study was undertaken to better understand the origin of AU19 by determining the genotype constellation of AU19. Upon nearly-full genome sequencing, AU19 had a G1-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H2 genotype constellation. Possession of I5 and A8 genotypes is indicative of its porcine rotavirus origin, whereas possession of H2 genotype is indicative of its DS-1 like human rotavirus origin. At the phylogenetic lineage level for the genome segments that share the genotype between porcine and human rotaviruses, the VP1-4, VP7, NSP3-4 genes were most closely related to those of porcine rotaviruses, but the origin of the NSP2 gene was inconclusive. As to the NSP5 gene, the lineage containing AU19 and the other three super-short human strains, 69M, 57M and B37, carrying the H2 genotype (H2b) clustered with the lineage to which DS-1- like short strains belonged (H2a) albeit with an insignificant bootstrap support. Taken all these observations together, AU19 was likely to emerge as a consequence of interspecies transmission of a porcine rotavirus to a child coupled with the acquisition of a rare H2b genotype by genetic reassortment probably from a co-circulating human strain. The addition of the AU19 NSP5 sequence to much homogeneous H2b genotypes shared by previous super-short rotavirus strains made the genetic diversity of H2b genotypes as diverse as that of the H2a genotype, lending support to the hypothesis that super-short strains carrying H2b genotype have long been circulating unnoticed in the human population.

Molecular epidemiology of Rotavirus A, causing acute gastroenteritis hospitalisations among children in Nha Trang, Vietnam, 2007–2008: Identification of rare G9P[19] and G10P[14] strains

Rotavirus A (RVA) causes acute diarrhea in children as well as animals. As part of a cross‐sectional study of children less than 5 years of age hospitalized for acute diarrhea in Vietnam during a 15‐month period (2007–2008), 322 (43.5%) of 741 fecal specimens contained RVA with 92% either G1P[8] or G3P[8]. This study was undertaken to further characterize strains that remained untypeable to complete the G and P genotypes of the 322 rotavirus‐positive specimens. While 307 (95.3%) strains possessed the common human RVA genotypes: G1P[8] (45.0%), G2P[4] (2.8%), G3P[8] (46.9%), and G9P[8] (0.6%), sequencing of initially untypeable specimens revealed the presence of two unusual strains designated NT0073 and NT0082 possessing G9P[19] and G10P[14], respectively. The genotype constellation of NT0073 (G9‐P[19]‐I5‐R1‐C1‐M1‐A8‐N1‐T7‐E1‐H1) and the phylogenetic trees suggested its origin as a porcine RVA strain causing diarrhea in a 24‐month‐old girl whereas the genotype constellation of NT0082 (G10‐P[14]‐I2‐R2‐C2‐M2‐A3‐N2‐T6‐E2‐H3) and the phylogenetic trees suggested its origin as an RVA strain of artiodactyl origin (such as cattle, sheep and goats) causing diarrhea in a 13‐month‐old boy. This study showed that RVA strains of animal host origin were not necessarily attenuated in humans. A hypothesis may be postulated that P[19] and P[14] VP4 spike proteins helped the virus to replicate in the human intestine but that efficient onward human‐to‐human spread after crossing the host species barrier may require the virus to obtain some additional features as there was no evidence of widespread transmission with the limited sampling performed over the study period. J. Med. Virol. 89:621–631, 2017.

Evolution of DS-1-like G1P[8] double-gene reassortant rotavirus A strains causing gastroenteritis in children in Vietnam in 2012/2013

Rotavirus A (RVA) strains, a leading cause of severe gastroenteritis in children worldwide, commonly possess the Wa or DS-1 genotype constellations. During a hospital-based study conducted in Hanoi, Vietnam, in the 2012-2013 rotavirus season, G1P[8] strains with a virtually identical short RNA migration pattern were detected in 20 (14%) of 141 rotavirus-positive samples. Two representatives of these strains were shown by whole-genome sequencing to be double-gene reassortants possessing the genotype constellation of G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Sequencing and a database search revealed that these Vietnamese G1P[8] double-gene reassortant strains shared an immediate ancestor with a locally circulating G2P[4] strain in all of the inner-capsid and non-structural protein genes, whereas they were more closely related in the VP7 and VP4 genes to a Chinese G1P[8] strain and a Chinese G3P[8] strain, respectively, than to locally circulating G1P[8] strains. Despite the marked similarity between Japanese and Thai G1P[8] double-gene reassortant strains, phylogenetic analysis suggested that the Vietnamese and Japanese/Thai G1P[8] double-gene reassortant strains originated from independent reassortment events. Clinically, children infected with Vietnamese G1P[8] double-gene reassortant strains experienced severe diarrhoea, but it was not more severe than that in children infected with ordinary G1P[8] strains. In conclusion, Vietnamese G1P[8] double-gene reassortant strains originated from a locally circulating G2P[4] strain and caused severe diarrhoea, but there was no evidence of increased virulence.

Whole genotype constellation of prototype feline rotavirus strains FRV-1 and FRV64 and their phylogenetic relationships with feline-like human rotavirus strains

Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.

Phylogenetic inference of the porcine Rotavirus A origin of the human G1 VP7 gene.

Rotavirus A (RVA) is an important cause of acute gastroenteritis in children worldwide. The most common VP7 genotype of human RVA is G1, but G1 is rarely detected in porcine strains. To understand the evolutionary relationships between human and porcine G1 VP7 genes, we sequenced the VP7 genes of three Japanese G1 porcine strains; the first two (PRV2, S80B) were isolated in 1980 and the third (Kyusyu-14) was isolated in 2001. Then, we performed phylogenetic and in-silico structural analyses. All three VP7 sequences clustered into lineage VI, and the mean nucleotide sequence identity between any pair of porcine G1 VP7 sequences belonging to lineage VI was 91.9%. In contrast, the mean nucleotide sequence identity between any pair of human G1 VP7 sequences belonging to lineages I-V was 95.5%. While the mean nucleotide sequence identity between any pair of porcine lineage VI strain and human lineage I-V strain was 85.4%, the VP7 genes of PRV2 and a rare porcine-like human G1P[6] strain (AU19) were 98% identical, strengthening the porcine RVA origin of AU19. The phylogenetic tree suggests that human G1 VP7 genes originated from porcine G1 VP7 genes. The time of their most recent common ancestor was estimated to be 1948, and human and porcine RVA strains evolved along independent pathways. In-silico structural analyses identified 7 amino acid residues within the known neutralisation epitopes that show differences in electric charges and shape between different porcine and human G1 strains. When compared with much divergent porcine G1 VP7 lineages, monophyletic, less divergent human G1 VP7 lineages support the hypothesis that all human G1 VP7 genes included in this study originated from a rare event of a porcine RVA transmitting to humans that was followed by successful adaptation to the human host. By contrast, AU19 represents interspecies transmission that terminated in dead-end infection.

Genomic constellation and evolution of Ghanaian G2P[4] rotavirus strains from a global perspective.

Understanding of the genetic diversity and evolution of Rotavirus A (RVA) strains, a common cause of severe diarrhoea in children, needs to be based on the analysis at the whole genome level in the vaccine era. This study sequenced the whole genomes of six representative G2P[4] strains detected in Ghana from 2008 to 2013, and analysed them phylogenetically with a global collection of G2P[4] strains and African non-G2P[4] DS-1-like strains. The genotype constellation of the study strains was G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Strains from the same season were highly identical across the whole genome while strains from different seasons were more divergent from each other. The VP7, VP4, VP2, NSP1, and NSP5 genes belonged to lineage IVa; the VP6, VP1, NSP2, and NSP3 genes belonged to lineage V, and all these genes evolved in the same fashion as the global strains. In the NSP4 gene, lineages V (2008) and X (2009) were replaced by VI (2012/2013) whereas in the VP3 gene, lineage V (2008/2009) was replaced by VII (2012/2013) and these replacements coincided with the vaccine introduction period (2012). The evolutionary rate of the NSP4 gene was 1.2×10-3 substitutions/site/year and was rather comparable to that of the remaining 10 genes. The multiple NSP4 lineages were explained by intra-genotype reassortment with co-circulating African human DS-1-like strains bearing G2[6], G3P[6], G6[6] and G8. There was no explicit evidence of the contribution of animal RVA strains to the genome of the Ghanaian G2P[4] strains. In summary, this study revealed the dynamic evolution of the G2P[4] strains through intra-genotype reassortment events leading to African specific lineages such IX and X in the NSP4 gene. So far, there was no evidence of a recent direct involvement of animal RVA genes in the genome diversity of African G2P[4] strains.

Whole genome analysis of Vietnamese G2P[4] rotavirus strains possessing the NSP2 gene sharing an ancestral sequence with Chinese sheep and goat rotavirus strains

Because imminent introduction into Vietnam of a vaccine against Rotavirus A is anticipated, baseline information on the whole genome of representative strains is needed to understand changes in circulating strains that may occur after vaccine introduction. In this study, the whole genomes of two G2P[4] strains detected in Nha Trang, Vietnam in 2008 were sequenced, this being the last period during which virtually no rotavirus vaccine was used in this country. The two strains were found to be >99.9% identical in sequence and had a typical DS‐1 like G2‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2 genotype constellation. Analysis of the Vietnamese strains with >184 G2P[4] strains retrieved from GenBank/EMBL/DDBJ DNA databases placed the Vietnamese strains in one of the lineages commonly found among contemporary strains, with the exception of the NSP2 and NSP4 genes. The NSP2 genes were found to belong to a previously undescribed lineage that diverged from Chinese sheep and goat rotavirus strains, including a Chinese rotavirus vaccine strain LLR with 95% nucleotide identity; the time of their most recent common ancestor was 1975. The NSP4 genes were found to belong, together with Thai and USA strains, to an emergent lineage (VIII), adding further diversity to ever diversifying NSP4 lineages. Thus, there is a need to enhance surveillance of locally‐circulating strains from both children and animals at the whole genome level to address the effect of rotavirus vaccines on changing strain distribution.

Porcine-like G3P[6] and G4P[6] rotavirus A strains detected from children with diarrhoea in Vietnam

Animal rotavirus A (RVA) strains can infect children and cause diarrhoea. We determined the full genome sequences of one G3P[6] strain (NT0001) and five G4P[6] strains (NT0042, NT0077, NT0205, NT0599, and NT0621) detected from children with diarrhoea in Vietnam in 2007-2008. Strain NT0001 had a genotype constellation of: G3-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, strain NT0042: G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1, strain NT0077: G4-P[6]-I5-R1-C1-M1-A8-N1-T7-E1-H1, and strains NT0205, NT0599, and NT0621: G4-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence divergence data and phylogenetic analysis showed that they were different porcine RVA strains that independently and directly crossed the host species barrier to infect children.