Introduction
Rotavirus A (RVA) is a leading cause of severe gastroenteritis in children worldwide [
1]. RVA, a member of the genus
Rotavirus within the family
Reoviridae, has a genome comprising 11 segments of double-stranded RNA that encode six structural viral proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP5/6) [
2]. Each of these genes is differentiated into genotypes according to a predefined nucleotide sequence identity cutoff value [
3‐
5]. This classification system denotes the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 genes as the descriptor Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx (where x represents a genotype number) [
3‐
5]. Most human RVA strains can be classified into three genotype constellations, Wa, DS-1 and AU-1, which are described as G1/3/4/9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3, respectively [
3,
6].
In 2012, novel double-gene reassortant strains with the genotype constellation G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 emerged in Japan [
7‐
9]. Upon whole-genome analysis of these double-gene reassortant strains, Fujii et al. [
9] concluded that the strains were of clonal origin and spread throughout the entire country with a detection rate of 31-63%. Similarly, in Thailand, three G1P[8] double-gene reassortant strains were sporadically detected in 2013 [
10]. All 11 genes of the Thai strains were reported to be closely related to each other and to those of Japanese G1P[8] double-gene reassortant strains, and they were considered to have originated from a recent common ancestor [
10].
In the 2012-2013 rotavirus season in Hanoi, Vietnam, where we conducted a rotavirus study, over a quarter of G1P[8] rotavirus-positive samples exhibited short RNA migration patterns upon polyacrylamide gel electrophoresis. As short RNA migration patterns are usually associated with the DS-1-like genotype constellation [
11], we determined the whole genotype constellation of representative Vietnamese G1P[8] strains to understand how they emerged and how they were related to the Japanese and Thai G1P[8] double-gene reassortant strains. In addition, it was explored whether the G1P[8] double-gene reassortant strains caused more-severe disease in children than ordinary G1P[8] strains
Materials and methods
Study specimens and case patients
A cross-sectional study was performed at Saint Paul Hospital and Bach Mai Hospital, Hanoi, Vietnam, from November 2012 to June 2013 (the 2012/2013 rotavirus season in Hanoi). Briefly, faecal specimens were collected from all children less than two years of age who were hospitalised for acute diarrhoea, which was defined as three or more looser-than-normal stool passages or watery diarrhoea during the preceding 24 hours. For each faecal specimen, a 10% suspension (w/v) was made in phosphate-buffered saline (pH 7.2), and tested for rotavirus antigen using an enzyme-linked immunosorbent assay (Premier Rotaclone, Meridian Bioscience, Inc., OH, USA) according to the manufacturer’s instructions.
Demographic data for the enrolled patients were collected together with information about signs and symptoms they presented at the time of hospitalisation in order to calculate the severity score of diarrhoea according to Vesikari’s 20 point scale [
12]. Informed consent was obtained from the parents or guardians of the enrolled patients, and the study was approved by the institutional review boards of the participating hospitals as well as the National Institute of Hygiene and Epidemiology, Vietnam, and Nagasaki University, Japan.
Viral RNA extraction, G and P genotyping, and electropherotyping
Viral RNA was extracted from 140 µL of supernatant obtained from 10% stool suspension (w/v) using a QIAamp Viral RNA Mini Kit (QIAGEN Sciences, Germantown, MD, USA) according to the manufacturer’s instructions. G and P genotyping was done by reverse-transcription PCR by using the primers designed by Gouvea et al. [
13] and Gunasena et al. [
14]. Genomic RNAs were separated for 16 hours at a constant current of 8 mA on a 10% polyacrylamide gel, and the electropherotype of each strain was determined after staining with silver nitrate as described previously [
15].
Whole-genome amplification and sequencing
Based on the results of the G and P genotyping combined with electropherotyping, five strains were selected for investigation of the whole genome. These included two G1P[8] strains with short RNA migration patterns (SP026 and SP071) and three G2P[4] strains with short RNA migration patterns (SP015, SP108, and SP355). The VP7, VP4 and NSP4 genes of two G1P[8] strains with long RNA migration patterns (SP110 and SP118) were sequenced. An AcessQuick Kit (Promega Corporation, Madison, WI, USA) was used with the gene-specific end primer pairs described previously [
3,
16] to generate cDNAs and the full-length amplicons for the 11 genes of SP015, SP026, SP071 and SP108. For strain SP355, the SuperScript III first-strand synthesis system for reverse transcription PCR (Invitrogen, Carlsbad, CA, USA) was used with random hexamers (Invitrogen) to generate the cDNAs, which were then amplified using the GoTaq Green Master Mix System (Promega Corporation) with gene-specific end primer pairs that allowed the generation of full-length amplicons [
3,
16]. PrimeSTAR GXL DNA Polymerase (Takara Bio, Inc., Shiga, Japan) was used together with primers designed by Fujii et al. [
16] to amplify larger genes that could not be amplified previously.
The amplified full-length genes were purified using an ExoSAP-IT purification kit (USB products, Cleveland, OH, USA) and sequenced from end to end in both the forward and reverse directions by the fluorescent dideoxy chain termination chemistry using a Big Dye Terminator Cycle Sequencing Ready Reaction Kit v3.1 (Applied Biosystems, Foster City, CA, USA). Nucleotide sequence reads were obtained with the aid of an ABI-PRISM 3730 Genetic Analyzer (Applied Biosystems).
Sequence and phylogenetic analyses
Nucleotide sequences were aligned using the SeqMan programme in the Lasergene 11 software package (DNASTAR, Inc. Madison, WI, USA). The genotype of each genome segment was determined by using the RotaC 2.0 automated genotyping tool for RVA [
17].
Multiple sequence alignment was carried out using the MUSCLE programme, and the genetic distances were calculated by the p-distance method implemented in MEGA ver. 6.06 [
18]. Nucleotide substitution model testing was carried out in MEGA ver. 6.06, and the best-fit evolutionary model for each gene was selected based on the lowest Bayesian information criterion score. Maximum-likelihood phylogenetic trees were constructed using MEGA ver. 6.06 [
18]. Trees were analysed by bootstrapping with 1000 replicates, and inferred by using the general time-reversible model (GTR) with gamma distribution (G) and invariant sites (I) for VP3; GTR+ I for VP1 and VP2; the Tamura-Nei model (TN93) + I for NSP3; the Tamura 3-parameter (T92) +G for VP7, VP4, VP6, NSP2, NSP4 and NSP5; and T92+I for NSP1 [
19].
Nucleotide sequence accession numbers
The nucleotide sequences were deposited under the accession numbers LC066147-LC066196, and LC174963-LC174973, and the lengths of these sequences are listed in Supplementary Table 1.
Discussion
The identification of G1P[8] double-gene reassortant strains in Vietnam in the 2012/2013 rotavirus season is reminiscent of the DS-1-like G1P[8] double-gene reassortant strains emerging in one rotavirus season earlier in Japan [
7‐
9] and the other strains emerging in the same rotavirus season in Thailand [
10]. In Thailand, the detection was limited to three sporadic cases accounting for 0.4% of the circulating strains [
10]. On the other hand, in Japan, they caused a country-wide spread and accounted for 31-62% of the strains, depending on the region [
9]. Based on high nucleotide sequence identities of 98.5-99.7% and co-clustering topology in phylogenetic trees for all 11 genes, Komoto et al. [
10] hypothesised that Japanese and Thai double-gene reassortant strains originated from a recent common ancestor.
While the determination of the whole genotype constellation showed that all of these DS-1-like G1P[8] strains were generated by genetic reassortment, previous studies failed to identify the G2P[4] strain that donated the DS-1-like background of the double-gene reassortant strains. In this study, determination of the whole genome sequence and phylogenetic analysis showed that the internal-capsid and non-structural protein genes of SP355 were 99.3-100% identical to those of SP026/SP071, and they clustered together in the same lineage in the phylogenetic trees.
With respect to the origin of the outer-capsid genes of the Vietnamese double-gene reassortant strains, two Chinese strains that had the highest nucleotide sequence identities (99.5% and 99.8% identical to the Vietnamese VP7 and VP4 genes, respectively) clustered with SP026 and SP071 with very high bootstrap support. The G1P[8] strains co-circulating during the study period belonged to the same cluster with the SP026/SP071 in the VP7 tree, but not in the VP4 tree. Nevertheless, when the geographical closeness of Vietnam and China and our limited strain sampling were taken into consideration, it is likely that locally circulating G1P[8] strains in Vietnam donated their VP7 and VP4 genes to the SP355-like strains, resulting in the emergence of the double-gene reassortants in Vietnam.
The results obtained in this study led us to hypothesise that the Vietnamese G1P[8] strains were most likely to have been generated in Vietnam through the genetic reassortment events in which a locally circulating G2P[4] strain acquired the outer-capsid genes from co-circulating G1 and P[8] strains. The generation of these double-gene reassortant strains is unlikely to be attributable to the events in which the Japanese and Thai double-gene reassortants were generated on the following grounds: First, the Vietnamese double-gene reassortant strains always clustered together with a locally-circulating G2P[4] strain, and this cluster, which was supported by a high bootstrap value, was clearly distinguished from the cluster comprising the Japanese/Thai double-gene reassortant strains. The Vietnamese G2P[4] strain (SP355) together with Thai G2P[4] strains (SKT138 and NP-M51) never clustered with the Japanese/Thai double-gene reassortant strains in any of the internal-capsid and non-structural protein genes, indicating that they were unlikely to be the donor strains that provided the backbone of the Japanese/Thai double-gene reassortant strains. Second, the outer-capsid genes that clustered together with those of the Vietnamese and Japanese/Thai double-gene reassortant strains were from different strains: the Chinese G1P[8] strain BX5 and G3P[8] strain R1604 for the former, and the American G1P[8] strain 2007719635 for the latter.
While the further evolution of any of the G1P[8] double-gene reassortant strains from Japan and Thailand is unclear, the emergence of G3P[8] double-gene reassortant strains in Australia [
20], Thailand [
21], and Spain [
22] deserves mention because they also possessed the DS-1-like internal-capsid and non-structural protein genes. Sporadically detected Thai G3P[8] double-gene reassortant strains, although co-circulating with the G1P[8] double-gene reassortant strains in the same rotavirus season, were described as having their nine genes having originated from G1P[8] double-gene reassortant strains [
21]. The G3P[8] strains that caused a local outbreak in the Basque country in northern Spain were hypothesised to have originated from the G3P[8] strains that had appeared earlier in the Asian Pacific region [
22].
In Vietnam, bovine-like G8P[8] strains possessing a DS-1-like backbone emerged in 2014 and became dominant in 2015 [
23], but their backbone genes as well as the P[8] VP4 gene were unlikely to have been direct descendants of the genes of the Vietnamese G1P[8] double-gene reassortant strains described in this study.
Few studies have addressed the question of whether recently emerging strains with unusual genotype constellations, such as G1P[8] double-gene reassortant strains, cause more-severe disease in children than ordinary RVA strains. In this study, it was shown that children infected with Vietnamese G1P[8] double-gene reassortant strains experienced severe diarrhoea (as the mean Vesikari score of 13.1 was categorised as severe diarrhoea) but slightly less-severe diarrhoea than those children infected with ordinary G1P[8] strains (Fig.
3). Since this study did not examine other enteric pathogens that might have co-infected the children under investigation, the observed difference may not simply be ascribed to the difference in the pathogenic potential of the strains compared. Thus, it can be concluded that the disease caused by G1P[8] double-gene reassortant strains was not more severe than that caused by common G1P[8] strains.
In conclusion, this study showed that apparently clonal G1P[8] double-gene reassortant strains emerged in Hanoi, Vietnam and accounted for 14% of the RVA-positive specimens recovered from infants and young children hospitalised for severe diarrhoea during the 2012/2013 rotavirus season. Whole-genome analysis showed that these Vietnamese strains were generated by genetic reassortment events in which a locally circulating G2P[4] strain acquired the VP7 and VP4 genes from strains similar to Chinese G1P[8] and G3P[8] strains, respectively. Despite the similarity in their emergence in time (2012/2013) and place (the Western Pacific region), the Vietnamese and Japanese/Thai G1P[8] double-gene reassortant strains were generated by different combinations of parental strains.