Full genome-based characterization of an Asian G3P[6] human rotavirus strain found in a diarrheic child in Japan: Evidence for porcine-to-human zoonotic transmission.

Human rotavirus strains having the unconventional G3P[6] genotype have been sporadically detected in diarrheic patients in different parts of the world. However, the full genomes of only three human G3P[6] strains from Asian countries (China, Indonesia, and Vietnam) have been sequenced and characterized, and thus the exact origin and evolution of G3P[6] strains in Asia remain to be elucidated. Here, we sequenced and characterized the full genome of a G3P[6] strain (RVA/Human-wt/JPN/SO1199/2020/G3P[6]) found in a stool sample from a 3-month-old infant admitted with acute gastroenteritis in Japan. On full genomic analysis, strain SO1199 was revealed to have a unique Wa-like genogroup configuration: G3-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1. VP6 genotype I5 and NSP1 genotype A8 are commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis demonstrated that all 11 genes of strain SO1199 were closely related to those of porcine and/or porcine-like human rotaviruses and thus appeared to be of porcine origin. Thus, strain SO1199 was shown to possess a porcine-like genomic backbone and thus is likely to be the result of interspecies transmission of a porcine rotavirus strain. Of note is that all 11 genes of strain SO1199 were phylogenetically located in clusters, distinct from those of the previously identified porcine-like human G3P[6] strains from around the world including Asia, suggesting the occurrence of independent porcine-to-human zoonotic transmission events. To our knowledge, this is the first report on full genome-based characterization of a human G3P[6] strain that has emerged in Japan. Our findings revealed the diversity of unconventional human G3P[6] strains in Asia, and provide important insights into the origin and evolution of G3P[6] strains.

Whole genome sequencing and evolutionary analysis of G8P [8] rotaviruses emerging in Japan.

Unusual DS-1-like intergenogroup reassortant rotaviruses with a bovine-like G8 genotype (DS-1-like G8P [8] rotaviruses) have emerged and rapidly spread in several countries. In this study, the nucleotide sequences of seven human rotavirus G8P [8] strains in 2017 and 2019 in Japan were determined using viral metagenomics. Its genomic constellation (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes) was defined as G8-P [8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Our genetic analysis revealed that the Japanese G8P [8] rotavirus strains in 2017 and 2019 were classified into the same lineages G8-5 and P [8]-3, but they were phylogenetically located on separate branches and belonged to distinct clusters. Our study is the first attempt to investigate the evolution of emerging rotavirus G8P [8] in Japan.

Rapid Spread in Japan of Unusual G9P[8] Human Rotavirus Strains Possessing NSP4 Genes of E2 Genotype.

The emergence of unusual G9P[8]-E2 human rotaviruses in the Tokyo metropolitan area, Japan, in 2018 has been reported. During rotavirus strain surveillance in different regions of Japan (Mie, Okayama, and Chiba prefectures), G9P[8]-E2 strains were detected in children with diarrhea from all three prefectures. Here, we characterized the whole genome of seven representative G9P[8]-E2 strains. In the full-genome-based analysis, the seven study strains exhibited a unique genotype configuration with the NSP4 gene of genogroup 2 in a genogroup 1 genomic backbone: G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1. This genotype constellation was shared by the Tokyo G9P[8]-E2 strains. Phylogenetic analysis showed that all 11 genes, except NSP4, of the seven study strains appeared to have originated from co-circulating Wa-like G9P[8]-E1 strains. In contrast, NSP4 appeared to have originated from the co-circulating DS-1-like G2P[4]-E2 strains. Thus, G9P[8]-E2 strains appear to be derived through reassortment between G9P[8]-E1 and G2P[4]-E2 strains in Japan. Notably, the seven study G9P[8]-E2 strains and Tokyo G9P[8]-E2 strains were revealed to have 11-segment genomes almost indistinguishable from one another in their sequences (99.3-100%), indicating all these G9P[8]-E2 strains had a common origin. To our knowledge, this is the first description of the rapid spread of G9P[8]-E2 strains across a country.

Whole genome sequence of an uncommon G9P[4] species A rotavirus containing DS-1-like (genotype 2) genes in Japan.

Species A rotaviruses (RVAs) have been recognized as one of the leading causes of acute gastroenteritis in humans worldwide. Here, the complete coding sequences of 11 RNA segments of an uncommon G9P[4] RVA strain, which was detected in feces of a diarrheal child in Japan, were determined by next-generation sequencing technology. Its genomic constellation, VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, was determined as G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This work reports the complete coding sequences of a G9P[4] RVA strain containing DS-1-like (genotype 2) genes that was isolated in Japan in 2013.

Genomic characterization of an African G4P[6] human rotavirus strain identified in a diarrheic child in Kenya: Evidence for porcine-to-human interspecies transmission and reassortment.

Human rotavirus strains having the unconventional G4P[6] genotype have been sporadically identified in diarrheic patients in different parts of the world. However, the whole genome of only one human G4P[6] strain from Africa (central Africa) has been sequenced and analyzed, and thus the exact origin and evolutionary pattern of African G4P[6] strains remain to be elucidated. In this study, we characterized the full genome of an African G4P[6] strain (RVA/Human-wt/KEN/KCH148/2019/G4P[6]) identified in a stool specimen from a diarrheic child in Kenya. Full genome analysis of strain KCH148 revealed a unique Wa-like genogroup constellation: G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1. NSP3 genotype T7 is commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis showed that 10 of the 11 genes of strain KCH148 (VP7, VP4, VP6, VP1-VP3, NSP1, and NSP3-NSP5) appeared to be of porcine origin, the remaining NSP2 gene appearing to be of human origin. Therefore, strain KCH148 was found to have a porcine rotavirus backbone and thus is likely to be of porcine origin. Furthermore, strain KCH148 is assumed to have been derived through interspecies transmission and reassortment events involving porcine and human rotavirus strains. To our knowledge, this is the first report on full genome-based characterization of a human G4P[6] strain from east Africa. Our observations demonstrated the diversity of human G4P[6] strains in Africa, and provide important insights into the origin and evolutionary pattern of zoonotic G4P[6] strains on the African continent.

Full genome-based characterization of G4P[6] rotavirus strains from diarrheic patients in Thailand: Evidence for independent porcine-to-human interspecies transmission events.

The exact evolutionary patterns of human G4P[6] rotavirus strains remain to be elucidated. Such strains possess unique and strain-specific genotype constellations, raising the question of whether G4P[6] strains are primarily transmitted via independent interspecies transmission or human-to-human transmission after interspecies transmission. Two G4P[6] rotavirus strains were identified in fecal specimens from hospitalized patients with severe diarrhea in Thailand, namely, DU2014-259 (RVA/Human-wt/THA/DU2014-259/2014/G4P[6]) and PK2015-1-0001 (RVA/Human-wt/THA/PK2015-1-0001/2015/G4P[6]). Here, we analyzed the full genomes of the two human G4P[6] strains, which provided the opportunity to study and confirm their evolutionary origin. On whole genome analysis, both strains exhibited a unique Wa-like genotype constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The NSP1 genotype A8 is commonly found in porcine rotavirus strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strains DU2014-259 and PK2015-1-0001 appeared to be of porcine origin. On the other hand, the two study strains consistently formed distinct clusters for nine of the 11 gene segments (VP4, VP6, VP1-VP3, and NSP2-NSP5), strongly indicating the occurrence of independent porcine-to-human interspecies transmission events. Our observations provide important insights into the origin of zoonotic G4P[6] strains, and into the dynamic interaction between porcine and human rotavirus strains.

Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Genomic characterization of a novel G3P[10] rotavirus strain from a diarrheic child in Thailand: Evidence for bat-to-human zoonotic transmission.

An unusual rotavirus strain with the G3P[10] genotype (RVA/Human-wt/THA/MS2015-1-0001/2015/G3P[10]) was identified in a stool sample from a hospitalized child aged 11 months with severe gastroenteritis in Thailand. In the current study, we sequenced and characterized the full genome of strain MS2015-1-0001. On full-genomic analysis, strain MS2015-1-0001 exhibited the following genotype configuration: G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is identical or closely related to those of bat and bat-like rotavirus strains (MYAS33-like). Furthermore, phylogenetic analysis revealed that all 11 genes of strain MS2015-1-0001 appeared to be of bat origin. Our findings provide evidence for bat-to-human interspecies transmission of rotaviruses and important insights into dynamic interactions between human and bat rotavirus strains.

Generation of recombinant rotaviruses from just 11 cDNAs encoding a viral genome.

Reverse genetics technology allows one to engineer replication-competent viruses from cloned cDNAs at will. Since the establishment of the initial reverse genetics system for species A rotaviruses (RVAs) requiring a helper virus in 2006, attempts have been successfully made to improve this technology. Efficient generation of replication-competent RVAs is now possible from just 11 T7-driven plasmids encoding an RVA genome when the quantity ratio of the two rescue T7-driven plasmids for the NSP2 and NSP5 segments is increased by 3-fold in relation to that of the other nine plasmids (11 plasmid-only system). Further, it is now possible to generate recombinant RVAs even with severely less efficient infectivity by using the 11 plasmid-only system, which has not been possible with the existing approaches. More importantly, the 11 plasmid-only system does not need any helper expression plasmid, and thus this simplest and robust system has a clear advantage over the existing systems in terms of safety. This 11 plasmid-only system should contribute to the development of safe next-generation vaccines and vaccine vectors.

Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics.

Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1-3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1-4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.

Full genome characterization of novel DS-1-like G9P[8] rotavirus strains that have emerged in Thailand.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

Characterization of an Unusual DS-1-Like G8P[8] Rotavirus Strain from Japan in 2017: Evolution of Emerging DS-1-Like G8P[8] Strains through Reassortment.

The emergence of unusual DS-1-like intergenogroup reassortant rotaviruses with a bovine-like G8 genotype (DS-1-like G8P[8] strains) has been reported in several Asian countries. During the rotavirus surveillance program in Japan in 2017, a DS-1-like G8P[8] strain (RVA/Human-wt/JPN/SO1162/2017/G8P[8]) was identified in 43 rotavirus-positive stool samples. Strain SO1162 was shown to have a unique genotype constellation, including genes from both genogroup 1 and 2: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that the VP1 gene of strain SO1162 appeared to have originated from DS-1-like G1P[8] strains from Thailand and Vietnam, while the remaining 10 genes were closely related to those of previously reported DS-1-like G8P[8] strains. Thus, SO1162 was suggested to be a reassortant strain that acquired the VP1 gene from Southeast Asian DS-1-like G1P[8] strains on the genetic background of co-circulating DS-1-like G8P[8] strains. Our findings provide important insights into the evolutionary dynamics of emerging DS-1-like G8P[8] strains.

Generation of Infectious Recombinant Human Rotaviruses from Just 11 Cloned cDNAs Encoding the Rotavirus Genome.

The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) was also generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses.IMPORTANCE Human group A rotavirus (HuRVA) is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs "to order" will be useful for next-generation vaccine production for this medically important virus and for the engineering of clinical vectors expressing any foreign genes.

Genomic characterization of uncommon human G3P[6] rotavirus strains that have emerged in Kenya after rotavirus vaccine introduction, and pre-vaccine human G8P[4] rotavirus strains.

A monovalent rotavirus vaccine (RV1) was introduced to the national immunization program in Kenya in July 2014. There was increased detection of uncommon G3P[6] strains that coincided temporally with the timing of this vaccine introduction. Here, we sequenced and characterized the full genomes of two post-vaccine G3P[6] strains, RVA/Human-wt/KEN/KDH1951/2014/G3P[6] and RVA/Human-wt/KEN/KDH1968/2014/G3P[6], as representatives of these uncommon strains. On full-genomic analysis, both strains exhibited a DS-1-like genotype constellation: G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that all 11 genes of strains KDH1951 and KDH1968 were very closely related to those of human G3P[6] strains isolated in Uganda in 2012-2013, indicating the derivation of these G3P[6] strains from a common ancestor. Because the uncommon G3P[6] strains that emerged in Kenya are fully heterotypic as to the introduced vaccine strain regarding the genotype constellation, vaccine effectiveness against these G3P[6] strains needs to be closely monitored.