Junsuke Shirai

Full genome analysis of bovine astrovirus from fecal samples of cattle in Japan: identification of possible interspecies transmission of bovine astrovirus

A viral metagenomics approach was used to investigate fecal samples of Japanese calves with and without diarrhea. Of the different viral pathogens detected, read counts gave nearly complete astrovirus-related RNA sequences in 15 of the 146 fecal samples collected in three distinct areas (Hokkaido, Ishikawa, and Kagoshima Prefectures) between 2009 and 2015. Due to the lack of genetic information about bovine astroviruses (BoAstVs) in Japan, these sequences were analyzed in this study. Nine of the 15 Japanese BoAstVs were closely related to Chinese BoAstVs and clustered into a lineage (tentatively named lineage 1) in all phylogenetic trees. Three of 15 strains were phylogenetically separate from lineage 1, showing low sequence identities, and clustered instead with an American strain isolated from cattle with respiratory disease (tentatively named lineage 2). Interestingly, two of 15 strains clustered with lineage 1 in the open reading frame (ORF)1a and ORF1b regions, while they clustered with lineage 2 in the ORF2 region. Remarkably, one of 15 strains exhibited low amino acid sequence similarity to other BoAstVs and was clustered separately with porcine astrovirus type 5 in all trees, and ovine astrovirus in the ORF2 region, suggesting past interspecies transmission.

Identification of novel bovine group A rotavirus G15P[14] strain from epizootic diarrhea of adult cows by de novo sequencing using a next-generation sequencer.

Abstract There are few reports describing diarrhea of adult cattle caused by group A rotaviruses. Here, we report the identification of a novel bovine group A rotavirus from diarrhea of adult cows. A group A rotavirus was detected from an epizootic outbreak of diarrhea in adult cows with a decrease in milk production in Japan in 2013. The comprehensive genomic analyses from fecal samples by viral metagenomics using a next-generation sequencer revealed that it had an unreported genotype combination G15P[14]. The genome constellation of this strain, namely, RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] was G15-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Each gene segment of Tottori-SG was most closely related to Japanese bovine group A rotaviruses suggesting that Tottori-SG might have derived from multiple reassortment events from group A rotavirus strains circulating among Japanese cattle. No other diarrhea pathogen of adult cattle was detected by routine diagnosis and metagenomics. Viral metagenomics, using a next-generation sequencer, is useful to characterize group A rotaviruses from fecal samples and offers unbiased comprehensive investigations of pathogen.

Effects of invert soap with 0.05% sodium hydroxide on infectious bursal disease virus.

The effects of invert soaps with sodium hydroxide on infectious bursal disease virus (IBDV) were studied. Didecyldimethylammonium chloride was most effective, followed by alkylbenzyldimethylammonium chloride and [mono-bis(tri-methylammonium-methylene chloride)]-alkyl (C9-15) toluene. Dilutions without NaOH had little effect on virus titer. Didecyldimethylammonium chloride was further tested for its effects on IBDV by varying temperature, concentration of invert soap, and pH of the dilution. The effect of the invert soap was strong at 40 C, moderate at room temperature, and weak at 4 C. The concentration of invert soap influenced its efficacy at room temperature but not at 4 C. At pH values below 12.9, the invert soap showed decreased efficacy.

H2 genotypes of G4P[6], G5P[7], and G9[23] porcine rotaviruses show super-short RNA electropherotypes.

During group A rotavirus (RVA) surveillance of pig farms in Japan, we detected three RVA strains (G4P[6], G5P[7], and G9P[23] genotypes), which showed super-short RNA patterns by polyacrylamide gel electrophoresis, in samples from a healthy eight-day-old pig and two pigs of seven and eight days old with diarrhea from three farms. Reverse transcription PCR and sequencing revealed that the full-length NSP5 gene of these strains contained 952 or 945 nucleotides, which is consistent with their super-short electropherotypes. Due to a lack of whole genome data on Japanese porcine RVAs, we performed whole genomic analyses of the three strains. The genomic segments of these RVA strains showed typical porcine RVA constellations, except for H2 NSP5 genotype, (G4,5,9-P[6,7,23]-I5-R1-C1-M1-A8-N1-T1-E1-H2 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes). In phylogenetic analyses, these porcine RVA strains clustered with porcine and porcine-like human RVA strains and showed a typical porcine RVA backbone, except for the NSP5 gene; however, intra-genotype reassortment events among porcine and porcine-like human RVA strains were observed. The NSP5 gene segments of these strains were clustered within the H2b genotype with super-short human RVA strains. The H2 genotype has to date only been identified in human and lapine RVA strains. Thus, to our knowledge, this report presents the first case of H2 NSP5 genotype showing a super-short RNA pattern in porcine RVA. These data suggest the possibility of interspecies transmission between pigs and humans and imply that super-short porcine RVA strains possessing H2 genotype are circulating among both asymptomatic and diarrheic porcine populations in Japan.

Viruses produced from complementary DNA of virulent and avirulent strains of swine vesicular disease viruses retain the in vivo and in vitro characteristics of the parental strain

SummaryA full-length cDNA copy of the genome of the pathogenic strain, J1’73, of swine vesicular disease virus (SVDV) was constructed and inserted into the plasmid pSVL to generate a recombinant plasmid pSVLSJ1. Infectious virus was produced following transfection of cultured mammalian cells with the plasmid. The recovered virus had the same in vitro properties as the parental strain with regard to antigenicity, plaque size on IBRS-2 cells and single-step growth. Pigs were experimentally infected with the parental virus, J1’73 strain, and viruses recovered from cells transfected with the plasmids pSVLSJ1 and pSVLS00 [Inoue T, Yamaguchi S, Saeki T, Sekiguchi K, J Gen Virol 71: 1 835–1 838 (1990)] corresponding to the pathogenic (J1’73) and non-pathogenic (H/3’76) Japanese strains of the SVDV, respectively. All pigs inoculated with the virus recovered from pSVLSJ1 produced clinical signs of similar severity to those inoculated with the parental J1’73 strain. In contrast, pigs inoculated with the virus recovered from pSVLS00 did not show any clinical signs. Viruses recovered from cells transfected with either pSVLSJ1 or pSVLS00 therefore retained the in vitro characteristics and the in vivo pathogenicity of their respective parental strains.

Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination

Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs.

Identification and complete genome analysis of a novel bovine picornavirus in Japan.

Abstract We identified novel viruses in feces from cattle with diarrhea collected in 2009 in Hokkaido Prefecture, Japan, by using a metagenomics approach and determined the (near) complete sequences of the virus. Sequence analyses revealed that they had a standard picornavirus genome organization, i.e. 5′ untranslated region (UTR) - L- P1 (VP4- VP3- VP2- VP1) - P2 (2A- 2B- 2C) - P3 (3A- 3B- 3C-3D) - 3′UTR- poly(A). They are closely related to other unclassified Chinese picornaviruses; bat picornaviruses group 1–3, feline picornavirus, and canine picornavirus, sharing 45.4–51.4% (P1), 38.0–44.9% (P2), and 49.6–53.3% (P3) amino acid identities, respectively. The phylogenetic analyses and detailed genome characterization showed that they, together with the unclassified Chinese picornaviruses, grouped as a cluster for the P1, 2C, 3CD and VP1 coding regions. These viruses had conserved features (e.g. predicted protein cleavage sites, presence of a leader protein, 2A, 2C, 3C, and 3D functional domains), suggesting they have a common ancestor. Reverse-transcription-PCR assays, using specific primers designed from the 5′UTR sequence of these viruses, showed that 23.0% (20/87) of fecal samples from cattle with diarrhea were positive, indicating the prevalence of these picornavirus in the Japanese cattle population in Hokkaido Prefecture. However, further studies are needed to investigate the pathogenic potential and etiological role of these viruses in cattle.

Whole-genome sequence analysis of G3 and G14 equine group A rotaviruses isolated in the late 1990s and 2009-2010

Equine group A rotavirus (RVA) G3P[12] and G14P[12] strains cause gastroenteritis in foals worldwide. Both of these strains have been co-circulating in Japan since G14P[12] strains emerged in the late 1990s. Although it is important to comprehensively understand the evolution of RVA strains, whole-genome sequence data on recent equine RVA strains in Japan are lacking. Therefore, in this study, whole-genome analysis of 23 equine RVA isolates from the late 1990s and 2009-2010 and the vaccine strain RVA/Horse-tc/JPN/HO-5/1982/G3P[12] (HO-5) was performed. The G3 strains, including strain HO-5, shared a G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 genotype constellation, and all of their 11 gene segments were highly conserved, regardless of the year of isolation. G14 strains also exhibited an identical genotype constellation (G14-P[12]-I2-R2-C2-M3-A10-N2-T3-E2-H7), but, phylogenetically, segregated into two lineages within the VP7-G14 and NSP4-E2 genotypes. G14 strains were closely related to G3 strains in their VP4, VP1-3, NSP1-3 and NSP5 gene segments. Interestingly, the NSP4 gene of all G3 and G14 strains isolated in the late 1990s branched into a bovine-RVA-like NSP4 gene cluster. These results indicate that, apart from VP7, VP6, and NSP4 genes, the Japanese equine RVA strains share a highly conserved genetic backbone, and that strains possessing a bovine-RVA-like NSP4 gene were predominant in the late 1990s in Japan.

Detection of Bovine Group A Rotavirus Using Rapid Antigen Detection Kits,RT-PCR and Next-Generation DNA Sequencing

ABSTRACT We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick ‘Eiken’ Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 100–103-fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes.

Whole genome sequences of Japanese porcine species C rotaviruses reveal a high diversity of genotypes of individual genes and will contribute to a comprehensive, generally accepted classification system.

Porcine rotavirus C (RVC) is distributed throughout the world and is thought to be a pathogenic agent of diarrhea in piglets. Although, the VP7, VP4, and VP6 gene sequences of Japanese porcine RVCs are currently available, there is no whole-genome sequence data of Japanese RVC. Furthermore, only one to three sequences are available for porcine RVC VP1-VP3 and NSP1-NSP3 genes. Therefore, we determined nearly full-length whole-genome sequences of nine Japanese porcine RVCs from seven piglets with diarrhea and two healthy pigs and compared them with published RVC sequences from a database. The VP7 genes of two Japanese RVCs from healthy pigs were highly divergent from other known RVC strains and were provisionally classified as G12 and G13 based on the 86% nucleotide identity cut-off value. Pairwise sequence identity calculations and phylogenetic analyses revealed that candidate novel genotypes of porcine Japanese RVC were identified in the NSP1, NSP2 and NSP3 encoding genes, respectively. Furthermore, VP3 of Japanese porcine RVCs was shown to be closely related to human RVCs, suggesting a gene reassortment event between porcine and human RVCs and past interspecies transmission. The present study demonstrated that porcine RVCs show greater genetic diversity among strains than human and bovine RVCs.