Ayako Miyazaki

Genetic polymorphism of the nsp2 gene in North American type- Porcine reproductive and respiratory syndrome virus

We determined the complete nucleotide sequence of EDRD-1, a Japanese strain of the North American type-Porcine reproductive and respiratory syndrome virus (PRRSV), and identified a novel 117-base deletion and 108-base insertion previously reported in the nsp2 gene of the SP strain, which contains the largest genome among PRRSV strains. Based on genetic analysis of the partial nsp2 gene in 30 additional Japanese isolates and 50 strains from various countries, we classified North American-type PRRSVs into three nsp2-types, represented by EDRD-1, which contains the 117-base deletion and 108-base insertion; prototypic VR-2332, which does not contain the deletion and insertion; and SP, which contains only the 108-base insertion. The three nsp2-types were phylogenetically separated, suggesting that these structural changes only occurred at earlier stages of viral evolution. In the nsp2 genes, we identified an additional 19 deletions ranging from 3 to 378 bases and 2 insertions of 3 and 21 bases which were not common within each nsp2-type, suggesting that these changes occurred at later stages of viral evolution. In addition, our results suggest that the three nsp2-types can be rapidly differentiated by RT-PCR using their polymorphisms as natural tags.

New porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs

From October 2013 to date, approximately 1,000 outbreaks of porcine epidemic diarrhoea virus (PEDV) have occurred in Japan. Porcine epidemic diarrhoea with non-lethal effects in piglets was identified in Tottori prefecture in October 2014. Complete genome analysis revealed that the causative pathogen, Tottori2, is a new PEDV variant with a large (582 nt) deletion in the spike gene. Phylogenetic analysis indicated that the Tottori2 PEDV strain might have been derived from the current PEDV strains circulating in domestic pigs. Moreover, the Tottori2 PEDV strain was successfully isolated in Vero cells by serial passage.

Genetic diversity and classification of the outer capsid glycoprotein VP7 of porcine group B rotaviruses

We determined the nucleotide sequences of the outer capsid glycoprotein (VP7) genes of 38 porcine group B rotaviruses (GBRs) from feces of pigs at 27 farms in Japan between 2000 and 2007. Substantial diversity among porcine GBR VP7 genes was observed, with up to 42.4% difference in nucleotides and 49.8% in amino acids. On comparison of VP7 genes, porcine GBRs were clearly distinct from the published corresponding genes from human, bovine and murine GBRs (53.7–70.8% identity in nucleotides and 45.8–73.4% identity in amino acids). Phylogenetic analysis showed that the VP7s of GBRs could be divided into five genotypes: the murine strain was genotype 1, human strains were genotype 2, bovine and some porcine strains were genotype 3, and other porcine strains belonged to genotype 4 or 5. In addition, GBR VP7s in genotypes 3 and 5 were further divided into four and five clusters, respectively. No relationship between VP7 genotype and double-stranded RNA migration patterns of porcine GBRs in polyacrylamide gel electrophoresis were observed. However, an antigen enzyme-linked immunosorbent assay using antiserum to recombinant bovine GBR VP6 did not react with fecal samples containing one cluster of genotype 5 of porcine GBRs. The abundant divergence of porcine GBR VP7 genes suggests that porcine species might be an original natural host of GBR infection and that different serotypes might exist among porcine GBRs. To our knowledge, this is the first report to describe the gene sequences and typing of porcine GBR VP7s.

Molecular characterization of pig epidemic diarrhoea viruses isolated in Japan from 2013 to 2014.

Since October 2013, approximately 1000 outbreaks of porcine epidemic diarrhoea (PED) have occurred, spanning almost all prefectures of Japan, after a period of seven years without a reported case. In order to consider occurrence factor of PED outbreaks, we determined the whole-genome sequences of 38 PED virus (PEDV) strains from diarrheal samples collected at swine farms in 18 prefectures between 2013 and 2014 using next-generation sequencing technology. Using these data, we investigated genetic variation among the recent Japanese PEDV strains and the genetic relationships between these strains and global PEDV strains isolated recently from multiple swine-industrial countries. Eleven out of 38 PEDV strains were isolated successfully on Vero cells with trypsin treatment and subjected to genome sequence analysis. In a comparative genome analysis, we detected two novel PEDV variants, TTR-2/JPN/2014 and MYG-1/JPN/2014, with large deletions in the spike and ORF3 genes, respectively. A phylogenetic analysis based on the spike gene showed that the 38 Japanese PEDV strains were classified into two PEDV types: the North American type with high virulence (n=34) and the INDEL type (n=4). In addition, the recent Japanese PEDV isolates had a close relationship to global PEDV strains isolated in recent years than to the classical PEDV strains detected in Japan the past decades ago. Moreover, the phylogenetic dendrogram of the complete genomes also indicated that the 38 Japanese PEDV strains, including the two novel PEDV variants discovered in this study, are closely related to the PEDV strains that were widespread in the United States and Korea in 2013-2014. These findings suggest that the re-emergence of PED outbreaks since the last reported case in 2006 was caused by the introduction of recent PEDV strains to Japan from overseas.

Detecting Avian Influenza Virus (H5N1) in Domestic Duck Feathers

To the Editor: Free-range domestic ducks can be a key factor in regional spreading of Asian subtype H5N1 avian influenza (AI) virus (1–3). Even asymptomatic domestic ducks can shed the virus continuously from the oral cavity and cloaca (3–5). Therefore, early detection of infected ducks that are shedding the virus would reduce the risk of spreading AI virus (H5N1) in a region where the virus has been endemic in domestic ducks. We previously reported that AI virus (H5N1) can replicate in feather epidermal cells in asymptomatic domestic ducks (6). Feathers are living tissues that are easily collectible from live birds with minimal damage. We now report the usefulness of feathers for virus detection in domestic ducks. An experimental infection study was conducted with Japanese domestic ducks (Anas platyrhynchos var. domestica) and influenza A virus (H5N1) A/chicken/Miyazaki/K11/2007 as previously described (6). Three 4-week-old domestic ducks (a–c) were inoculated intranasally with 107 50% egg infectious doses (EID50) of 0.1 mL. All experimental procedures were approved by the Ethics Committee of the National Institute of Animal Health in Japan. Inoculated ducks did not show any clinical signs except for persistent corneal opacity on day 3 or later. We collected 3–5 contour feathers, plucked from the body, and 2 sets of oropharyngeal and cloacal swabs from each duck at 24-hour intervals from days 2 through 10 postinoculation (pi). Samples were examined by rapid tests, virus isolation, and reverse transcription–PCR (RT-PCR). Feathers were also examined by immunohistochemical testing. On-site rapid tests were performed with a commercial kit, QuickVue Influenza A+B (Quidel Corp., San Diego, CA, USA), which can detect influenza virus nucleoprotein. The first set of swabs was used for rapid tests according to the manufacturer’s instructions. We also tested 1–2 sticks of the feather calamus (≈15–30 mg per stick) for rapid tests (Appendix Figure, panel A). Briefly, we put the calamuses into the test tube containing attached reagent solution (340 μL) and chopped them into small pieces with an iris scissor and then placed the test strip in the tube. We obtained the following results: feathers tested positive for influenza A virus from days 3 through 6 pi in 1 duck (a), and on days 3 and 4 pi in 2 ducks (b and c), whereas all oropharyngeal and cloacal swabs were negative (Appendix Figure, panel B). For virus isolation, we used a second set of swabs placed in 1 mL of phosphate-buffered saline containing antimicrobial drugs and the remaining 2–3 feather calamuses. Virus titers of swabs and 10% (wt/vol) feather homogenate supernatants were calculated with 10-day-old embryonated chicken eggs and expressed as EID50/mL. Viruses were isolated from the oropharyngeal swabs, cloacal swabs, and feathers of all birds, and feathers tested positive for the virus for a longer period than did the swabs (Table). Although the feather samples used for virus isolation differed from those used in rapid tests, the higher virus titers of feathers in each bird corresponded to the positive period of feathers for rapid tests. Table Results of virus isolation and RT-PCR in 3 domestic ducks inoculated with influenza A virus (H5N1)* One-step RT-PCR was performed on the total RNA extracted from the same samples as in virus isolation to detect the H5 AI virus gene (SuperScript One-Step RT-PCR System; Invitrogen, Carlsbad, CA, USA). The 1:10 dilution of RNA templates was used for feathers. The primers used were H5–248–270F and H5–671–647R; the expected product was 424 bp (7). The sensitivity of RT-PCR was slightly higher than that of virus isolation except for the results with cloacal swabs (Table). Immunohistochemical testing was performed to detect influenza virus nucleoprotein in the feather tissue by using a rabbit polyclonal antibody (ab22285; Abcam Ltd., Cambridge, UK). Virus antigens were detected in feather epidermal cells from days 3 through 6 pi, and in a few stromal cells in the feather pulp on days 3 and 4 pi (Appendix Figure, panel C). Our results indicate that larger amounts of viruses can be isolated for a longer time from feathers than from swabs. Therefore, feathers can be considered useful samples for surveillance or diagnostic examination of AI virus (H5N1) in domestic ducks. The epidermis, the outer layer of the feather, is a tissue that has poor host immune response against viral replication (8). As has been observed in virus isolation, viruses may be able to survive longer in differentiated epidermal tissue such as contour feathers. The sensitivity of the rapid test was not adequate for swabs, a finding similar to that of other studies (9,10). However, positive results for rapid tests of feather samples only may shed light on the on-site field detection of AI (H5N1) in asymptomatic domestic ducks. When virus shedding from domestic ducks is maintained at a low level of viral load during the infection, selecting the sample with higher viral load and antigens in tissues, such as feathers, can increase the detection rate obtained from on-site examination. Our results show the potential of feathers as candidates for early AI virus (H5N1) detection.

First Isolation of Cytopathogenic Bovine Torovirus in Cell Culture from a Calf with Diarrhea

ABSTRACT A cytopathogenic virus (designated the Aichi/2004 strain) was isolated in a human rectal adenocarcinoma cell line (HRT-18) from the ileum contents of a calf with diarrhea. Oval and elongated particles, approximately 100 to 170 nm in diameter, with club-shaped projections were seen in the infected culture supernatant, and torovirus-like (tubular and torus nucleocapsid) structures were seen in the infected cells by electron microscopy. An antiserum against bovine torovirus (BToV) reacted with the infected cells by immunofluorescence and neutralized the isolate. However, antisera against bovine coronavirus (BCV) failed to react with the infected cells by immunofluorescence or did not neutralize the isolate. Further, the isolate was positive for BToV by reverse transcription-PCR (RT-PCR) targeting fragments of the nucleocapsid (N), membrane (M), and spike (S) genes. Comparison of the nucleotide sequences of the PCR products with those of the published N, M, and S genes (476 to 497, 672, and 687 to 690 nucleotides, respectively) of toroviruses showed high sequence identities (up to 99.4%, 98.7%, and 94.9% for the N, M, and S genes, respectively) between the isolate and BToVs. In contrast, the isolate was negative for BCV by RT-PCR. In a serological survey of serum samples from 355 calves at 33 farms, 92% of calves were positive for neutralizing antibodies to the isolate. These results indicate that the isolate in this study was BToV and that BToV infection might be common in cattle in Japan. To our knowledge, this is the first isolation of BToV in tissue culture.

Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity.

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhoea, has recently caused significant economic damage worldwide. Porcine aminopeptidase N (pAPN) has been reported to be the receptor for PEDV, although robust evidence is lacking. In the present study, we explored whether pAPN functions as a receptor for PEDV. Human HeLa cells expressing pAPN and pAPN-positive porcine CPK cells failed to support PEDV infection, but were susceptible to infection by transmissible gastroenteritis virus (TGEV), which utilizes pAPN as a functional receptor. In contrast to TGEV, PEDV did not bind soluble porcine aminopeptidases (pAPs) and infection was not inhibited by the soluble form of pAPs. However, overexpression of pAPN in porcine CPK cells (CPK-pAPN cells) slightly increased the production of PEDV, and the increased replication in CPK-pAPN cells was inhibited by bestatin, an inhibitor of the protease activity of aminopeptidase N. These results suggest that pAPN is not a functional receptor for PEDV, but promotes the infection of PEDV through its protease activity.

Annual changes in predominant genotypes of rotavirus A detected in the feces of pigs in various developmental stages raised on a conventional farm.

The goal of the present study was to improve understanding of the ecology of porcine rotavirus A (RVA) infection in pigs raised on a conventional farrow-to-finish farm. We collected 145 fecal samples over a 3-year period from suckling pigs and their dams, and pigs at 30, 60, 90, 120, and 150 days of age. Reverse transcriptase-polymerase chain reaction analysis revealed that 29 samples (20%) were positive for the viral VP7 gene. The detection rate of VP7 sequences was highest in 30-day-old pigs (67%), followed by suckling pigs (43%), lactating sows (17%), and 120-day-old pigs (7%). At least five different combinations of G and P genotypes were identified (G4P[13], G5P[6], G5P[13], G9P[6], and G9P[13]), and their appearance varied with time; three to four different combinations of G and P genotypes were detected in samples taken during each year, and predominant genotypes differed between suckling and 30-day-old pigs and changed annually. While the VP7 and VP4 sequences of isolates belonging to the same G or P genotype were highly similar with only two exceptions, some were combinations of different P or G genotypes, suggesting that gene reassortment occurred. Further, viral sequences carrying the same combinations of G and P genotypes were also identified in pigs of different ages in different years. Our findings here show a wide distribution of genetically diverse porcine RVA sequences that vary annually with respect to predominant genotype and according to developmental stage. These findings enhance our understanding of how RVA infections persist among farm-raised pigs.

Phylogenetic characterization of VP6 gene (inner capsid) of porcine rotavirus C collected in Japan

Porcine rotavirus C (RVC) has been often detected in sporadic cases or outbreaks of diarrhea in suckling and weaned pigs. Previous surveillance studies using both enzyme-linked immunosorbent assays and reverse-transcription polymerase chain reaction in some countries including Japan and the United States have demonstrated a high prevalence of porcine RVCs. In order to understand the phylogenetic relatedness of RVCs, we performed genetic analysis of VP6 gene encoding inner capsid protein by using 22 porcine RVC strains collected in Japan from 2002 to 2010. Comparative analyses of the VP6 nucleotide and amino acid sequences from these porcine RVCs exhibited lower sequence identities than those from human and bovine RVCs. The phylogenetic analysis of VP6 gene of RVC indicated the presence of seven clusters (tentatively assigned I1-I7) according to host species with cut-off values of 87% at the nucleotide level, and VP6 genes of porcine RVCs were divided into five genotypes. These findings indicate that multiple porcine RVC strains with distinctive genotypes are broadly spreading and circulating among farms in Japan. Our data may provide important insights in understanding evolutionary dynamics of RVCs.

Sequence and phylogenetic analyses of nonstructural protein 2 genes of species B porcine rotaviruses detected in Japan during 2001–2009

Porcine rotavirus B (RVB) has been often detected in diarrhea of suckling and weaned pigs. Because it is difficult to serially cultivate RVBs in cell culture, the number of available sequence data for RNA segments other than VP7 and NSP1 in especially porcine RVBs is still limited. We performed genetic analysis focusing on nonstructural protein 2 (NSP2) using several porcine RVB strains, which were detected in diarrheic feces collected around Japan during 2001-2009. Comparison of NSP2 nucleotide and deduced amino acid sequences from porcine RVB strains exhibited low identities (64.0-99.9% in nt and 66.7-100.0% in aa) to those of other RVB strains. Phylogenetic analysis of RVB NSP2 revealed the presence of four clusters (N1-N4) including human plus murine, bovine and porcine clusters with cut-off values of 75% at the nt and 85% at the aa level. Furthermore, the NSP2 genes of porcine RVBs were divided into three genotypes, of which some porcine RVBs belonged into bovine-cluster. PB-70-H5 and PB-70-H3, which belonged to same pig farm, might be identical in NSP2 gene as shown sequence identity of 99.9%, nevertheless both had different VP7 genes each other. Thus, this data demonstrates the occurrence of gene reassortment among porcine RVBs derived from same pig farm. Our findings presented here would provide more valuable information to elucidate evolution of RVBs.