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Here, we report the genomic characterization of a fowl adenovirus serotype 4 strain isolated from a chicken with hydropericardium syndrome in Japan. The viral genome of FAdV-4 strain JP/LVP-1/96 was found to be 45,688 bp long. Amino acid substitutions at position 219 (G to D) in the fiber-2 protein and at position 188 (I to R) in the hexon protein, which are commonly found in virulent FAdV-4 strains, were also found in the JP/LVP-1/96 strain. Additional specific amino acid substitutions commonly found in virulent FAdV-4 strains were found in ORFs 4 and 43, which are present only in members of the species Fowl adenovirus C. Phylogenetic analysis based on complete hexon protein gene sequences showed that strain JP/LVP-1/96 belongs to a different genetic cluster from the strains circulating in neighboring countries.

The complete nucleotide sequence of the S1 glycoprotein gene of the Japanese infectious bronchitis virus (IBV) strains was determined and genetically analyzed. A total of 61 Japanese IBV strains were classified into seven genotypes, namely GI-1, 3, 7, 13, 18, 19, and GVI-1 using the classification scheme that was proposed by Valastro et al, with three exceptions. These genotypes practically corresponded to those defined in Japan, namely Mass, Gray, JP-II, 4/91, JP-I, JP-III, and JP-IV, which have been identified through their partial nucleotide sequences containing hypervariable regions 1 and 2. In addition, three exceptive strains were considered to be derived from recombination within the S1 gene of IBV strains G1-13 and GI-19. By analyzing the amino acid polymorphism of the S1 glycoprotein among Japanese genotypes, a diversity was observed based on the genotype-specific amino acid residue, the proteolytic cleavage motif at the S1/S2 cleavage site, and the position of the potential N-glycosylation sites.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Bovine papillomavirus type 9 (BPV9) is a causative agent of severe teat papillomatosis. Considering the lack of efficient BPV culture methods, recombinant proteins such as virus-like particles developed through genetic engineering may serve as a useful tool for developing effective vaccines against BPV infection. In this study, we successfully produced immunogenic particles composed of recombinant L1 protein of BPV9 (rBPV9-L1), using a baculovirus expression system. rBPV9-L1-immunized mice produced BPV9-specific IgG, which did not cross-react with BPV type 6, which is another causative agent of teat papillomatosis. Hence, immunogenic rBPV9-L1 is potentially applicable as a vaccine candidate for teat papillomatosis.

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Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.

Background The type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has spread throughout countries of southeast Asia, where it has caused severe economic losses. Even countries presently free of PRRSV are at high risk for infection and spread of this virus. Some of these countries, including Japan, have broad epidemics of the local type 2 PRRSV, creating chronic pathogenicity in the domestic pig population. The present study aimed to evaluate the protective efficacy of immunity by infection with a Japanese field isolate, EDRD1, against heterologous challenge with a Vietnamese HP-PRRSV field strain. To this end, four groups of PRRSV-negative crossbreed piglets were used for a challenge study. Groups 1 and 2 were inoculated with EDRD1 via the intranasal route. After 26 days, Groups 2 and 3 were inoculated with HP-PRRSV via the same route. Group 4 served as an uninfected control. Blood and oral fluid samples were taken every 3–4 days after HP-PRRSV challenge; on day 16 post-challenge, all pigs were euthanized, and examined pathologically. Results The nucleotide sequence analysis of nonstructural protein 2 gene of EDRD1 and comparison with Vietnamese HP-PRRSV showed that the 39 amino acid deletion sites of EDRD1 was nearly in the same region as the 29 amino acid deletion sites of HP-PRRSV. Immunity conferred by inoculation with EDRD1 dramatically reduced viral load in the sera and tissues besides viral shedding (Group 2) compared with those in pigs infected only with HP-PRRSV (Group 3). The clinical signs and rectal temperature were significantly reduced, and the average daily weight gain was significantly improved in the EDRD1-inoculated pigs (Group 2) compared with the Group 3 pigs. Notably, no viral RNA was detected in various organs of the Group 2 pigs 16 days post-infection with HP-PRRSV, except in one pig. Therefore, the immunity induced by EDRD1 and its genetically close field isolates may play a role in reducing viremia caused by HP-PRRSV. Conclusions The results of the present study demonstrate that pigs are highly protected against heterologous Vietnamese HP-PRRSV challenge by immunity against a Japanese local strain, EDRD1.

•BPV6-VLP were produced in silkworm pupae using a baculovirus expression system.•BPV6-VLP were affinity-purified by heparin column chromatography.•BPV6-VLP-immunized mice produced a specific IgG to BPV6.•BPV6-specific IgG recognized BPV6 antigen with high sensitivity in teat papilloma legion. Bovine papillomaviruses (BPVs) are the causative agent of bovine teat papillomatosis, which can lead to severe economic losses in dairy cattle. Among the 14 identified BPV genotypes, BPV type 6 (BPV6) is the most frequently detected in teat papilloma lesions, and is therefore thought to play a major role in teat papillomatosis. To develop an effective vaccine against BPV6 infection, we produced virus-like particles of BPV6 (BPV6-VLP) in silkworm (Bombyx mori) pupae and purified these by heparin affinity chromatography using a single column. About 0.7mg purified BPV6-VLP was obtained from one pupa. BPV6-VLP-immunized mice produced a specific IgG to BPV6 that recognized BPV6 antigen with high sensitivity in an immunohistochemical analysis. Thus, silkworm pupae are a useful bioreactor for the production of BPV6-VLP, which can potentially be used as a vaccine for bovine teat papillomatosis.

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.

A one-step multiplex reverse transcription ( RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: bovine group A rotavirus (GAR), bovine group B rotavirus (GBR), bovine group C rotavirus (GCR), bovine coronavirus (BCV), and bovine torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 10 2 , 10 0 , 10 1 , and 10 2 TCID 50 /ml, respectively, and that for GBR was 10 6 copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.