Genomic properties of 62 field isolates of bovine viral diarrhea virus (BVDV) collected from 1974 to 1999 in Japan were investigated. The 5' untranslated region (UTR) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and the 244 to 247 base nucleotide sequences were determined. Serological properties were also characterized by the cross-neutralization test using antisera against the representative strain of the classified genotype. Using phylogenetic tree analysis, BVDV 1 was subdivided into two major clusters, BVDV-1a (29 isolates) and BVDV-1b (27 isolates). In group 1a, 3 differed from the other viruses, and were classified in a branch assigned as 1a'. However, 4 isolates (So CP/75, 190 CP, 190 NCP and KS86-1-NCP) could not be assigned to group 1a or 1b. In comparison with the published sequence data, KS86-1-NCP, 190 CP and 190 NCP were similar to the Southern Africa isolates that have recently been proposed as BVDV 1c. The 5' UTR sequence of So CP/75 was unique among those of BVDV 1, suggesting that the isolate should be classified into a new genotype. Only 2 out of 62 isolates collected in 1989 and 1990 were identified as BVDV 2. The results of the cross-neutralization test strongly supported these data, suggesting a close correlation between the 5' UTR sequence and the antigenicity of BVDV.

An epizootic outbreak of diarrhea occurred in adult cows on a dairy farm in Hokkaido, Japan. One colostrum-fed calf inoculated with pooled feces of the 5 affected cows, developed mild diarrhea, and shed rotavirus-like particles which reacted with antiserum to group B rotavirus in immune electron microscopy. Cell culture immunofluorescence tests, RNA-polyacrylamide gel electrophoresis and RT-PCR confirmed that this virus was bovine group B rotavirus, which was designated the Nemuro strain. Additional 2 colostrum-deprived calves inoculated with feces of the first calf also developed diarrhea and shed virus, suggesting that this group B rotavirus might be the etiological agent of the outbreak of adult cow diarrhea. The identities of the nucleotide (nt) and deduced amino acid (aa) sequences of the Nemuro VP7 gene were high (93-95% in nt and 96-97% in aa) and low (61-63% in nt and 49-61% in aa) compared to those of the published corresponding genes from 3 bovine and 2 other mammalian (human and rat) strains of group B rotaviruses, respectively. To our knowledge, this is the first report on the presence of bovine group B rotavirus in Japan.

A total of 65 equine group A rotaviruses (GAR) isolated from diarrheal foals at 48 farms in Hokkaido, Japan, between 1996 (29 isolates) and 1997 (36 isolates) were characterized for their VP7 and VP4 serotypes by PCR, nucleotide sequencing, and virus neutralization (VN) tests. By PCR VP7 typing, all isolates were classified as G3 or G 14, and the predominant serotype in each year was G3 (86%) in 1996 and G14 (53%) in 1997. VN tests with these 20 isolates randomly selected confirmed the specificity of PCR on the bases of complete agreement of the results in these methods (9 G3 and 11 G14), and revealed that all 9 G3 isolates were subtype G3B. There were five differing amino acid residues in three VP7 antigenic regions between subtypes G3A and G3B. Antiserum to a baculovirus recombinant that expressed P[12] VP4 neutralized all isolates and P[12] reference strains. These results suggest that genotype P[12] GAR belong to a single VP4 serotype, and that one VP4 and two VP7 serotypes (G3B and G14) of GAR were predominant in the equine population in Japan.

Isolates of the Salmonella enterica serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.Recently, Salmonella enterica serotype Typhimurium multidrug-resistant strain definitive phage type 104 (DT104) has emerged and spread over many countries (4,9,14,15). The organism has a core pattern of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. Previously, we used fluorescent amplified-fragment length polymorphism fingerprinting (FAFLP) analysis for molecular epidemiological investigation of serotype Typhimurium (13). Among 120 isolates from cattle, there were 17 FAFLP profiles that formed four distinct clusters (A to D). The isolates belonging to cluster A, in which all of the isolates of DT104 were included, have become increasingly common since 1992 in the northernmost island of Japan. The sequence of a polymorphic marker that is common to the strains of FAFLP cluster A has homology with the segment of the eae gene of phage P22. In this study, we isolated prophage ST104, which is common to isolates of DT104, and determined the whole sequence of this phage. The genomic architecture is similar to that of P22, and a number of regions are very similar to those of P22.Isolation of prophage common to serotype Typhimurium DT104. Forty-two serotype Typhimurium strains, including the 12 isolates of DT104 used in this study, were described previously (13). To investigate whether lysogenic prophages are present in serotype Typhimurium, we cultured the strains in the presence of mitomycin C at a concentration of 0.5 g/ml as previously described (17). Thirty-four out of 42 strains released phages that produced plaques on lawns of serotype Typhimurium strain LT2. To characterize the isolated phages, the restriction patterns of their DNAs were compared. Endonuclease digestion with EcoRI revealed five DNA types, designated a1, a2, b, c, and d (Fig. 1). All of the phages isolated from strains that belong to FAFLP cluster A (13), including 12 isolates of DT104, show the same restriction pattern, type a1 (Fig. 1). We have named this phage ST104. The restriction patterns of the prophages from strains NET25 and NET26, both of which belong to FAFLP cluster B, are similar to that of ST104; however, an additional EcoRI site was observed (Fig. 1). Therefore, the DNA type of these phages was designated a2. The restriction patterns of the phages isolated from strains belonging to FAFLP cluster B or C are different from those of ST104. No phage was detected in the two strains that belong to FAFLP cluster D. Schicklmaier et al. (10) suggested that prophage restriction patterns in natural isolates of serotype Typhimurium could serve as markers for the epidemiologic classification of pathogenic strains. Our resul...

ABSTRACT. During the period from 2001 to the following year, progenital diseases had been epidemic among the draft stallions and mares pastured together in Iwate Prefecture, the northeastern district of Japan. A stallion and 8 of 31 mares were affected in 2001, and 1 of 2 stallions and 10 of 36 mares in 2002. The clinical symptoms consisted of the formation of papules, pustules, ulcers and scabs on the progenital skin and mucosa in stallions and mares. In 2002, Equine herpesvirus 3 (EHV3) was isolated from 2 mares and the glyco protein G gene of the virus detected from a stallion and 4 mares by polymerase chain reaction. Serum neutralizing tests showed that 12 of 38 horses, 10 clinically and 2 subclinically affected, changed to be positive for the EHV3 antibody. The results suggest that the horses were affected with equine coital exanthema (ECE) through coitus. Five mares with the antibody at the pre-pastured period may have been the possible origins of EHV3 infection in 2002, although the exact origin in 2001 remains unknown. The artificial insemination was performed for the prevention of ECE spreading through coitus on the pasture in 2003. There was no epidemic of the disease in 31 mares, although 3 mares with the antibody at the pre-pastured period showed the significant increase in the titers during the pastured period.

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ABSTRACT. To get basic information to control persistent virus infection among domestic animals by cytokines, the antiviral activity of four natural human cytokines against bovine viral diarrhea virus (BVDV) was evaluated. Normal bovine peripheral blood mononuclear leukocytes (PBML) and fetal bovine muscular cells (FBMC) were treated with varying doses of human interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α and TNF-β. The antiviral activity in treated cells was measured by the titration of virus infectivity in comparison with non-treated controls. IFN-α significantly suppressed virus growth in both PBML and FBMC. The growth of two cytopathogenic and two noncytopathogenic strains was suppressed in the presence of more than 10 3 u/ml of IFN-α. Addition of either TNF-α or TNF-β to IFN-α did not potentiate the suppressive effect. IFN-α also suppressed the replication of BVDV in PBML from cattle persistently infected with BVDV. -KEY WORDS: bovine viral diarrhea virus, cytokine, interferon, persistent infection, pestivirus.

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