Detection of Equine Herpesvirus (EHV) -1, -2, -4 and -5 in Ethiopian Equids with and without Respiratory Problems and Genetic Characterization of EHV-2 and EHV-5 Strains.

Infections with equine herpesviruses (EHVs) are widespread in equine populations worldwide. Whereas both EHV-1 and EHV-4 produce well-documented respiratory syndromes in equids, the contribution of EHV-2 and EHV-5 to disease of the respiratory tract is still enigmatic. This study describes the detection and genetic characterization of EHVs from equids with and without clinical respiratory disease. Virus-specific PCRs were used to detect EHV-1, -2, -4 and -5. From the total of 160 equids with respiratory disease, EHV-5 was detected at the highest prevalence (23.1%), followed by EHV-2 (20.0%), EHV-4 (8.1%) and EHV-1 (7.5%). Concurrent infections with EHV-2 and EHV-5 were recorded from nine (5.2%) diseased horses. Of the total of 111 clinically healthy equids, EHV-1 and EHV-4 were never detected whereas EHV-2 and EHV-5 were found in 8 (7.2%) and 18 (16.2%) horses, respectively. A significantly higher proportion of EHV-2-infected equids was observed in the respiratory disease group (32/160, 20.0%; P = 0.005) compared to those without disease (8/111; 7.2%). EHV-2-positive equids were three times more likely to display clinical signs of respiratory disease than EHV-2-negative equids (OR 3.22, 95% CI: 1.42-7.28). For EHV-5, the observed difference was not statistically significant (P = 0.166). The phylogenetic analysis of the gB gene revealed that the Ethiopian EHV-2 and EHV-5 strains had a remarkable genetic diversity, with a nucleotide sequence identity among each other that ranged from 94.0 to 99.4% and 95.1 to 100%, respectively. Moreover, the nucleotide sequence identity of EHV-2 and EHV-5 with isolates from other countries acquired from GenBank ranged from 92.9 to 99.1% and 95.1 to 99.5%, respectively. Our results suggest that besides EHV-1 and EHV-4, EHV-2 is likely to be an important contributor either to induce or predispose equids to respiratory disease. However, more work is needed to better understand the contribution of EHV-2 in the establishment of respiratory disease.

Distribution and characteristics of bovine leukemia virus integration sites in the host genome at three different clinical stages of infection.

Bovine leukemia virus (BLV) is an oncogenic retrovirus closely related to human T-cell lymphotropic virus. BLV-infected cattle are categorized as asymptomatic carriers or as having persistent lymphocytosis or enzootic bovine leukemia, depending on the clinical stage. We investigated the BLV integration site distribution at three BLV clinical stages and examined genome sequence features around the integration sites. In all, 264 BLV integration sites, at various locations on each chromosome, were identified in 28 cattle by inverse PCR and BLAST searches. Approximately one-third of BLV proviruses were independently integrated within transcriptional units, and approximately 10 % were integrated near transcription start sites. Moreover, less than 7 % of BLV integration sites were located near CpG islands. BLV did not preferentially integrate into transcriptionally active regions during any of the clinical stages. At the nucleotide level, regions around BLV integration points were significantly A/T rich with weak sequence consensus. BLV preferentially integrated within long interspersed nuclear repeat elements. Although BLV integration sites may not be associated with disease progression, integration is selective at the nucleotide level.

Small Indian mongooses and masked palm civets serve as new reservoirs of Bartonella henselae and potential sources of infection for humans.

The prevalence and genetic properties of Bartonella species were investigated in small Indian mongooses and masked palm civets in Japan. Bartonella henselae, the causative agent of cat-scratch disease (CSD) was isolated from 15.9% (10/63) of the mongooses and 2.0% (1/50) of the masked palm civets, respectively. The bacteraemic level ranged from 3.0 × 10(1) to 8.9 × 10(3) CFU/mL in mongooses and was 7.0 × 10(3) CFU/mL in the masked palm civet. Multispacer typing (MST) analysis based on nine intergenic spacers resulted in the detection of five MST genotypes (MSTs 8, 14, 37, 58 and 59) for the isolates, which grouped in lineage 1 with MST genotypes of isolates from all CSD patients and most of the cats in Japan. It was also found that MST14 from the mongoose strains was the predominant genotype of cat and human strains. This is the first report on the isolation of B. henselae from small Indian mongooses and masked palm civets. The data obtained in the present study suggest that these animals serve as new reservoirs for B. henselae, and may play a role as potential sources of human infection.

Identification of bovine leukocyte antigen class II haplotypes associated with variations in bovine leukemia virus proviral load in Japanese Black cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. Bovine leukocyte antigen (BoLA) is strongly involved in the subclinical progression of BLV infections. Recent studies show that the BoLA-DRB3 gene might play a direct role in controlling the number of BLV-infected peripheral B lymphocytes in vivo in Holstein cattle. However, the specific BoLA class II allele and DRB3-DQA1 haplotypes determining the BLV proviral load in Japanese Black cattle are yet to be identified. In this study, we focused on the association of BLV proviral load and polymorphism of BoLA class II in Japanese Black cattle. We genotyped 186 BLV-infected, clinically normal cattle for BoLA-DRB3 and BoLA-DQA1 using a polymerase chain reaction-sequence-based typing method. BoLA-DRB3*0902 and BoLA-DRB3*1101 were associated with a low proviral load (LPVL), and BoLA-DRB3*1601 was associated with a high proviral load (HPVL). Furthermore, BoLA-DQA1*0204 and BoLA-DQA1*10012 were related to LPVL and HPVL, respectively. Furthermore, we confirmed the correlation between the DRB3-DQA1 haplotype and BLV proviral load. Two haplotypes, namely 0902B or C (DRB3*0902-DQA1*0204) and 1101A (DRB3*1101-DQA1*10011), were associated with a low BLV proviral load, whereas one haplotype 1601B (DRB3*1601-DQA1*10012) was associated with a high BLV proviral load. We conclude that resistance is a dominant trait and susceptibility is a recessive trait. Additionally, resistant alleles were common between Japanese Black and Holstein cattle, and susceptible alleles differed. This is the first report to identify an association between the DRB3-DQA1 haplotype and variations in BLV proviral load.

Identification and diversity of bovine major histocompatibility complex class II haplotypes in Japanese Black and Holstein cattle in Japan.

Bovine leukocyte antigen (BoLA), the major histocompatibility complex of cattle, is one of the most polymorphic gene clusters. We genotyped a population of 109 Japanese Black and 39 Holstein cattle to analyze their BoLA class II haplotypes, BoLA-DRB3 locus, 5 BoLA-DQA loci, and 5 BoLA-DQB loci. We identified 26 previously reported DRB3 alleles, 22 previously reported and 3 new DQA alleles, and 24 previously reported and 6 new DQB alleles. A dendrogram was constructed based on the predicted amino acid sequences of the α1 or ß1 domains encoded by BoLA-DQA or -DQB alleles, which revealed that DQA alleles were clustered into 5 loci, whereas DQB alleles could not be clearly assigned to specific DQB loci. The BoLA-DRB3-DQA-DQB haplotypes were sorted by sequential analytical processes, and 42 distinct haplotypes, including 11 previously published haplotypes and 31 novel haplotypes, were defined. Strong linkage disequilibrium was present in the BoLA genes. We also compared DRB3-DQA1 haplotype frequencies between 507 Japanese Black and 143 Holstein cattle. Thirty-nine DRB3-DQA1 haplotypes were identified, including 29 haplotypes from Japanese Black and 22 haplotypes from Holstein cattle. The majority of the haplotypes could be identified in both breeds, although several haplotypes were identified in only a single breed. This is the first report presenting a detailed study of the BoLA class II haplotype in Japanese Black and Holstein cattle in Japan.

Distribution and superinfection of bovine leukemia virus genotypes in Japan.

A study to investigate the types and distribution of bovine leukemia virus (BLV) was conducted on about eight hundred cattle drawn from 53 farms found in 16 prefectures in Japan. Agar gel immunodiffusion (AGID) tests of serum samples and nested-PCR to detect BLV provirus, in peripheral blood leukocytes were performed. To identify genotypes, restriction fragment length polymorphism (RFLP) was performed with a PCR-amplified 444 bp fragment of the env gene using endonucleases. Three genotypes (1, 3, and 5) were dominant in Japan, and were found in 48.3%, 32.7%, and 16.9% of PCR positive cattle, respectively. Of the cattle infected with genotype 1, 84.7% were strongly positive in the AGID test. Similarly, in cattle with genotype 3, 78.9% were strongly positive. However, only 59.1% of cattle with genotype 5 were strong positive. Three cattle showed unusual RFLP patterns and they were found to be infected with more than one genotype. These results suggest that some BLV infected cattle can not induce effective immune reactions and suffer from superinfection by BLV in the field.

Genomic analysis of some Japanese isolates of Getah virus.

Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.

Mucosal disease induced in cattle persistently infected with bovine viral diarrhea virus by antigenically different cytopathic virus.

Four cattle persistently infected with non-cytopathic (NCP) bovine viral diarrhea (BVD) virus were challenged with cytopathic (CP) BVD virus that was antigenically different from the persistent virus. Two of the animals were injected with dexamethasone (DM) and then challenged. They developed mucosal disease on days 21 and 33 post-challenge. CP-BVD viruses were isolated from their lymph nodes but not from the sera. The isolates were antigenically different from the persistent virus and the nucleotide sequence of a 787 base region in the E2 gene was markedly different. One of the isolates was indistinguishable from the challenge virus by virus neutralization tests and the nucleotide sequence showed high homology with that of the challenge CP-BVD virus. The other two cattle, challenged with the CP-BVD virus without DM treatment, developed mucosal disease at 30 and 264 days post-inoculation. CP-BVD virus was isolated from the sera as well as the lymph nodes of the cattle and was antigenically and genetically similar to the persistent virus and different from the challenge CP-BVD virus. The present results indicate that cattle persistently infected with NCP-BVD virus can develop mucosal disease induced by antigenically different CP-BVD viruses when their cellular immunity is suppressed, although they are not immunotolerant to the virus.

Serological survey of parapoxvirus infection in wild ruminants in Japan in 1996-9.

The prevalence of parapoxvirus infection was examined in free-ranging wild ruminants in Japan, Japanese serow (Capricornis crispus) and Japanese deer (Cervus nippon centralis), in 1996-9. We collected a total of 151 serum samples from 101 Japanese serows and 50 Japanese deer and tested for antibodies against parapoxvirus by an enzyme-linked immunosorbent assay and an agar gel immunodiffusion test. Overall seroprevalences among Japanese serows were 5/25 (20.0%) in 1996, 4/14 (28.6%) in 1997, 5/32 (15.6%) in 1998 and 2/30 (6.7%) in 1999, respectively. The seroprevalence increased with age but was not affected by sex. No antibodies were detected from any of 50 serum samples taken from Japanese deer. Our results in this study suggest that parapoxvirus infection is widespread among the population of Japanese serows, however, Japanese deer appear to be still free of the disease.

Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field.

Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337-V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test. Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombinant antigens were essentially the same as those with the virion antigen prepared from horse cell cultures both in the AGID test and ELISA. The reactivities of the antigens were further compared using serum samples collected from horses in 1999 in certain districts of Mongolia where equine infectious anemia has been prevalent, and from horses in Japan in 1973 when EIA had not been eliminated completely from Japanese horses. These results were completely concurrent. Generally, recombinant antigens have high specificity but low cross reactivity to heterologous strains. However, the present study showed that the recombinant EIAV p26 antigen has cross reactivity to the heterologous strain and is useful for diagnosis of EIA in the field.

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.

Anti-viral effect of recombinant bovine interferon gamma on bovine leukaemia virus.

The antiviral activity of recombinant bovine interferon gamma (rbIFN-gamma) against bovine leukaemia virus (BLV) was evaluated by an in vitro assay. rbIFN-gamma was prepared using a baculovirus expression system and replication of BLV was measured by syncytium assay. Antiviral effects were observed when bovine and sheep cells were used as target cells or effector cells and treated with 0.1 unit/ml of rbIFN-gamma. Formed syncytium numbers were reduced less than 1/20 when these cells were treated with 10 units/ml of rbIFN-gamma. However, the antiviral effects on cells of heterologous species were decreased and more than 1000 units/ml of rbIFN-gamma were required to induce an anti-BLV effect on the combination of CC81 cells as target cells and Bat2Cl6 cells as effector cells, which originated from the cat and bat, respectively. When the degree of BLV production was estimated by reverse transcriptase (RT) activity, no antiviral effect of rbIFN-gamma was induced soon after the treatment, but it was evident in the cells persistently infected with BLV. These results showed that rbIFN-gamma suppresses the replication of BLV in vitro, but has effective biological activity on cells of homologous species.

Mutations occurring during serial passage of Japanese equine infectious anemia virus in primary horse macrophages.

An attenuated equine infectious anemia virus (EIAV), named V26, was previously obtained after 50 passages of the Japanese virulent strain V70 in primary macrophage culture. To clarify the differences between both viruses, their full-length sequences were determined. There were higher mutations in S2 (6.15% amino acid difference) and LTR (10.7% nucleotide difference). The presumed initiation codon of the S2 gene was absent from the sequence of V26. There was a large insertion within the long-terminal repeat (LTR) U3 hypervariable region of V26. In addition, there were minor mutations in gag (1.22% amino acid difference), pol (1.05% amino acid difference) and env (1. 65% amino acid difference) regions, but no mutation in tat region. No mutations were observed in the principal neutralizing domain in the gp90. Thus, the mutations in the S2 and LTR might be the major target sites of mutation in EIAV during serial passages in vitro.

Simple preparation of parapoxvirus genome DNA for endonuclease analysis.

We compared five methods for improved extraction of very-large parapoxvirus DNA from infected cells: (i) alkaline-lysis procedure followed by phenol extraction; (ii) modified Hirt procedure, which was a neutral lysis procedure followed by phenol extraction; (iii) Hirt procedure; (iv) method used for extraction of vaccinia virus DNA; and (v) standard procedure using virus purification with an ultracentrifuge and protease-sodium dodecyl sulfate-phenol treatment. The alkaline-lysis procedure was more rapid, inexpensive and simpler than the other methods. Moreover, with this method it is not necessary to prepare any special facilities, reagents and kits. Although the extracted DNA was still crude, we could reproducibly prepare viral DNA from 2 X 10(6) infected cells in less than 2 hr and it could be readily digested by restriction endonuclease. This method will aid rapid genetic classification of parapoxvirus.

Survey on antibody against parapoxvirus among cattle in Japan.

A seroepidemiological survey was performed on antibody against parapoxvirus among cattle in Japan using the agar gel immunodiffusion test and enzyme-linked immunosorbent assay. A total 1,819 sera were collected from cattle in various parts of Japan for the survey. The positive rates were in the range of 40 to 98%, and the reactors increased gradually in number with advancing age. These results indicate that parapoxvirus infection is already prevalent among cattle in Japan. It remains to be elucidated, however, whether the antibodies detected have been produced by infection of the same virus or other viruses that belong to the genus parapoxvirus.

Detection and diagnosis of parapoxvirus by the polymerase chain reaction.

The genus Parapoxvirus includes four members, bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), orf virus (ORFV) and parapoxvirus of red deer in New Zealand (PVNZ). A set of primers for polymerase chain reaction (PCR) was designed to detect viral DNA from cells infected with each of the four parapoxviruses. The set of primers resulted in the amplification of appropriately sized products from cells infected with BPSV, PCPV, ORFV and PVNZ, respectively. The PCR method was applied for the detection of seven field isolates of parapoxvirus from cattle, sheep and free-ranging wild Japanese serows. The expected size of DNA was amplified from cells infected with each of the seven isolates. No specific PCR products were detected from vaccinia virus-, fowlpox virus- and mock-infected cells. Moreover, by a semi-nested PCR with an inner primer and Southern blot analysis, viral DNA was detected from lesions of clinically affected cattle, sheep and Japanese serows. These results suggested that the PCR method used in this study was specific for the detection of parapoxviruses and thus useful for diagnosis of parapoxvirus infections, especially in discrimination from diseases with similar clinical symptoms.

Characterization of a bovine herpesvirus type 4 isolated from the spinal cord of a cow with astasia.

A bovine herpesvirus (BHV) strain designated B11-41, was recently isolated from the spinal cord of a cow with astasia. By BHV type specific polymerase chain reaction (PCR), a BHV-4 specific fragment was amplified from the DNA of strain B11-41. The nucleotide sequence of the PCR product revealed high homologies (98.4% and 99.8%) with those of two BHV-4 strains (Movar 33/63 and 86-068). The BamHI restriction endonuclease cleavage patterns of the B11-41 were more similar to those of BHV-4 than those of other types of BHV. BHV-4 is divided into two groups, European and American. The EcoRI, and BamHI, and HindIII restriction endonuclease analysis demonstrated that strain B11-41 was closely related to the American type of BHV-4. It was concluded that B11-41 was identified as a BHV-4 that belongs to the American group. Additionally, the results of this study may indicate that the nervous system is one of the sites of viral latency in natural infection.

Expression of porcine endogenous retrovirus in peripheral blood leukocytes from ten different breeds.

The expression of porcine endogenous retroviruses (PERV) was investigated in primary porcine peripheral blood leukocytes (PBL) of ten different pig breeds. The data suggest that PERV exists in all porcine PBL. A new retroviral element, a foamy-like pol-related sequence, was also detected in PBL. Three types of PERV were detected in almost every animal. The breeding of PERV-free pigs is likely to be difficult. Further studies are required to assess the infectious disease risks associated with xenotransplantation.

Replication ability in vitro and in vivo of equine infectious anemia virus avirulent Japanese strain.

An attenuated equine infectious anemia virus (EIAV), V26, was previously prepared by 50 passages of the Japanese virulent strain V70 in primary horse macrophage culture. The horses inoculated with this V26 virus were shown to raise neutralizing antibodies against V70 without any viremia. Here, we investigated the in vitro and in vivo replication ability of V26. Comparison of the long-terminal repeat (LTR) sequences between V26 and V70 revealed a large insertion within the LTR U3 hypervariable region of V26. V26 with the mutation in the LTR showed much higher promoter activity in vitro than V70. This is consistent with the much higher replication rate of V26 in horse primary macrophage cultures compared with V70. In sharp contrast, we failed to identify the V26-specific LTR sequence by PCR, at least in sequential samples of plasma or peripheral blood mononuclear cells derived from three horses until day 62 after V26 inoculation. In contrast, antibody responses to EIAV were observed in all horses. The results suggest that the replication ability of V26 in vivo is extremely low. When one of the horses was subsequently challenged with cell-associated V70, it was found that the horse became PCR positive for EIAV. There was no LTR mutation in EIAV genome in samples periodically prepared from the V70-challenged horse. Thus it was suggested that the LTR mutation in EIAV, which occurs during serial passage in vitro, affects EIAV replication in vitro and in vivo.

Isolation of parapoxvirus from a cow treated with interferon-gamma.

A virus was isolated from peripheral blood leukocytes of a cow which was kept in an isolated pen after it was injected with recombinant bovine interferon-gamma. The virus was identified as a member of genus Parapoxvirus in the family Poxviridae on the basis of electron microscopic observations and serological tests. Parapoxvirus has seldom been isolated other than from papular lesions, the characteristic sign of parapoxvirus infection. This is the first report of parapoxvirus isolation from the peripheral blood of a cow without any clinical signs. These results show that parapoxviruses are capable of causing persistent infection in cattle without clinical signs and can be activated by stress factors that induce modification of immune reactions. Relationships between the isolated virus and other parapoxviruses isolated previously from cattle in Japan were investigated and discussed.