Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation after disappearance of amyloid depositions in mice.

Amyloid A (AA) amyloidosis is characterized by extracellular pathogenic deposition of insoluble fibril protein in various body organs. Deposited amyloid generally remains in a variety of organs for long periods, but its disappearance has been reported after the precursor protein is diminished. The kinetics of AA deposition are not completely understood and, in particular, the roles of cells and cytokines in the deposition and clearance of amyloid remain unclear. In this study, we investigated the disappearance of amyloid depositions in mice over a 1-year period. AA amyloidosis was induced experimentally in mice by injecting amyloid-enhancing factor (AEF) and silver nitrate. Mice were killed at different time-points to examine the occurrence and disappearance of amyloid depositions. Maximum levels of amyloid depositions were observed at 20 days after inoculation. Clearance of amyloid depositions was observed from the 40th day onwards, with only minute traces of amyloid present by 240 days. A second inflammatory stimulus consisting of AEF and silver nitrate was given at 330 or 430 days, after amyloid depositions had disappeared almost completely. After that, serum amyloid A was overproduced and redeposition of amyloid was observed, indicating that all mice were primed for aggressive amyloid depositions. After administration of the inflammatory stimuli, the proinflammatory environment was found to have increased levels of interleukin (IL)-6, while anti-inflammatory conditions were established by IL-10 as regression of amyloid deposition occurred. These results suggest that the proinflammatory and anti-inflammatory status have key roles in both amyloid deposition and clearance.

AA amyloidosis in vaccinated growing chickens.

Systemic amyloid-A (AA) amyloidosis in birds occurs most frequently in waterfowl such as Pekin ducks. In chickens, AA amyloidosis is observed as amyloid arthropathy. Outbreaks of systemic amyloidosis in flocks of layers are known to be induced by repeated inflammatory stimulation, such as those resulting from multiple vaccinations with oil-emulsified bacterins. Outbreaks of fatal AA amyloidosis were observed in growing chickens in a large scale poultry farm within 3 weeks of vaccination with multiple co-administered vaccines. This study documents the histopathological changes in tissues from these birds. Amyloid deposits were also observed at a high rate in the tissues of apparently healthy chickens. Vaccination should therefore be considered as a potential risk factor for the development of AA amyloidosis in poultry.

Genetic characterization of Protostrongylus shiozawai from Japanese serows (Capricornis crispus).

Protostrongylus shiozawai (Nematoda: Strongylida) infection is known to occur in the lungs of wild Japanese serows (Capricornis crispus) and, to date, has been classified only by its morphological characteristics, as well as host specificity. To characterize P. shiozawai genetically, a partial sequence of the second internal transcribed spacer (ITS-2) region was determined and compared with those of related nematodes. Our subsequent classification of P. shiozawai based on phylogenetic analysis was consistent with the current classification. Phylogenetic analysis also showed that P. shiozawai is more closely related to Protostrongylus stilesi than to Protostrongylus rufescens. PCR using genetic markers in the ITS-2 region should provide a useful tool for expanded studies of P. shiozawai and other Protostrongylus species.

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.

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.

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.

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.

Serosurvey for selected virus infections of wild carnivores in Taiwan and Vietnam.

Serum samples from two leopard cats (Felis bengalensis) and four Formosan gem-faced civets (Paguma larvata taivana) in Taiwan, September 1995, and nine leopard cats in Vietnam, August and December 1997, were examined for the prevalence of antibodies against feline parvovirus, feline herpesvirus type 1, feline calicivirus and feline immunodeficiency virus. All civets and nine of 11 leopard cats were shown to have antibodies against feline parvovirus (FPV), and FPV's were isolated from mononuclear cells in the peripheral blood of the six leopard cats.

An epidemic of parapoxvirus infection among cattle: isolation and antibody survey.

A disease characterized by papules, nodules, vesicles and, rarely, pustules and ulcers on teats was seen among cattle on a farm in Chiba Prefecture, Japan. A virus was isolated by inoculation of fetal bovine lung cell cultures from a vesicle on a teat of an infected cow. The virus was subsequently passaged in fetal bovine lung and muscle cells in which it produced complete cytopathic changes. The virus was identified by physicochemical examinations and electromicroscopic observation as a parapoxvirus. A seroepidemiological survey was performed on antibody to the isolated virus by the agar gel immunodiffusion test. The isolated virus formed a precipitation line which cross reacted with other parapoxviruses isolated previously in Japan. The positive rate was more than 50% among cattle in the Kanto district. The positive rate increased with age. It was suggested that parapoxvirus infection might have already been prevalent among cattle in Japan.

Use of protein AG in an enzyme-linked immunosorbent assay for screening for antibodies against parapoxvirus in wild animals in Japan.

Using protein AG in an enzyme-linked immunosorbent assay (ELISA), we tried to detect antibodies against parapoxvirus in 9 species of wild animals in Japan: the Japanese badger (Meles meles anakuma), Japanese black bear (Ursus thibetanus japonicus), Japanese deer (Cervus nippon centralis), Japanese monkey (Macaca fuscata), Japanese raccoon dog (Nyctereutes procyonoides viverrinus), Japanese serow (Capricornis crispus), Japanese wild boar (Sus scrofa leucomystax), masked palm civet (Paguma larvata), and nutria (Myocastor coypus). A total of 272 serum samples were collected over the period from 1984 to 1995 and were tested by the protein AG-ELISA, the agar gel immunodiffusion test, and an indirect immunofluorescence assay. The protein AG-ELISA was effective in a serological survey for parapoxvirus in wild animals, and antibodies were detected only in Japanese serows. A total of 24 of 66 (36.4%) Japanese serows reacted positively, and they were found in almost all prefectures in all years tested. These results suggest that epizootic cycles of parapoxvirus exist widely in Japanese serows and that they could be reservoirs for the virus in the field in Japan. Moreover, it is probable that they might carry the virus to domestic animals such as cattle, sheep, and goats.

In vivo functions of the auxiliary genes and regulatory elements of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a widespread lentivirus of domestic cats that causes an acquired immunodeficiency syndrome (AIDS)-like disease similar to human AIDS caused by human immunodeficiency virus. FIV has a complex genome structure including structural, enzymatic and auxiliary genes and regulatory elements. In this article, we review the in vivo roles of some of these FIV auxiliary genes and regulatory elements, especially focusing on the dUTPase, vif, and ORF-A genes and AP-1 binding site in the enhancer region of the long terminal repeat, by comparison with those of other non-primate lentiviruses. These genes and elements are considered to be important for viral replication, immunological response and pathogenesis in cats.

The roles of vif and ORF-A genes and AP-1 binding site in in vivo replication of feline immunodeficiency virus.

To examine the in vivo roles of auxiliary genes and regulatory elements of feline immunodeficiency virus (FIV), the provirus load in various tissues of cats infected with each of the mutant viruses (delta vif, delta ORF-A and delta AP-1) was studied. Although all mutant viruses could infect various tissues, provirus loads in various tissues especially those in cats infected with delta vif virus were lower than those with the wild-type virus. Our results indicate the significance of vif and ORF-A genes and AP-1 binding site of FIV for efficient viral replication and full pathogenicity in cats.

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus.

We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301 ddlTK-gag was confirmed to express the FIV Gag precursor protein in the cells.