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.

Regulatory properties of the integrated long terminal repeat of the feline immunodeficiency virus.

To examine the regulatory properties of feline immunodeficiency virus (FIV) long terminal repeat (LTR) integrated into host chromatin, Crandell feline kidney cells were stably transfected with the FIV LTR that directs the bacterial chloramphenicol acetyltransferase (CAT) gene. Using these cells, we examined the effects of treatment with several chemical agents, infection with feline viruses, or transfection with effector plasmids expressing FIV gene products on FIV LTR-directed gene expression. Among them, treatment with the phorbol ester (a strong activator of protein kinase C), forskolin (an inducer of cyclic-AMP), 5-azacytidine (a DNA methylation antagonist), or infection with feline herpesvirus type 1 (FHV-1), resulted in induction of CAT activity in the cells. These results suggest that the integrated FIV LTR is stimulated by cellular transcriptional factors induced by phorbol ester, forskolin and FHV-1, and is also inactivated by DNA methylation. Furthermore, this permanent cell line can be used as a screening system of activators of the FIV LTR.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus long terminal repeat.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were examined by gel-mobility-shift assays using oligonucleotides designed to represent putative AP-1 or ATF motif from the FIV LTR. Infection with FIV led to less nuclear proteins binding to the AP-1 and ATF sites, suggesting that proteins binding to the sites were consumed or suppressed by FIV-replication in FIV-infected cells. Nuclear proteins that bind to the AP-1 or ATF site were examined by using extracts from Crandell feline kidney (CRFK) cells treated with TPA (a phorbol ester; a strong activator of protein kinase C) or forskolin (an inducer of cyclic-AMP), or infection with feline herpesvirus type 1 (FHV-1). Although TPA or forskolin treatment moderately increased the level of both proteins that bound to AP-1 and ATF sites, FHV-1 infection markedly changed the protein-binding patterns of the sites. Furthermore, FHV-1-induced proteins that bind adjacent to the transcriptional initiation site of FIV promoter were also observed in FHV-1-infected CRFK cells, suggesting that the FHV-1-induced-proteins affects the transcription of FIV through the AP-1, ATF and leader sequences.

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.

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan.

Feline immunodeficiency virus was isolated from four cats from Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C.

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.

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.

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.

Immunological and histological disorders in cats experimentally infected with feline immunodeficiency virus subtype B (TM2 stain).

Three conventional cats were experimentally infected with subtype B (TM2 strain) of FIV, and two conventional cats served as controls. The infected cats were examined immunologically 99-176 weeks post FIV inoculation (wpi) and histologically at 130 wpi. Two of the three infected cats exhibited lower CD4/CD8 T cell ratios and hypergammaglobulinemia compared with two control cats. Further, all the infected cats showed morphological changes in popliteal lymph nodes such as lymphoid depletion, atrophy and plasma cell hyperplasia. In addition, apoptosis was induced in peripheral blood mononuclear cells (PBMC) from the FIV-infected cats after in vitro culture for one or two days, but not in PBMC from uninfected cats. These observations indicate that FIV subtype B has the potential to induce some immunological and histological disorders in cats.

In vitro attenuation of highly virulent infectious bursal disease virus: some characteristics of attenuated strains.

Some strains of highly virulent infectious bursal disease virus (HV-IBDV) were adapted through serial passage in embryonated eggs. The embryonated egg-adapted HV-IBDV was successfully adapted to grow in chicken embryo fibroblast (CEF) cell cultures showing a cytopathic effect by preparing the CEF cells from the virus-infected embryos. The embryonated egg- and cell culture-adapted strains significantly reduced their pathogenicity to, and did not kill any, young chickens in experimental infection. The bursal lesions of the adapted strain-infected chickens were similar to those observed in classical strain-infected chickens. Cross-virus neutralization analysis showed antigenic diversity between the cell culture-adapted HV-IBDV strains and classical strains. In immunization tests, the adapted strain-immunized chickens showed good protection against the fatal infection of HV-IBDV. Especially, in case of challenge at 3 days postimmunization, the adapted strains showed effective immunogenicity. The adapted strains appear to provide a new and effective live vaccine against HV-IBDV infection.

Growth properties of a feline immunodeficiency virus mutant which lacks an AP-1 binding site in primary peripheral blood mononuclear cells.

Thirty-one base pairs (bp) containing putative AP-1 and AP-4 binding sequences in the U3 region of feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted from an infectious molecular clone of FIV for construction of a mutant virus, and the replication rate and the cytopathogenic activity of the virus were compared with those of the wild type virus in concanavalin-A (Con-A) stimulated primary feline peripheral blood mononuclear cells (fPBMCs). It was found that the replication rate and cytopathogenic activity of the mutant were almost the same as those of the wild type. The deletion of the mutant virus was stable during the infection experiments. From these data, we concluded that the 31 bp fragment in the LTR is not required for the replication of FIV in Con-A stimulated primary fPBMC.

Quantification of feline immunodeficiency virus (FIV) proviral DNA in peripheral blood mononuclear cells of cats infected with Japanese strains of FIV.

The polymerase chain reaction (PCR) method was applied for measurement of the proviral DNA copy number of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMC) of cats experimentally and naturally infected with FIV. In experimentally infected cats except one cat infected with the Petaluma strain, FIV-specific DNAs were efficiently amplified with the PCR method under the conditions used in this study. In the naturally FIV-infected cats, the specific DNAs were also amplified. We established a quantitative method for measurement of proviral DNA copy number in PBMC from cats infected with TM2-type of FIV strains, and found that the number was variable among the six cats examined, ranging from 10(4.0) to 10(5.7) copies per 10(5) PBMCs. This method can be applicable to cats naturally infected with FIV of TM2-type. Proviral DNA quantitation developed here could be useful as an additional parameter to evaluate the relationships among the proviral load, immune response and development of the clinical symptoms, and to monitor efficacy of antiviral therapy in vivo.

Stable expression of the cDNA encoding the feline CD8 alpha gene.

The stable expression of the alpha chain of the feline cytotoxic T cell differentiation antigen (fCD8 alpha) on Crandell feline kidney cells (CRFK) was carried out by using an expression vector which contains the Rous sarcoma virus long terminal repeat and a neo resistant gene. After three rounds of cloning under G418 selection for over two months, the expression of the feline polypeptide was detected by human monoclonal antibody OKT8.

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.

Comparison of the Rev transactivation of feline immunodeficiency virus in feline and non-feline cell lines.

The Rev protein of feline immunodeficiency virus (FIV) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in cytoplasm via the Rev response element (RRE) at the end of env. To investigate the role of rev gene of FIV for the virus life cycle and cell tropism, we constructed the Rev expression plasmids, and functional activity of the Rev was assayed by using chloramphenicol acetyltransferase (CAT) assay system in feline and non-feline cell lines. Although the FIV Rev protein showed high transactivity to result in enhanced CAT production in a feline cell line, the productions of the CAT in non-feline cell lines were significantly lower than that in the feline cell line. These results indicate that specific cellular factor(s) present in feline cell line is required for the FIV Rev full-action and also suggest that the Rev action plays one of the important roles in determining the FIV cell tropism.

Seroepidemiological survey of feline retrovirus infections in cats in Taiwan in 1993 and 1994.

In order to investigate the prevalence of infections with three feline retroviruses feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and feline syncytial virus (FSV) in Taiwan, we collected a total of 75 blood samples from cats from veterinary hospitals, a breeding cattery and a homeless shelter in 1993 and 1994. We examined the presences of anti-FIV and FSV antibodies and FeLV-p27 antigen in these samples by the indirect immunofluorescence and/or enzyme-linked immunosorbent assays. All of the serum samples positive for FIV were obtained from homeless cats and the overall FIV positive rate was 4%. The overall positive rates of FSV and FeLV were 28% and 1.3%, respectively. From these results, together with previous seroepidemiological surveys by others, it was revealed that the prevalence of FIV and FeLV infections appeared to be lower in Taiwan than in the United States or Japan. In contrast, the prevalence of FSV infection in Taiwan was as high as that in Japan.

The feline herpesvirus type 1 ICP4 down-regulates feline immunodeficiency virus long terminal repeat (LTR)-directed gene expression via the C/EBP site in the LTR.

We investigated effects of feline herpesvirus type 1 (FHV-1) ICP4 on feline immunodeficiency virus (FIV) long terminal repeat (LTR)-directed gene expression by transient transfection assay in Crandell feline kidney cells. We demonstrated that FHV-1 ICP4 significantly stimulates the FIV LTR after introduction of site-specific mutation of the C/EBP site in the LTR, and the C/EBP site is sufficient to confer inhibitory effects by FHV-1 ICP4 on a heterologous promoter. These results indicate that FHV-1 ICP4 possesses both ability to transactivate FIV LTR-directed gene expression and to down-regulate the FIV LTR via the C/EBP site.

Persistence of high virus neutralizing antibody titers in cats experimentally infected with feline immunodeficiency virus.

The development of virus neutralizing (VN) antibody is one of the most effective host defense mechanisms against virus infection. In the present study, we developed a new VN assay against feline immunodeficiency virus (FIV) using a feline T-lymphoblastoid cell line, MYA-1 cells, based on inhibition of viral reverse transcriptase production. This assay is applicable to strains of FIV which can not infect CRFK cells. By using the assay, we examined long-term responses of VN antibody in cats experimentally infected with FIV. VN antibody titers increased progressively during first 30 weeks post inoculation and remained at high titers thereafter for 7 years of observation periods.

Eight-year observation and comparative study of specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) subtypes A and B: terminal acquired immunodeficiency syndrome in a cat infected with FIV petaluma strain.

Three specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) strains Petaluma, TM1 and TM2, respectively were observed for over 8 years. Without showing any significant clinical signs of immunodeficiency syndrome (AIDS) for 8 years and 4 months of asymptomatic phase, the Petaluma-infected cat exhibited severe stomatitis/gingivitis, anorexia, emaciation, hematological and immunological disorders such as severe anemia, lymphopenia, thrombocytopenia, and decrease of CD4/CD8 ratio to 0.075, and finally died with hemoperitoneum at 8 years and 8 months post-infection. Histopathological studies revealed that the cat had systemic lymphoid atrophy and bone marrow disorders indicating acute myelocytic leukemia (aleukemic type). Plasma viral titer of the cat at AIDS phase was considerably high and anti-FIV antibody titer was slightly low as compared with the other FIV-infected cats. In addition, immunoblotting analysis using serially collected serum/plasma samples of these cats revealed that antibodies against FIV proteins were induced in all the infected cats, however in the Petaluma-infected cat anti-Gag antibodies disappeared during the asymptomatic period. These results suggested that plasma viral load and anti-FIV Gag antibody response correlated with disease progression, and supported FIV-infected cats as a suitable animal model of human AIDS.

Serological evidence of Coxiella burnetii infection in wild animals in Japan.

One hundred and thirty-four (26%) of 511 sera from 11 wild animal species in eight prefectures in Japan had antibody titers to Coxiella burnetii by the enzyme-linked immunosorbent assay. High prevalences were observed in Japanese black bears (Ursus thibetanus) (78%), Hokkaido deer (Cervus nippon yesoensis) (69%), Japanese hares (Lepus brachyurus) (63%), Japanese deer (Cervus nippon centralis) (56%), and to some extent in Japanese monkeys (Macaca fuscata) (28%). A low prevalence (13%) was observed in nutrias (Myocastor coypus). Japanese serows (Capricornis crispus), wild rats (Muroides sp.), raccoon dogs (Nyctereutes procyonoides viverrinus), wild pigs (Sus scrofa leucomystax), and masked palm civets (Paguma larvata) had no detectable antibodies to C. burnetii. Thus, six of 11 wild animal species in Japan were exposed to C. burnetii. Based on the high prevalences in some species, they may be a potential source of infection to both domestic animal and human populations.