Masahiko Kishi

Demonstration of human Borna disease virus RNA in human peripheral blood mononuclear cells

BDV naturally infects horses and sheep, and causes sporadic neurological disease. Serological evidence suggests an association of BDV, or a related virus, with specific psychiatric diseases in humans. Here, by using a nested RT‐PCR technique, we demonstrate that human BDV RNA is present in the PBMC of psychiatric patients. In an examination of a total of 60 patients from 5 wards of a hospital in Japan, the detection rate differed within each ward, ranging from 8% to > 50% (37% on the average). Of particular note was the finding that the human derived BDV sequences, which included deleted forms in about 23% of the positive samples, were slightly different from those derived from horse BDV. These results suggest urgent consideration of the measures to be taken to cope with the effects of blood transfusion. In addition, the detection of a high level of BDV in the PBMC of patients will help our understanding of the pathogenesis in the disease.

Prevalence of Borna disease virus RNA in peripheral blood mononuclear cells from blood donors

The presence of Borna disease virus (BDV) in peripheral blood mononuclear cells (PBMC) of 100 blood donors from Sapporo and 72 blood donors from Tokyo was examined using nested reverse transcriptase/polymerase chain reaction amplification with specific-primers for BDV p24. Anti-BDV p24 antibodies in the plasma of the 100 blood donors from Sapporo also were studied by enzyme-linked immunosorbent assay and by Western blot. BDV RNA was detected in 3 (4.2%) of the 72 PBMC samples from Tokyo, and in 5 (5%) of the 100 PBMC samples from Sapporo. In contrast, anti-p24 antibodies were found in only 1 (1%) of the donors from Sapporo. These results suggest that BDV infection in humans may be more widespread than previously thought.

Demonstration of Borna disease virus RNA in peripheral blood mononuclear cells from healthy horses in Japan

Borna disease (BD) is a progressive poliomeningoencephalomyelitis which occurs naturally in horses and sheep. Here, peripheral blood mononuclear cells (PBMC) derived from 57 healthy horses in Japan were examined by a nested reverse transcription-polymerase chain reaction to determine the prevalence of BD virus (BDV) infection. Seventeen (29.8%) of the samples were positive by this examination and the specificity of the amplified product was confirmed by hybridization with authentic oligomer probes. About 60% of the BDV RNA-positive individuals also showed seropositivity by Western blotting. This report is the first for the demonstration of BDV RNA in PBMC of healthy horses, as well as the first on the BDV infection in horses in Japan. Thus, BDV may be more widespread in healthy horses over the world as well as in Japan and the detection of BDV RNA in PBMC at a high rate indicates that the disease might develop in a part of the carriers only after long-incubation period.

Detection of anti-Borna Disease Virus (BDV) antibodies from patients with schizophrenia and mood disorders in Japan

The relationship between infection with the Borna Disease Virus (BDV) and the clinical symptoms of schizophrenia and mood disorders (DMS-IV) was investigated. Western blotting techniques were used to examine anti-p10-BDV antibodies in serum from 32 patients with schizophrenia and 33 patients with mood disorders in Japan. The results showed that 1 out of 25 controls (4.0%), 7 out of 32 patients with schizophrenia (21.9%) and 9 out of 33 patients with mood disorders (27.3%) were positive for anti-BDV-p10 antibodies. Compared with levels of anti-BDV-p10 antibodies in controls, the production of anti-BDV-p10 antibodies failed to show a statistically significant relationship with schizophrenia but did show a significant relationship with mood disorder. The subgroup of schizophrenia patients with positive syndromes had a non-significantly higher frequency of anti-BDV-p10 antibodies than the subgroup of patients with negative syndromes. Similarly, the production of anti-BDV-p10 antibodies was non-significantly higher among patients with the unipolar subtype of mood disorder than in those with the bipolar subtype.

Cells surviving infection by human immunodeficiency virus type 1 : vif or vpu mutants produce non-infectious or markedly less cytopathic viruses

Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non-infectious or less cytopathic virus.

Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells

Long‐term, persistent infection by HIV‐1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV‐1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV‐1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi‐quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV‐1‐seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus‐infected PBMC. These data are consistent with a mechanism of HIV‐1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.

Characterization of the P Protein-Binding Domain on the 10-Kilodalton Protein of Borna Disease Virus

ABSTRACT The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S3 and L16 are important for the interaction.

Human immunodeficiency virus type 1vif, vpr, andvpu mutants can produce persistently infected cells

SummaryA series of human immunodeficiency virus type 1 (HIV-1) mutants invif, vpr, vpu, andnef were constructed from an infectious plasmid (pNL 432) containing the full-length HIV-1 DNA by frameshift mutations. The capacities for replication and cell killing of these mutant viruses were examined in a clonal cell line (M 10) isolated from HTLV-I-transformed MT-4 cells. In all cases, the mutant viruses replicated, expressed HIV-1 antigens, and induced drastic cytopathic effects. However, some M 10 cells survived infection withvif, vpr, andvpu mutant viruses and became persistently HIV-1-infected, whereas no cells survived infection with thenef mutant as well as the wild-type virus. The HIV-1 particles produced from the surviving cells after infection with thevif, vpr, orvpu mutant viruses were fully replicative in M 10 cells without apparent cytopathic effects.

Sequence Analysis of an Asian Isolate of Minute Virus of Canines (Canine Parvovirus Type 1)

Minute virus of canines (MVC), also known as canine parvovirus (CPV) type 1, is an autonomous parvovirus that infects domestic dogs worldwide and responsible for clinical problems of neonates and pregnant bitches. It was preliminary described that MVC is antigenically as well as genetically different from CPV type 2 that emerged later. However, much of MVC is still obscure since only a limited number of MVC isolates have been available for the study. MVC infections of Japanese and Korean dogs were epidemiologically studied in the previous study, and several MVC isolates could be cultivated in vitro. In the present study an almost full-length nucleotide sequence of a Korean MVC strain HM-6 genome was obtained and comparatively analyzed with those of the American MVC strain GA3 characterized recently. Genome structure of the HM-6 strain was similar to the GA3 strain showing 96.4% identity at the nucleotide sequence. Each of the deduced amino acid sequences of NS1, NP-1, and VP1/2 showed homology of 96.5%, 92.5%, and 97.5% between the HM-6 and GA3 strains. When compared with other parvovirus species, the HM-6 strain was most closely related to bovine parvovirus (BPV) as described for the GA3 strain. These results suggest that MVC together with BPV can possibly be classified into a new clade in the Parvovirinae subfamily.

STRUCTURE AND EXPRESSION OF THE MOUSE S10 GENE

We previously reported the cloning of a human S10 cDNA which encodes a small GTP-binding protein belonging to the Rab subfamily. Here we describe a mouse S10 cDNA and its genomic structure. Mouse S10 is 92.3% homologous at the nucleotide level and 98.3% identical at the amino acid level compared to human S10. The mouse S10 gene is comprised of two exons and a single intron. Northern blotting of tissue RNAs indicates that the S10 gene is predominantly expressed in brain.