Hajime Tsujimoto

Sequence homology of the simian retrovirus genome with human T-cell leukemia virus type I.

A retrovirus highly related to human T-cell leukemia virus type I (HTLV-I) was isolated from a T-cell line established from a seropositive pig-tailed monkey and the provirus genome was molecularly cloned using HTLV-I as a probe. The monkey virus (STLV) had the genomic structure of the LTR-gag-pol-env-pX-LTR. Analysis of the env-pX-LTR region revealed the 90% homology of the nucleotide sequence with that of HTLV-I in each region. This high homology of the sequence indicates that STLV is a member of the HTLV family, but apparently different from HTLV-I. This suggests that the possibility of recent interspecies transmission from monkeys to humans in the endemic area is very small. From its similarity to HTLV, STLV should be useful as an animal model in studies on natural HTLV infection and leukemogenesis of HTLV in humans.

Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin).

Inactivated influenza vaccine was administered intranasally to BALB/c mice together with an adjuvant (cholera toxin B subunit [CTB] supplemented with a trace amount of the whole toxin, CTB*) and its ability to induce innate immunity and confer protection against influenza was examined. Nasal wash virus titres 3 days after inoculation of homologous viruses were measured as an index of the ability of the vaccine to confer protection in mice immunized with either CTB*-combined vaccine or CTB* alone 1-21 days previously. The results were as follows. (1) Partial but significant reduction of the nasal-wash virus titres (prevention) was detected beginning 3 days after the vaccination, that is, 2 days earlier than the appearance of both virus-specific antibody-forming cells (AFCs) in the nasal-associated lymphoid tissue (NALT) and virus-specific IgA antibody responses in the nasal washes of mice immunized with the CTB*-combined vaccine. (2) The protection, detected on day 3 and peaking on day 5 but lost by day 21, was also conferred in mice immunized with CTB* alone. (3) The non-specific prevention was detected at doses of more than 0.3 microg of CTB*/mouse. (4) The nonspecific protection beginning 3 days after the immunization involved the enhanced expression of cytokine mRNAs (IL-15 and IL-18), considered responsible for natural killer (NK) cell activation, by the non-T cell populations in the NALT. (5) Normal NALT cells, when cultured in vitro with CTB*, secreted IL-1beta within a few hours in culture. These results demonstrate that the CTB*-combined vaccine, when given intranasally into mice, can confer nonspecific protection against influenza beginning 3 days after the vaccination and that CTB* also possessed this ability to confer protection non-specifically and temporarily by inducing the secretion of IL-1beta, one of the most important cytokines that initiates both innate and adaptive immunity, and also NK cell activity.

Detection and characterization of simian retroviruses homologous to human T-cell leukemia virus type I

Lymphoid cell lines were established from five different species of monkeys which were positive in antibodies cross-reactive with human T-cell leukemia virus type I (HTLV-I) and were shown to contain provirus sequences homologous to HTLV-I. Gene-specific probes of HTLV-I, gag, pol, env, pX, and LTR, hybridized efficiently with monkey DNAs from these cell lines under stringent conditions, indicating that the proviruses are very similar to HTLV-I along with whole viral genomes. However, the preliminary restriction mapping turned out the difference between simian retroviruses and HTLV-I and also among simian retroviruses. These findings suggest a common ancestor of simian and human retroviruses, but exclude the recent interspecies transmission between monkeys and humans.

Molecular Analysis of Chitin Synthase 1 (CHS1) Gene Sequences of Trichophyton mentagrophytes Complex and T. rubrum

Abstract. Nucleotide sequences of chitin synthase 1 (CHS1) gene of dermatophytes, Arthroderma benhamiae, A. simii, A. vanbreuseghemii, Trichophyton mentagrophytes var. interdigitale (T. interdigitale), and T. rubrum were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these five dermatophytes showed more than 90% similarity between the species. The phylogenetic analysis of their sequences revealed that A. benhamiae, A. simii, A. vanbreuseghemii, and T. rubrum were genetically distinct from one another, but T. interdigitale was genetically very close to A. vanbreuseghemii. On the other hand, a specific restriction endonuclease site of HinfI was present in the CHS1 gene fragment of T. rubrum but not in those of A. benhamiae, A. simii, A. vanbreuseghemii and T. interdigitale. The molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution.

Phylogenetic analysis of 8 dermatophyte species using chitin synthase 1 gene sequences

Summary. Nucleotide sequences of chitin synthase 1 (CHS1) gene of eight species of dermatophytes, Arthroderma benhamiae, A. fulvum, A. grubyi, A. gypseum, A, incurvatum, A. otae, A. simii and A. vanbreuseghemii were obtained and analysed for their phylogenetic relationship. A 600‐bp genomic DNA fragment of the CHS1 gene was amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these eight dermatophyte species showed more than 85% similarity between the species. The phylogenetic analysis of their sequences revealed three clusters, the first cluster consisting of A. benhamiae, A. simii and A. vanbreuseghemii, the second cluster consisting of A. fulvum, A. gypseum and A. incurvatum, and the third cluster consisting of A. grubyi and A. otae. The phylogenetic analysis of CHS1 gene in this study will provide useful information for classification and understanding the evolution of these dermatophyte species.

Molecular cloning of a feline leukemia provirus integrated adjacent to the c-myc gene in a feline T-cell leukemia cell line and the unique structure of its long terminal repeat☆

This paper reports the molecular cloning of a rearranged c-myc region from the FT-1 cell line, which was derived from a spontaneous feline T-cell leukemia carrying the feline leukemia virus (FeLV). An abnormal c-myc EcoRI fragment of about 18 kilobases, detected by Southern blotting, was molecularly cloned from the DNA of the FT-1 cell line. The c-myc rearrangement in FT-1 was due to direct integration of the FeLV provirus genome immediately upstream of the c-myc gene in the opposite transcriptional orientation. Nucleotide sequencing showed that the LTR of this provirus had three copies of an enhancer-like sequence, unlike the sequences of FeLVs reported previously, which have only a single copy of this enhancer-like sequence.

Genetic heterogeneity of env gene of feline immunodeficiency virus obtained from multiple districts in Japan

Feline immunodeficiency virus (FIV) infection is widespread in many countries. FIV isolates have been classified into five distinct subtypes, A, B, C, D and E based on their env gene sequences. Several reports indicate that most of the FIVs isolated in Japan belong to subtype B which includes the first Japanese isolate, TM2 strain. To examine the distribution of FIV subtypes in Japan, proviral DNA sequences of the env gene were directly amplified by nested PCR from FIV-infected cats that had been kept in multiple districts throughout Japan. Phylogenetic analysis of the 11 strains showed that four FIV subtypes, A, B, C and D, were present in Japan. Among these subtypes, subtypes B and D were the two most common subtypes in Japan, and they were mainly distributed in the eastern and western parts of Japan, respectively. The present study provides information that is fundamental for development of a vaccine to protect against FIV infection in cats.

The First Isolation of Arthroderma benhamiae in Japan

A clinical isolate of Trichophyton mentagrophytes from rabbit was examined by polymerase chain reaction (PCR) analysis and a mating experiment. The species‐specific primers designed from the nucleotide sequences of the chitin synthase 1 (CHS1) gene in the teleomorph of Arthroderma benhamiae amplified a fragment from genomic DNA samples of A. benhamiae and the clinical isolate but not from those of A. simii and A. vanbreuseghemii. On the other hand, the species‐specific primers of A. simii and A. vanbreuseghemii did not amplify any fragment from the genomic DNA of the clinical isolates. When the isolate was respectively crossed with (+) or (‐) tester strains of A. benhamiae, A. simii and A. vanbreuseghemii, ascospores were produced in the crossing with the A. benhamiae (+) strain. Therefore, the isolate was identified to be A. benhamiae (‐), confirming the result of molecular analysis. This is the first report on the isolation of A. benhamiae in Japan.

IgE sensitivity and cross-reactivity to crude and purified mite allergens (Der f 1, Der f 2, Der p 1, Der p 2) in atopic dogs sensitive to Dermatophagoides mite allergens.

The present study investigates IgE-reactivity to crude and purified mite allergens by intradermal skin test (IDST), Immunodot method, and ELISA in atopic dogs sensitive to mite allergens, as well as the allergenic cross-reactivity between Dermatophgoides (D) farinae (DF) and D. pteronyssinus (DP) in dogs by IgE-ELISA inhibition. IDST and Immunodot method for crude mite allergens were performed for atopic dogs and 16 atopic dogs showed sensitivity to mite allergens. Of the 16 dogs, all dogs had anti-DF IgE and 11 had anti-DP IgE. We measured specific IgE to purified major allergens (Der f 1, Der f 2, Der p 1, Der p 2). Of the 16 atopic dogs, six had anti-Der f 1 IgE and seven had anti-Der f 2 IgE. Similarly, of the 16 dogs, six had anti-Der p 1 IgE and seven had anti-Der p 2 IgE. However, eight dogs had no specific IgE to these mite allergens. These dogs may be sensitive to other major mite allergens except Der 1 and Der 2. In the dogs that had both anti-DF and DP IgE, IgE binding to DF was greatly inhibited by DP, and reciprocal inhibition was observed. Based on these data, it appears that there is a strong cross-reactivity between DF and DP in dogs. Similarly, a cross-reactivity between DF and DP in purified allergens was also observed. IDST and Immunodot method are useful methods for the diagnosis of atopic diseases in dogs, and ELISA is a useful method for further investigation of IgE-reactivity for the allergens.

High prevalence of Borna disease virus in domestic cats with neurological disorders in Japan

A total of 15 (T-1-T-15) domestic cats with neurological disorders in Tokyo area were examined for association with Borna disease virus (BDV). None had detectable antibodies to feline immunodeficiency virus (FIV), feline leukemia virus, feline infectious peritonitis virus and Toxoplasma gondii, and only cat T-8 had detectable antibody to FIV. Serological and molecular epidemiological studies revealed a significantly high prevalence of BDV infection in these cats: antibodies against BDV p24 and/or p40 proteins in 10/15 (66.7%) and p24 and/or p40 RNA in peripheral blood mononuclear cells in 8/15 (53.3%). Further, in situ hybridization and immunohistochemistry analyses of the autopsied brain samples derived from one of the cats (T-15) revealed BDV RNA predominantly in neuronal cells in restricted regions, such as olfactory bulb and medulla of cerebrum. Thus, BDV is present in Japanese domestic cats with neurological disorders at a high prevalence.