ウマヘルペスウイルス1型感染症の遺伝子診断とウイルスDNAの解析に関する研究 Genetic diagnosis for equine herpesvirus-1 (EHV-1) infection and viral genomic analysis of EHV-1 isolates in Japan and attenuated EHV-1 strains

博士論文

Equine herpesvirus-1 (EHV-1), a member of the subfamily Alphaherpesvirinae, has long been causally implicated in the occurrence of abortion respiratory disease, neonatal deaths and neurological disorders in horses. Antigenically related equine herpesvirus-4 (EHV-4), a member of the same subfamily, is primarily a causative agent of respiratory disease and, occasionally, abortion. Outbreaks of abortion due to EHV-1 have been observed almost every year and causes severe economic losses in the horse breeding industry. In attempts to prevent EHV-1 infection, inactivated vaccine was applied to pregnant mares but with little efficacy. A safe and effective vaccine is necessary to protect horses from diseases associated with EHV-1, particularly abortion. The purpose of the present study was to establish polymerase chain reaction (PCR) technique as rapid and sensitive method to the detection and identification of EHV-1 and EHV-4 to control the spread of the infections, and to investigate the electropherotype of Japanese field isolates of EHV-1 before and after vaccine use and to determine the variable regions of the viral genome among the viruses. In addition, the genomes of virulent and attenuated EHV-1 strains were compared to delineate the architectural differences as a step toward elucidating the potential relationship between pathogenicity and passage-induced alterations incurred at the genomic level and an estimated virulence factor was investigated by constructions of recombinant EHV-1 viruses for a live vaccine development. The results of the experiments were summarized as follows: 1. Detection and identification of EHV-1 and EHV-4 by PCR. A rapid method for detection and identification of EHV-1 and EHV-4 was developed using PCR. Primers for PCR were designed from aligned nucleotide sequences of glycoprotein B genes of both viruses to amplify specific regions for EHV-1 or EHV-4. By using type specific primer mixture, amplified fragments were identified as EHV-1 or EHV-4 in a one-step reaction. This technique was applied on specimens from aborted fetuses. There was complete accordance between the results of PCR and virus isolation. The PCR system could differentiate the two virus types rapidly in a one-step reaction. 2. The genomic diversity among EHV-1 strains isolated in Japan. The DNAs from nine field isolates of EHV-1 in Japan were analyzed by digestion with the restriction endonuclease Bam HI and Southern hybridization. EHV-1 DNA was cleaved to 20 fragments (fragments A - T) by Bam HI digestion. Comparing restriction profiles among the EHV-1 strains, there was no considerable difference between isolates before and after vaccine application, but some minor variations in the mobility of Bam HI fragments were observed. Eight fragments (Bam HI A, D, E, N, P, QR, S and T) were identified as variable fragments in molecular weight among the nine isolates by Southern blot analysis. The variability of the A fragment has not been reported yet. 3. Comparison of the genomes of attenuated EHV-1 strains with their parent virulent strain. To elucidate the virulence factor of EHV-1, the genomic architecture of the HHI strain, the first Japanese isolate of EHV-1, was compared with that of its four descendant strains obtained by serial passage in bovine kidney (BK) cells, i.e., BK77, BKI61, BK271 and BK343. In preliminary studies high-passaged BK271 and BK343 1ost virulence in colts, and in the present study digestion with restriction endonuclease Bam HI showed that the DNAs of BK271 and BK343 differed markedly from the DNA profiles of HHI, BK77 and BKI61. BK343 involved nine variable fragments, i.e., Bam HI A, B, D, E, N, P, QR, S and T. Among the genes located in the variable fragments, three genes (1, 24 and 71) were found to have changed via serial passage in the BK cells. Nucleotide sequence analysis revealed that the three genes contained a different number of copies of repeat sequences compared to those of the genes of HHI. In addition, the gene 71 had two point mutations which affected the amino acid sequences. Thus these three genes are thought to play an important role on the pathogenicity of EHV-1. 4， Characterization of gene 71 mutants of EHV-1 in cell cultures and the lungs of mice. The direct relationship of the gene 71 of EHV-1 with virulence was analyzed by constructing three recombinant EHV-1 viruses by homologous recombination. The gene 71 of EHV-1 HHI strain was replaced by the mutant gene 71 of BK343, and designated 3F-71. The β-galactosidase (β-gal) gene of Escherichia coli was inserted within the gene 71 coding sequences of HHI and designated β-71, Revertant virus, Res－71, was constructed by replacing the fusion gene 71 of β-71 with the gene 71 of HHI. Virus replications of these recombinant viruses in cell cultures were not affected but release of β-71 from infected cells was partially affected compared to the other viruses. After intranasal infection of mice with the viruses, lung virus titers were determined. The growth abilities of β-71 were reduced to the level of BK343 and the titers were about 100 times lower than those of 3F-71, Res-71 and HHI. These results indicated that inactivation of the gene 71 affected the virulence of EHV-1. However the mutant gene 71 of BK343 alone did not confer the attenuated nature to EHV-1.