Type 2 dengue virus 野生株,並びに野生株より分離したClone株の性状解析について

Abstract

Wild type strains of type 2 dengue virus which were isolated from viremic human sera of Dengue hemorrhagic fever (DHF) or Dengue schock syndrome (DSS) patients in Burma, and proto-type strain were investigated these virulence, plaque distribution, temperature sensitivity and influence of characteristics by passage with mouse and Vero cells to find out available marker for evaluation of pathogenicity.Additionaly, pathomorph ology of suckling mouse brains after intracerebranl (ic) or intraperitoneal (ip) inoculation were also investigated to get indicator which determines the severity of DHF and DSS was discussed. And following results were obtained.1. The virulence of proto-type strain Tr 1751 to mouse was 108LD50/ml after ic inoculation. The virulence of parent strains of BR 006 and BR 116 were less than 102LD50/ml after ic inoculation. However, virulence of wild type viruses to mouse was increased stepwise and 50%lethal dose was increased up to 4 logo ic LD50/m1 after 10th passage in mouse brain. And virulence of 2 log10 ip LD50/ml was showed after same passage level.2. Wild type strains BR 006 and BR 116 were genetically mixed, th at is, wild type viruses were demonstrated large (L), medium (M) and small (S) size plaques, while proto-type strain Tr 1751 was demonstrated uniform small plaques by plaque assay method with Vero cells.3. The plaque distribution of Tr 1751 was demonstrated as uniform small plaques at every passage level, while BR 006 and BR 116 were demonstrated that 90% of plaques showed S size after 10th passage in mouse brain.4. The virulence to mouse followi ng ic and ip inoculation were compared between L and S clone which were purified from parent strain of BR 006. Maximum titer of S clone strain was higher (2 log10 ic LD50/ml) than L clone strain.5. Plaque size of S cloned virus was not influe nced by passage with mouse and Vero cells but plaque size of L cloned virus became small, and approximately 100% of plaques were S size more than 5th passage.6. L clone strain was not indicated temperature sensitive (Ts), but S clone strains were sensitive. This was suggested that mouse virulent, S size plaque forming and Ts positive virus may be a mutant strain, which developed from parent strain of wild type viruses.7. Infected suckling mouse brain following ic inoculation with wild type viruses showed that mononuclear cells infiltration in subarachnoidal space and marked edema and necrosis formation in piamater and cortex after 10th passage by hematoxylin-eosin staining.8. Electron micrographic study of Vero cells and suckling mou se brains following ic and ip inoculation with Tr 1751, BR 006 and BR 116 showed the same findings that large numbers of enveloped electrondense 50 nm virus particles were observed in distended cisterna of endoplasmic reticulum mostly in the perinuclear region. Type 2 dengue viruses acquired their envelope from the host cell membrane and mature virions were completed through the budding.From these results, followings are concluded. Type 2 dengue virus wild type s trains, isolated from DHF or DSS patients were originaly composed of L plaque virus, which was not temperature sensitive and avirulent to mouse. And passage with suckling mouse and Vero cells, which are different from the original host, was not available for virological analysis of virulence to human being and severity of DHF and DSS.