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1 School of Biological Sciences, University of Surrey, Guildford, GU2 5XH, UK
and2 Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0605, USA
Of four wild-type strains (Nakayama-original, SA14, 826309 and Beijing-1) of Japanese encephalitis (JE) virus that were passaged six times in HeLa cells (HeLa p6), two (Nakayama-original and 826309) became attenuated for mice. In the case of strain Nakayama-original, the virulence for mice was markedly reduced and attenuation was retained on passage in primary chicken embryo fibroblast, LLC-MK2 and C6/36 cells. The binding of non-HeLa-passaged Nakayama virus to mouse brain membrane receptor preparations could be differentiated from binding by Nakayama HeLa p6 virus, suggesting that the envelope (E) protein is involved in the attenuated phenotype. Both of the attenuated viruses can be distinguished from the virulent non-HeLa-passaged parental viruses by examination with E protein reactive vaccine and wild-type-specific monoclonal antibodies (MAbs). The vaccine-specific MAb V23, which is only reactive with the SA14 series of live vaccine viruses, recognized the HeLa cell-attenuated Nakayama-original and 826309 viruses, whereas two wild-type-specific MAbs (MAbs K13 and K39) lost reactivity. Comparison of the nucleotide sequences of the structural protein genes of the 826309 and Nakayama-original virulent parent and attenuated HeLa p6 viruses revealed that the viruses differed by 37 and 46 nucleotides coding for eight and nine amino acid mutations, respectively. However, other than one amino acid in the E protein, the membrane and E protein amino acid sequences of the two attenuated HeLa p6 viruses were identical.
* Author for correspondence. Fax +1 409 772 3606. e-mail ABARRETT@BEACH.UTMB.EDU
Present address: Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, Victoria, British Columbia, Canada VW8 3P6.
Present address: Department of Medicine, University of Cambridge Clinical School, Addenbrookes Hospital, Cambridge CB2 2QW, UK.
Present address: Bangladesh Livestock Research Institute, PO Box Savar Dairy Farm, Dhaka, Bangladesh.
Received 7 February 1995;
accepted 30 June 1995.
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