Passage of Japanese encephalitis virus in HeLa cells results in attenuation of virulence in mice.

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.

Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis.

The virulence of a yellow fever (YF) virus (P-16065) isolated from a fatal case of vaccine-associated viral encephalitis was investigated. P-16065 appeared identical to its parent vaccine virus (17D-204 USA, lot 6145) when examined with monoclonal antibodies except that YF wild type-specific MAb S24 recognized P-16065 but not 17D-204 USA 6145. Thus, a mutation of at least one epitope on the envelope (E) protein had occurred. Unlike 17D-204 USA 6145 and other 17D vaccine viruses, P-16065 was neuroinvasive and virulent for mice after intranasal inoculation, and neurovirulent for monkeys after intracerebral inoculation. The E protein of P-16065 differed from 17D-204 USA by two amino acids at positions 155 and 303. Changes at amino acid position 155 are found in other YF vaccine viruses that are not neurovirulent, and it is therefore postulated that the change at position 303 is involved in the alteration of the phenotype of P-16065 and may be important for virulence of YF virus.

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.

Immunogenicity of experimental live attenuated Japanese encephalitis vaccine viruses and comparison with wild-type strains using monoclonal and polyclonal antibodies.

A total of 105 hybridomas secreting anti-Japanese encephalitis (JE) virus monoclonal antibodies (mAbs) were generated from six fusions against four strains of JE virus: wild-type strains SA14 and G8924 and live attenuated vaccines SA14-5-3 and SA14-14-2 (PDK-9). Most of the mAbs (87%) elicited haemagglutination inhibition activity while only a minority (24%) elicited neutralization. None of the mAbs prepared against SA14-5-3, parent of SA14-14-2, elicited neutralization while the only mAbs prepared against SA14-14-2 that elicited neutralization recognized flavivirus cross-reactive epitopes. In comparison, mAbs raised against wild-type strains showed that a spectrum of epitopes with different specificities, including JE type-specific epitopes, elicited neutralizing activity. Two mAbs, prepared against SA14-5-3 virus, were found to be vaccine-specific and five, prepared against strains SA14 and G8924, were wild-type-specific.

Yellow fever 17DD vaccine virus is temperature sensitive when grown in mosquito C6-36 cells.

Twenty-three yellow fever (YF) vaccine viruses and three wild-type YF viruses were propagated independently in human adenocarcinoma (SW13) cells and mosquito Aedes albopictus C6-36 cells. The three YF 17DD vaccine viruses were found to be slightly temperature sensitive (ts) at 39.5 degrees C versus 37.0 degrees C (efficiency of plaquing 0.04 to 0.1) following propagation in C6-36 but not in SW13 cells. A plaque-purified preparation of the 17DD vaccine manufactured in Brazil was ts when grown in C6-36 cells and remained ts when passaged back into the SW13 cell line.

Antigenic characterization of the live attenuated Japanese encephalitis vaccine virus SA14-14-2: a comparison with isolates of the virus covering a wide geographic area.

In China, a live attenuated Japanese encephalitis (JE) vaccine, based on strain SA14-14-2, has been derived from the wild-type strain SA14 as an alternative to the current inactivated vaccines such as Nakayama-NIH and P-3. SA14-14-2 has been characterized using monoclonal antibodies derived in mice, using HAI and neutralization tests, and compared with other Chinese live vaccine clones and 14 wild-type strains of JE virus. Wild-type strain SA14 was found to be a poor immunogen and antigenically distant from all other viruses examined. The vaccine derivatives SA14-5-3 was more immunogenic than its wild-type parent, while SA14-14-2 (derived from SA14-5-3) was more immunogenic than SA14-5-3 and elicited a cross neutralizing antibody response. Our studies indicated that JE virus strain Nakayama elicited as good a neutralizing antibody response in hyperimmunized mice as the SA14-14-2 vaccine clones grown in either primary hamster kidney (PHK) or primary dog kidney (PDK) cells. A single dose of live SA14-14-2 (PHK) also elicited a good antibody response. Antigenic variation between wild-type and vaccine clones of JE virus were detected but were not considered significant in terms of controlling JE virus infections by vaccination.

In vitro homotypic and heterotypic interference by defective interfering particles of West Nile virus.

Defective interfering (DI) particles of the flavivirus West Nile (WN) were generated after as few as two high multiplicity serial passages in Vero and LLC-MK2 cells. Six cell lines (Vero, LLC-MK2, L929, HeLa, BHK-21 and SW13) were used to assay interference by DI particles in a yield reduction assay. Interference was found to vary depending on the cell type used. The highest levels of interference were obtained in LLC-MK2 cells, whereas no detectable effect was observed in BHK-21 and SW13 cells. The ability of DI virus to be propagated varied depending on the cell line used; no detectable propagation of DI virus was observed in SW13 cells. Optimum interference was obtained following co-infection of cells with DI virus and standard virus at a multiplicity of 5. Interference between DI and standard viruses occurred only when they were co-infected or when cells were infected with DI virus 1 h before standard virus. Investigation of heterotypic interference by DI particles of WN virus strains from Sarawak, India and Egypt revealed that interference was dependent on the strain of WN virus or flavivirus used as standard virus. A measure of the similarity between five strains of WN virus and other flaviviruses was made on the basis of interference by DI viruses, and was found to be similar to that based on haemagglutination inhibition tests using a panel of monoclonal antibodies.