Elucidation of the full genetic information of Japanese rubella vaccines and the genetic changes associated with in vitro and in vivo vaccine virus phenotypes.

Rubella is a mild disease characterized by low-grade fever, and a morbilliform rash, but causes congenital defects in neonates born from mothers who suffered from rubella during the pregnancy. After many passages of wild-type rubella virus strains in various types of cultured cells, five live attenuated rubella vaccines were developed in Japan. An inability to elicit anti-rubella virus antibodies in experimentally infected animals was used as an in vivo marker phenotype of Japanese rubella vaccines. All Japanese rubella vaccine viruses exhibit a temperature-sensitive (ts) phenotype, and replicate very poorly at a high temperature. We determined the entire genome sequences of three Japanese rubella vaccines (Matsuba, TCRB19, and Matsuura), thereby completing the sequencing of all five Japanese rubella vaccines. In addition, the entire genome sequences of three vaccine progenitors were determined. Comparative nucleotide sequence analyses revealed mutations that were introduced into the genomes of the TO-336 and Matsuura vaccines during their production by laboratory passaging. Analyses involving cellular expression of viral P150 nonstructural protein-derived peptides revealed that the N1159S mutation conferred the ts phenotype on the TO-336 vaccine, and that reduced thermal stability of the P150 protease domain was a cause of the ts phenotype of some rubella vaccine viruses. The ts phenotype of vaccine viruses was not necessarily correlated with their inability to elicit humoral immune responses in animals. Therefore, the molecular mechanisms underlying the inability of these vaccines to elicit humoral responses in animals are more complicated than the previously considered mechanism involving the ts phenotype as the major cause.

Protease and helicase domains are related to the temperature sensitivity of wild-type rubella viruses.

Wild-type rubella viruses grow well at 39°C (non-temperature sensitivity: non-ts), while vaccine strains do not (temperature sensitivity: ts). Histidine at position 1042 of the p150 region of the KRT vaccine strain was found to be responsible for ts, while wild-type viruses had tyrosine at position 1042 (Vaccine 27; 234-42, 2009). The point-mutated virus (Y1042H) based on the wild-type unexpectedly showed little reduction in growth at 39°C. In this report, several recombinant viruses were characterized, and point-mutated Y1042H together with the p90 region of KRT significantly reduced virus growth, compared to the parental wild-type virus. There was one amino acid difference at position 1497 of the helicase domain in the p90 region. Double mutation involving both positions 1042 and 1497 markedly reduced virus growth at 39°C, but single substitution at 1497 did not. The other vaccine strain (TO-336vac) was investigated, and serine at position 1159 of the protease domain in p150 was a crucial amino acid for ts and non-ts characteristics among four amino acid substitutions between TO-336vac and the wild-type. Our results suggest that protease and helicase domains in non-structural protein were consistent with ts phenotype, possibly related to the attenuation process of wild-type viruses.

Rubella virus-induced cytopathic effect in vitro is caused by apoptosis.

Rubella virus (RV) generally causes a mild disease but it is highly teratogenic when infection occurs during the first trimester of gestation. Under in vitro conditions, RV induces characteristic cytopathic changes on several cell lines, e.g. cell detachment from the monolayer and condensation of chromatin. The purpose of this study was to characterize RV-induced cell death and to determine the factors that might be involved in this process. Both acutely and persistently infected cells exhibited alterations characteristic of apoptosis, including DNA fragmentation, annexin V staining and reduced DNA content. UV-inactivated RV did not induce apoptotic cell death and expression of RV structural proteins in a transfected cell line was not sufficient to induce apoptosis, supporting the interpretation that replicating virus is necessary to provoke apoptosis. Both persistently infected and 24S-transfected cells retained their susceptibility to undergo apoptosis in response to either staurosporine or camptothecin. This indicates that RV does not block chemically induced apoptosis. The signals involved in RV-associated apoptosis appear to be independent of p53 and of the Bcl-2 gene family.

Photodynamic inactivation of rubella virus enhances recombination with a latent virus of a baby hamster kidney cell line BHK21.

Rubella virus is very sensitive to photodynamic action. When tested with 1.2 X 10(-5) M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m2, inactivation kinetics showed a linear single hit curve with a k value of 1.48 min-1. Photodynamic inactivation of rubella virus greatly enhanced recombination with a latent virus (R-virus) of baby hamster kidney BHK21 cells. In contrast, no hybrids were detected in lysates of the cells infected with either UV-treated or untreated rubella virus. Therefore, hybrid viruses were readily detected only in lysates of BHK21 cells infected with photodynamically treated rubella virus. Photodynamic damage of rubella virus genomes generated a new hybrid type (hybrid type 3) in addition to a previously described type 2 hybrid (formerly designated as HPV-RV variant). Although both of these hybrid types carry the CF antigens of rubella virus, plaque forming ability of type 3 hybrid is neutralized neither by anti-rubella serum nor by anti-latent virus serum while type 2 hybrid is neutralized by anti-latent virus serum.

In vitro effects of Rubella virus, strain RA 27/3, on human lymphocytes. II. Specific antigen stimulation in relation to Rubella haemagglutination inhibition antibodies.

Investigation has been made of the specific in vitro stimulation of human lymphocytes by live or inactivated rubella virus of the strain RA 27/3. The thymidine incorporation of lymphocytes from blood donors with different serological immunity against rubella was measured following incubation with the viral antigens. UV-inactivated rubella virus caused a moderate degree of stimulation of lymphocytes from seropositive individuals. On the other hand, live rubella virus stimulated lymphocytes from donors with low titres of anti-rubella HI-antibodies but not lymphocytes from donors with high antibody titres. Possible explanations for this discrepancy are discussed.

Interferon production in lymphocyte cultures after rubella infection in humans.

When stimulated with rubella virus irradiated with ultraviolet light, cultures of lymphocytes from individuals immune to rubella responded with production of 12-14 times more interferon than did cultures of lymphocytes from individuals susceptible to rubella. This property may be retained throughout life, since it was seen in individuals who had had rubella in their recent and remote pasts. Peak levels of interferon were detected six days after stimulation of lymphocytes with rubella virus antigen. In contrast, phytohemagglutinin induced production of interferon equally well in cultures of lymphocytes from persons immune and persons susceptible to rubella, with levels peaking three days after stimulation. Since there was a positive correlation among titers of hemagglutination-inhibiting antibody to rubella virus, incorporation of [14C]thymidine, and production of interferon after stimulation of lymphocyte cultures by rubella virus, interferon synthesis may be considered to be another measurable parameter of cell-mediated immunity in rubella.

Effects of actinomycin D and ultraviolet and ionizing radiation on Pichinde virus.

Actinomycin D (0.05 mug/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with (3)H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 x 10(6) to 8 x 10(6) daltons. This value is greater than the 3.2 x 10(6) daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods.