Antigenic variation of respiratory syncytial virus may play an important role in the development of disease in newborn seropositive infants and allow reinfection of older children to occur. Random genetic and antigenic heterogeneity among circulating RSV isolates may be an important obstacle to vaccine development. In addition live attenuated viruses used as vaccines are susceptible to the same type of instability and production of these vaccines could result in virus strains with altered immunogenic properties when they are compared to the parent or seed virus. Previously, this laboratory demonstrated the ability to amplify RSV-F gene sequences by the polymerase chain reaction. Direct sequencing of the PCR amplified cDNA proved to be a rapid, reliable and accurate method for determining the F gene sequences and mutations in the F glycoprotein of 14 monoclonal antibody resistant mutant viruses were identified. The biological activity of the MARMs in vivo was also determined as a part of this project. Replication of the MARMs in the upper and lower respiratory tract of cotton rats was compared to the replication of the the parental A2 strain. Several of the F MARMs were attenuated in the lower respiratory tract suggesting that specific mutations in the F glycoprotein may contribute to the attenuation phenotype of these viruses. Furthermore, these viruses can be considered as candidates for a live attenuated viral vaccine. In addition some of the attenuated MARMs had altered reactivity with G specific monoclonal antibodies in ELISA suggesting that mutations in this glycoprotein might also contribute to the attenuation phenotype. In order to identify mutations in the G glycoprotein of these viruses, the sequence for the G gene of the parental A2 strain has been determined using G specific primers and the methods described above. During the coming year we hope to more completely characterize the attenuated MARMs. First, the sequence for the G glycoprotein will be determined. Second, in collaboration with Dr.Brian Murphy, LID, NIAID, replication of the MARMs in primates will be evaluated.
