Human respiratory syncytial virus (HRSV) is a negative sense, single strand RNA virus that causes respiratory tract infection with common cold-like symptoms, which can be severe in children, immunocompromised, and the elderly. Even with 60 years of research, the need for vaccine and effective treatment has not been met. In this work, recombinant viruses have been generated which will be valuable in gaining a better understanding of HRSV subtypes, glycoprotein evolution, and the polymerase localization, which would contribute to HRSV vaccine and therapeutics development. The differences in the fitness of A and B antigenic subtypes of HRSV and how it affects the regional circulation pattern is not well understood. To study and compare the two subtypes, it is important to use clinically relevant recombinant viruses and to use animal models that best represent human infection. Using a wild-type virus strain (A11 and B05) from each HRSV subtype, a wild-type like recombinant (r) virus, rHRSVA11, and recombinant viruses expressing fluorescent proteins, rHRSVA11EGFP(5) and rHRSVB05dTom(5), were generated. Characterization of rB05 viruses demonstrated that the differences in the fluorescent protein expressed did not affect virus growth kinetics. To prepare for an experiment in cotton rats, recombinant HRSVs generated were used to infect cotton rat lung cells in vitro. With confirmation of infection of cotton rat lung cells by rHRSV, cotton rat co-infection experiment was planned for the recombinant A11 and B05 viruses and a microneutralization assay was developed for post-infection processing of the in vivo samples. The BA genotype of HRSV B subtype is a strain of HRSV B subtype containing a 60 nucleotide duplication in the glycoprotein (G) gene. HRSV BA genotype was first isolated in 1998 and has quickly become the predominant genotype circulating globally. Although a role of immune evasion by the strains of BA genotype has been suggested to explain this phenomenon, few studies have supported this hypothesis. To compare the HRSV B subtype virus with and without the duplication, rB05 virus lacking the duplication, rHRSVB05EGFP(5)GΔ60b, and containing an epitope tag within the duplication, rHRSVB05EGFP(5)Gmycb, were generated. A serial passage experiment was set up using rHRSVB05EGFP(5) and rHRSVB05EGFP(5)GΔ60b to understand the mutations that accumulate in the G protein gene of each virus. This will be valuable in setting up a similar experiment in the presence of immune pressure to understand the advantage that is conferred to the virus containing the duplication. Expression of Gmyc was confirmed in rHRSVB05EGFP(5)Gmyc infection, which validated that this virus can be used to study the HRSVB05 G protein and modifications in the duplicated region. The HRSV large (L) protein is essential in HRSV transcription and replication, but is difficult to study due to lack of immunologic reagents and challenges with purification. Recombinant viruses expressing reporter and polymerase fusion proteins have been generated and used for studying various other viral polymerases. Expression plasmids for HRSV L protein containing a reporter protein in its variable region 2 have been published. However, the modification resulted in downregulation in the function of the protein and rHRSV expressing modified L protein have not yet been published. In this study, rHRSVB05LVenus was generated to study the effects of modification of HRSV L protein variable region and the localization of HRSV L protein. LVenus protein in rHRSVB05LVenus infected cells was visualized by confocal laser scanning microscopy and the expression levels were examined by immunoblotting. rHRSVB05LVenus was compared to rHRSVB05EGFP(5) with unmodified L protein to show that modification of HRSV L protein had no effect on virus replication. Viruses had equivalent growth kinetics and were equally sensitive to ribavirin, a known HRSV inhibitor. The recombinant viruses generated in this study are valuable tools in answering questions that are difficult to pursue without clinically relevant recombinant viruses. Characterization of the rHRSVs demonstrated that these viruses will have many applications. In this study, viruses were characterized for the basic growth kinetics, expression of proteins of interest, and assay development. With these validated tools, questions such as the cause of the epidemiological pattern observed for HRSV A and B subtypes, the role of host immune response in advantage conferred to HRSV BA genotype, and the effects of inhibitors to formation of HRSV polymerase complex can be addressed. / 2018-10-31T00:00:00Z