Identification and characterization of Boone Cardiovirus, a novel virus of laboratory rats

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cardioviruses are members of the Picornaviridae family capable of causing several forms of disease including myocarditis, encephalitis, diabetes, fetal death, and neuron degeneration. A wide range of hosts are susceptible to cardioviruses with rodents frequently being suspected as the viral reservoir following outbreaks of infection. In this study we discovered a novel Cardiovirus, provisionally designated Boone Cardiovirus (BCV). BCV was identified in the feces of laboratory rats using a pan picornavirus-PCR assay. Phylogenetic analysis revealed that BCV is a new species within the Cardiovirus genus distinct from both Encephalomyelitis Virus (EMCV) and Theilovirus. With these two species, BCV shares [less-than]45% amino acid identity in the polyprotein region and [less-than]50% amino acid identity in the capsid proteins. To assess the prevalence of BCV in laboratory rodents a sensitive hemi-nested RT-PCR assay was developed to screen fecal samples. Screening revealed that 20% of rat samples and 30% of research institutions tested positive for BCV. During the screening process a second isolate of BCV was also identified. Phylogenetic analysis of the second isolate determined it shared 91% amino acid identity with the original strain in the polyprotein region. Identification of additional BCV strains aids in the understanding of BCV variability and provides additional information for the development of comprehensive PCR and serologic screening assays. As no definitive clinical disease has been observed with BCV, a quantitative RT-PCR (qRT-PCR) assay was developed to screen rat tissues for sites of replication. BCV was found predominately localized to the gastrointestinal tract with the highest titers in the duodenum. Screening animals of various ages also revealed that BCV causes persistent infections in laboratory rats. In addition, to the RT-PCR and qRT-PCR assays the VP2 protein was expressed and purified for use in Western blot analysis of rat serum for preliminary serologic data on BCV. Collectively, the RT-PCR and Western blot assays provide a foundation for BCV detection and will enable researchers to screen animals prior to experiments. These assays will also make it possible to establish BCV-free rat colonies as virally infected research animals can confound and invalidate research findings. Preliminary data from infections of nude rats suggest that BCV is capable of replication and the immune system of immunocompetent animals plays a role in modulating infections as once T-cell are eliminated viral titers are approximately 4 logs higher. Furthermore, the discovery of BCV may lead to the establishment of research models that can provide valuable information including host-viral interactions during persistent infections.