|SILVERIA, SIMONE - Universidade Federal Do Rio Grande Do Norte|
|WALZ, PAUL - University Of Alabama|
|CANAL, CLAUDIO - Universidade Federal Do Rio Grande Do Norte|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/31/2019
Publication Date: 8/1/2019
Citation: Silveria, S., Falkenberg, S.M., Dassanayake, R.P., Walz, P.H., Ridpath, J.F., Canal, C.W., Neill, J.D. 2019. In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay. Virology. 536:101-109. https://doi.org/10.1016/j.virol.2019.07.029.
Interpretive Summary: Bovine viral diarrhea virus (BVDV) can be separated into two viral species, BVDV type 1 and BVDV type 2, due to it’s genetic variability. The two viral species can be further segregated into several subgenotypes, but the most commonly observed in the US are BVDV-1a, 1b, and 2a. It is important to determine if the genetic diversity of BVDV isolates is leading to increased prevalence or emergence of specific subgenotypes. Two BVDV studies using simultaneous exposure to calves persistently infected with either BVDV-1a, 1b or 2a subgenotypes reported that the 2a strain was most frequently isolated from exposed animals. A laboratory model was developed that allows comparison of infection at the single cell level for BVDV-1a, 1b and 2a to compare viral competition between strains. Similar to the exposure studies in the field, when cell cultures were simultaneously inoculated with multiple BVDV strains, BVDV-2a outcompeted BVDV-1a and BVDV-1b strains. This technique provides the opportunity to evaluation of interactions between strains as a model for predicting which strains could predominate in the field. This is an important tool to help determine potential BVDV vaccine targets.
Technical Abstract: Ruminant pestiviruses are globally distributed pathogens that are responsible for a broad range of clinical presentations that lead to substantial economic losses. Previous observations in simultaneous in vivo experimental challenge and transmission studies suggest that competitive exclusion exists among pestivirus strains. The aim of this study was to determine if an in vitro model could be developed that mimics these in vivo observations. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in two cultured cell lines was compared to the results from a previous in vivo transmission study that used the same viral strains. A PrimeFlow RNA assay, immunofluorescent microscopy, RT-qPCR and DNA sequencing were used to compare viral strain infectivity rates. Similar results were observed in this in vitro study, as was observed in the earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains in dual and triple infections, for both cell lines used. The PrimeFlow RNA assay allowed the multiplex detection of pestivirus RNA at single cell level. Further, this assay proved to be an effective tool for the studying virus competition, the dynamics of viral co-infections, the potential interactions among strains in the field, and differences in replication dynamics of viruses in different cell types. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.