TOWARDS AN UNDERSTANDING OF PRV TRANSPORT AND SPREAD USING DUAL-COLORED VIRIONS

Abstract

Pseudorabies virus (PRV) is an alphaherpesvirus that is closely related to human herpes simplex virus. Although PRV infects many mammals, no human cases of PRV infection have been reported. PRV is therefore an ideal candidate for laboratory research of alphaherpesvirus infections. The mechanisms involved in the PRV infectious cycle have not been completely resolved. We performed two sets of in vitro experiments to further elucidate the dynamics of PRV axonal transport and spread in epithelial cells. We modified a single-color system for tracking of neuronal PRV infections to distinguish entering from egressing virus particles. We allowed the capsid-tagged strain PRV 180 to replicate on PK15 cells expressing mNeonGreen-VP26, yielding PRV 180G. As PRV 180G has a non-genetically encoded, secondary fluorescent tag, it can be distinguished from its red progeny. We see up to 92% of exocytosed particles incorporate mNG-VP26 at 14 hpi. However, integration of mNG-VP26 into PRV affects axonal transport efficiency. Dual-colored PRV shows promise as a tool for detailed analysis of PRV transport and for investigation of the fate of virion components during replication. In accordance with prior research, we find PRV 151 (diffusible GFP) plaques spread through PK15 monolayers 36% faster than the ~4.5 hour replication cycle would permit.

To gain insight into routes of enhanced spread, we studied PRV transport through PK15 cell monolayers. We detected PRV 151 in the medium below non-permeable PK15 cells layers as early as one hour post-infection. These results suggest that productive PRV transmission through epithelial cells can occur through a non-replicative and active mechanism, which greatly impacts our understanding of PRV infection in vivo.