In vitro and in vivo infection of snakehead rhabdovirus (SHRV) and recombinant chimeric SHRVs in Penaeus vannamei to evaluate availability as a vector for heterologous protein expression

Abstract

The development of viral vectors for heterologous protein expression in shrimp has been limited by the absence of stable shrimp cell lines capable of supporting consistent viral replication. This study addresses this critical challenge by exploring snakehead rhabdovirus (SHRV) as a potential vector that can propagate in fish-derived cell lines and simultaneously infect shrimp. Recombinant SHRVs (rSHRVs) expressing foreign proteins, including chimeric variants with glycoprotein substitutions from vesicular stomatitis virus (VSV) or spring viremia of carp virus (SVCV), were rescued through reverse genetic technology. In vitro experiments demonstrated that both native and chimeric rSHRVs effectively infected primary cultured lymphoid organ cells from Penaeus vannamei, resulting in the successful expression of reporter proteins. In vivo bioluminescence imaging (BLI) confirmed systemic expression of the foreign protein, with rSHRV-ALuc2 showing efficient infection and spread in shrimp tissues. Among the chimeric variants, rSHRV-ALuc2-G_VSV exhibited rapid initial infectivity but did not significantly outperform native rSHRV in prolonged replication. The use of SHRV, which propagates robustly in fish-derived EPC cells while maintaining infectivity in shrimp, represents a significant step towards overcoming the lack of shrimp cell lines for the development of heterologous protein expression tools using recombinant viruses. In the non-invasive route, while oral administration was unsuccessful partially due to viral instability in feed, reverse gavage demonstrated partial success with detectable viral loads but no bioluminescence signals. This study highlights the potential of SHRV as a versatile viral heterologous protein expression vector for shrimp. Further research is required to enhance infection and expression efficiencies and develop practical, non-invasive methods for broader application in aquaculture.