Viral diseases are a bottleneck in the growth and sustainability of shrimp aquaculture worldwide. Despite the enormous economic losses caused by viral diseases, our understanding of the molecular basis of viral pathogenesis in shrimp is often rudimentary. The lack of an infectious viral clone has been a major hurdle to genetically manipulate the viral genome, study viral pathogenesis and develop viral vector to deliver therapeutic molecules like RNAi, microRNA etc. This project was initiated to use reverse genetics approach to construct an infectious cDNA clone of Laem Singh virus (LSNV) that can be genetically manipulated to develop a replication incompetent viral vector to deliver antiviral therapeutic molecules. LSNV causes growth retardation in Penaeus vannamei and P. monodon and contributed to major economic losses in Thailand. The disease has now spread to other countries in Asia. LSNV genome contains bi-segmented single-stranded RNA of 2206 and 1846 bases in length. RNA1 encodes an RNA-dependent RNA polymerase (RdRp), and RNA2 encodes a viral capsid protein. It was hypothesized that due to a relatively small genome and a simple genomic architecture, LSNV is a good model virus for genetic manipulation, studying viral pathogenesis, and developing viral vectors.
The full-length cDNAs of LSNV RNA1 and RNA 2 were cloned in a dual baculovirus expression vector, and bacmid DNA was transfected to insect cells, Sf9, to produce infectious virions. Upon transfection, LSNV RNA was detected by RT-PCR in Sf9 cells, and a transmission electron micrograph (TEM) revealed both recombinant baculovirus as well as mature virions of LSNV (Fig. 1). In order to prove infectivity, Sf9 cell homogenate containing infectious LSNV was mixed with a commercial diet and fed to juvenile P. vannamei at a rate of 7.5% of the biomass, once a day for 7 days. Using the RT-PCR, LSNV could be detected in gill tissue, a target tissue of LSNV at three- and six weeks post-viral feeding in the challenged shrimp.
The data revealed that the reverse genetics approach can be used to engineer infectious LSNV clone using a baculovirus expression vector and Sf9 cells. This opens up avenues to manipulate the viral genome to study pathogenesis at a molecular level and to develop replication incompetent LSNV-based viral vector by replacing the RdRp gene to deliver therapeutic molecules, such as RNAi and microRNA for antiviral therapy.