Engineering an Infectious cDNA Clone of an RNA Virus, Laem Singh virus Infecting Shrimp (Penaeus vannamei)

Abstract

Shrimp farming has been expanding rapidly due to a high demand of seafood and declining wild fisheries for overfishing. Today, viral disease is the primary impediment in the growth and sustainability of shrimp farming worldwide. There are over thirty viral diseases known to infect shrimp. While there are some that cause lethal infection resulting in a large-scale mortality, there are others that cause apparently benign infection, but result in significant growth retardation and production loss. Laem Singh virus (LSNV) is one of those viruses that cause growth retardation and major losses in yield. LSNV is known to be associated with Monodon Slow Growth Syndrome (MSGS) in Black Tiger shrimp, P. monodon in Thailand from 2000 to 2002. The disease caused an economic losses of about 300 million USD (Chayaburakul et al., 2004a) and led to the introduction of an exotic species of penaeid shrimp, Pacific white shrimp, Penaeus vannamei for farming to replace P. monodon in Thailand. MSGS eventually spread from Thailand to other shrimp producing countries in Asia and there is no therapeutic to control MSGS. Preventing introduction of the disease through biosecurity remains the only means to control MSGS. There is a need to understand the host-virus interactions in MSGS with a goal to develop an antiviral therapy. In this project, an infectious cDNA clone of LSNV was engineered using a baculovirus expression system and insect cell, Sf9. Availability of an infectious cDNA clone will be immensely beneficial to manipulate the viral genome to study viral pathogenesis and developing viral vectors for delivering therapeutic molecules to control MSGS and potentially other viral diseases in shrimp. In chapter 1, the status of MSGS and LSNV is summarized. The literature review gave an overview of MSGS since it was first reported from Thailand in 2000 and follow up research that led to the elucidation of LSNV as the causal agent of MSGS. The review also presents the association of a RNA element with LSNV to cause MSGS, geographic distribution of MSGS, disease biology, clinical signs, susceptible hosts and life stages, disease transmission, and characterization of the etiologic agent. Finally, the current diagnostic tools in preventing the spread of MSGS and controlling the disease are also discussed. Chapter 2 describes the engineering of an infectious cDNA clone of Laem Singh virus (LSNV) infecting shrimp (Penaeus vannamei). The full-length cDNA of LSNV was cloned in a dual baculovirus expression vector, and a recombinant baculovirus expressing LSNV was generated in insect cells, Sf9, to produce infectious virions. The RT-PCR results showed that full-length LSNV was expressed in insect cells, and transmission electron microscopy photograph displayed the present of LSNV virion in insect cells. To determine the infectivity of recombinant LSNV, two bioassays were conducted by mixing the cell culture homogenate containing LSNV to a commercial diet and feeding to juveniles P. vannamei. The infectivity of the recombinant LSNV clone was determined by detecting viral nucleic acid in experimentally challenged shrimp by RT-PCR and examining the cellular changes in target tissues by histopathology. In chapter 3, a TaqMan real-time RT-PCR assay was developed for the detection and quantification of LSNV. Two sets of primers and TaqMan probes (one targeting RNA 1 and another targeting RNA 2) were selected after screening five sets of primers and probes. The limits of detection of the selected primers/ probes were found to be 10 copies for RNA1 and 100 copies for RNA 2. The described assay will be very useful in MSGS screening and in studying the expression viral RNA in different hosts. To summarize, the availability of an infectious viral clone will enable us to genetically manipulate the viral genome and study LSNV-host interaction. This project has opened up a new avenue to engineer RNA viruses in shrimp using a baculovirus expression system and insect cells, thus bypassing the need of a crustacean cell line. In addition, this will open up a future application to develop a viral vector for delivering therapeutic molecules like RNAi and microRNA.