In situ hybridization demonstrates that litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to Infectious Myonecrosis Virus (IMNV) KATHY F.J. TANG, CARLOS R. PANTOJA, RITA M. REDMAN AND DONALD V. LIGHTNER1 Outbreaks of a necrotic disease that has caused mortality in farmed populations of Pacific white shrimp Litopenaeus vannamei in Brazil have been attributed to a double-stranded RNA totivirus, IMNV (infectious myonecrosis virus). This virus infects shrimp skeletal muscle causing multifocal to diffuse necrotic lesions in the tissue (Lightner et al. 2004). The major clinical sign of this disease is the appearance of white, opaque areas in the tail musculature (Figure 1). As the disease progresses, infected shrimp become lethargic, and many eventually die. The virus can cause mortalities up to 40-60 percent of shrimp populations in affected ponds. The prevalence of this disease in Brazil has had severe economic consequences for shrimp producers in that country. Nunes et al. (2004) estimated that in 2003 alone, loss from this disease in Brazil was US$20 million. Diagnosis of IMNV infection has proven problematic, and current methods rely on clinical and histological examinations. Diagnosis based on clinical signs is not reliable, as a number of factors, including hypoxia, crowding, or sudden changes in temperature or salinity can cause similar symptoms. Diagnosis by histological examination is not specific or sensitive. In this paper, we report the development of a new molecular diagnostic method, based on in situ hybridization (ISH) that is both highly specific and sensitive. This technique is used routinely for the diagnosis of other shrimp viruses. We developed the method, tested its specificity, and then employed it in laboratory bioassays. We conducted a bioassay involving experimental IMNV infections in three penaeid shrimp species to compare their susceptibility to the virus. Although the shrimp industry of Brazil is based primarily on the production of L. vannamei, 18 MARCH 2007 Fig. 1. Subadult shrimp display clinical signs of IMNV: appearance of whitish, opaque, lesions in the skeletal muscle. other species of marine shrimp can be exposed to this virus, inasmuch as live L. vannamei are frequently exported to areas where the species is not indigenous. In this bioassay, we compared the susceptibility of L. vannamei and two other farmed species of shrimp (L. stylirostris and Penaeus monodon) to infection with IMNV IMNV Diagnosis with in situ Hybridization To develop the diagnostic method, we first needed to obtain a gene probe for IMNV to be used for in situ hybridization (ISH). The first step was to construct a cDNA library complementary to the IMNV RNA genome. In this standard procedure, cDNA fragments are generated enzymatically, inserted into plasmid vectors and amplified by propagating the vectors in bacterial (E. coli) cultures. In this way we generated approximately 60 cDNA clones; and,
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