| Infectious myonecrosis, caused by Infectious myonecrosis virus(IMNV), is an emerging disease of cultured penaeid shrimps, it was first observed from Brazil in August 2002 in Litopenaeus vannamei, and had spread to Asia in 2006. This disease brought significant economic losses in shrimp farming industries and had not been controlled.IMNV is a non-enveloped icosahedral virus with a diameter of 40nm and a double-stranded RNA genome of 7560bp, with a buoyant density of 1.366g/ml in caesium chloride(GenBank accession no. AY570892). It contains two extended open reading frames(ORF1 and ORF2). The 5′ORF1 encoded a putative RNA-binding protein and a capsid protein. The 3′ORF2 encoded a putative RNA-dependent RNA polymerase(RdRp) with motifs characteristic of Totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus(GLV, GenBank accession no. L13218). Muscles show severe necrosis, grossly seen as opaque, whitish discolorations in muscles of the distal abdominal segments and the tail fan, and the appearance of the affected muscle similar to the color of cooked shrimp. Histological examination reveals severe coagulative necrosis of skeletal muscles that may be accompanied by edema, hemocytic infiltration, and fibrosis. Four shrimp species, L. vannamei, Litopenaeus stylirostris, Penaeus monodon, Farfantepenaeus subtiltis have been shown to be susceptible to IMNV, but L. vannamei is the most susceptible host. In L. vannamei, striated muscles are the principal target organ, but gills and lymphoid organ can also be affected. Typically, the disease progressed slowly, with low mortality rates that persisted throughout the growing season. Our study was aimed at established some methods such as RT-PCR, dot blot hybridization and In situ hybridization(ISH) to detecte IMNV. The results are as follows:1. At first, total RNA were extracted from IMNV-infected shrimps. Two pairs of primers were designed from the IMNV genomic sequence AY570892(GenBank). In this study, annealing temperature was 55℃to determine the optimal RT-PCR for the established system. The sensitivity of primer to IMNV cDNA was 10~4 copies. In this study, a rapid method has been established to detect IMNV infections on shrimp, increase the sensitivity of method and provide a technical help. 2. Under the optimized PCR conditions, IMNV(infectious myonecrosis virus, IMNV) probe labeled by digoxigenin was synthesized by PCR, and the probe used for dot blot hybridization and in situ hybridization(ISH) test. The size of the DIG- labeled fragment was 123bp. Through dot blot hybridization, the sensitivity of this probe to IMNV cDNA was 10~8 copies and IMNV cDNA PCR product was 1 copy. An effective diagnostic tool is thus provided for screening shrimp for IMNV infections, diagnosing epidemic disease and specific pathogen free(SPF) shrimp breeding.3. Histologically the skeletal muscle and lymph tissue were examined by hematoxylin and eosin(H and E)staining, coagulative necrosis of skeletal muscle accompanied by haemocytic infiltration and fibrosis, dark basophilic inclusion bodies in existence of the muscle cells, different from the nucleus of muscle fiber. In situ hybridization, the IMNV digoxigenin-labeled probe reacted positively in tissue section, IMNV ISH signals that showed purple blue were predominantly located in the striated abdominal muscles and lymphoid organ spheroids, there is no any signal in healthy samples.4. The experiment surveyed L. vannamei samples with RT-PCR and ISH. The samples from Fujian were tested positive. The results indicated that the infection possibility of L. vannamei with IMNV in Fujian Province was high. |
