Studies On Pathogenicity Of Ranavirus GIV-R-SY1301 To Epinephelus Lanceolatus

Epinephelus lanceolatus, commonly known as giant grouper, is an important aquaculture species with great market value. However, with the enlargement of the cultivation scale, the frequency of the disease increased markedly and caused lots of economic losses. In July 2013, a ranavirus designated as GIV-R-SY1301 was reportedly isolated from the diseased farmed groupers, Epinephelus hybrids(blotchy rock cod, Epinephelus fuscoguttatus ♀× giant grouper, E. lanceolatus ♂), in Sanya, Hainan. Ranavirus was well known as causative agents of serious systemic diseases among reptiles, amphibians and fish in the worldwide, which could cause severe disease to many grouper species and has been detected frequently in cultured grouper. It was very important to monitoring, prevention and treatment of the disease to understand the pathogenicity, clinical signs and pathology of ranavirus to E.lanceolatus. So, in the present study, infection experiments were performed to examine the susceptibility, clinical signs and blood biochemical parameters of E. lanceolatus to GIV-R-SY1301 infection. By means of H&E, TEM, immunohistochemistry, in situ hybridization and q PCR, we discussed the histopathological changes and distribution of GIVR-SY1301 in different tissues.In this study, susceptibility of farmed juvenile giant grouper E. lanceolatus to GIV-R-SY1301 was determined by intraperitoneally injection. The cumulative mortality reached to 81% at 5 day post infection. Histologically, severe degeneration with massive pycnotic nuclei in spleen and kidney tissues was observed,and some small-size inclusion body-bearing cells(IBCs) existed in spleen. Hemorrhage and infiltration of inflammatory cells were presented in gill, liver and heart along with tissue degeneration and necrosis of varying severity. The results of immunohistochemistry analysis showed that the strongest immunolabellings were obtained from the kidney and spleen tissues, while intermediate intensity signals were observed in the heart, stomach, gill and liver tissues, and the weakest signals were obtained from the intestine and brain, but no signal was obtained in eyes. Electron microscopy revealed that spleen of moribund fish contained many viral particles in cytoplasm. Interestingly, in surviving fish, abnormal hypertrophic cells were observed in both splenic corpuscle and renal corpuscle, while no hypertrophic cell was observed in the other parts of spleen and kidney tissues. Moreover, immunolabellings only stained the hypertrophic cells in splenic corpuscle and renal corpuscle. This indicated that splenic corpuscle and renal corpuscle play an important role in GIV-R-SY1301 infection and replication.Secondly, tissue distribution of GIV-R-SY1301 in experimentally infected farmed E. lanceolatus was studied by in situ hybridization and real-time PCR. The GIV-R-SY1301 was widespread in all tissues of the infected ?sh. Strong hybridization signals were detected from the kidney and spleen tissues, while intermediate intensity signals were observed in the stomach, gill and liver tissues. The weakest signals were obtained from the heart and no signal existed in brain and eye. The real-time PCR results showed that the viruses copy number was large in spleen, kidney and heart and was between 4.5×104~6.85×107 copies·μg-1 DNA in different tissues.At last, by sampling at different time-point within 10 d after infection, the effect of GIV-R-SY1301 to E.lanceolatus’ blood biochemical parameters was studied by intraperitoneal injection of viral culture and PBS buffer solution respectively. The results showed that contents of ACP, AKP and ALT decreased significantly(P<0.01) in 1-2dpi. Contents of AST, LDH and GLU decreased significantly within 2dpi and increased markedly after 4dpi. Generally speaking, contents of TP, GLB, ALB and TBL decreased first and subsequently, recovered to the level similar to the control group. Contents of UA and CK generally decreased significantly. The results indicated that the physiological and metabolic functions of liver, kidney, heart, and spleen was damaged by infection of GIV-R-SY1301, and thus caused the blood biochemical parameters of E. lanceolatus changed remarkably.