One-step Reverse Transcription PCR Assay, Host Range And Procambarus Clarkii Experimental Infection Of Scylla Serrata Reovirus

Scylla serrata is one of important commercial species in China’s mariculture. A disease nominated as sleeping disease or waterclear disease, which caused by Scylla serrata reovrirus (SSRV), have emerged in Scylla serrata aquaculture and resulted in significant economic losses. So far, the diseases have no effective means of treatment.In this thesis, a one-step reverse transcription PCR (RT-PCR) assay for SSRV detection was established by using a pair of primers corresponding to RNA polymerase gene sequence of SSRV. Both cDNA synthesis by reverse transcription and polymerase chain reaction amplification were carried out in one reaction tube. And the sensitivity of one-step RT-PCR assay for SSRV is up to10-8μg purified SSRV dsRNA. The assay took one step to perform cDNA synthesis and PCR amplification in one tube that can reduce pollution brought by operational work and decrease false positive in the assay. In conclusion, it is reliable to the SSRV detection, the screening of seedlings and the host range survey.To investigate the host range of SSRV, a number of marine species were collected in Ningbo city of Zhejiang Province. Total RNAs were isolated and purified from the suspension of tissues from these marine species. The one-step RT-PCR was proceeded to detect SSRV in these marine species. It was found that several species such as Portunus trituberculatus, Palaemon carincauda Holthuis Ridgepail prawn, Oratosquilla oratoria de Haan, Sinonovacula constricat canarck, Ruditapes philippinarum, Busycon canaliculatu, Mytilus edulis, Tegillarca granosa are SSRV positive by one-step RT-PCR. The species Portunus trituberculatus, Ruditapes philippinarum, Tegillarca granosa and Busycon canaliculatu possessed higher SSRV positive rates, which is86.7%,90%,90%and70%respectively. And SSRV positive rate in Palaemon carincauda Holthuis Ridgepail prawn, Sinonovacula constricat canarck, Oratosquilla oratoria de Haan and Mytilus edulis was38.1%,30%,50%and50%respectively. Furthermore, one sample out of eleven unclassified trash fish samples as the feed of Scylla serrata aquacultured was SSRV positive, also. It is suggested that Scylla serrata should be aquacultured in the circumstances isolated from those marine species of SSRV positive so as to cut off the transmission route of SSRV, eliminate sources of contagium and avoid outbreak of Scylla serrata reovirus disease.In order to multiply the SSRV and to explore pathogenic mechanisms of SSRV, SSRV suspension were prepared from hepatopancreas tissues of SSRV positive Scylla serrata detected by PAGE and one-step RT-PCR and then intramusculary-inoculated into the third telson abdomen of health Procambarus clarki, a member of Arthropoda, Crustacea, Decapoda in taxonomy. Every two days a number of inoculated crayfish were taken to dissect into hepatopancreas, gill, muscle and intestinal tissues, and the SSRV infection and multiplication in these tissues were checked by polyacrylamide gel electrophoresis (PAGE), one-step RT-PCR assay, microscopy of tissue section stained with hematoxylin-eosin staining (HE staining) and electron microscopy detection of ultrathin tissue section negative-stained. PAGE of RNAs in the suspension from hepatopancreas, gill, muscle and intestinal tissues of the inoculated crayfish showed characteristic SSRV dsRNA pattern, one-step RT-PCR assay suggested that theses tissues are SSRV positiv, microscopy of HE stained section revealed the typical cytoplasmic viral inclusions in gill tissue from the inoculated crayfish, Electron microscopy presented typical crystal structure of reoviruses in the hepatopancreas of the inoculated crayfish, sugesting that SSRV experimental infection of Procambarus clarki was primarily achieved. SSRV multiplication in Procambarus clarki will produce enough amount of SSRV for its biology and molecular biology studies. Moreover, SSRV infection of Procambarus clarki will be helpful to inquire into SSRV pathogenic mechanism, in order to search the treatment target and screen medicine for the diseases caused by Scylla serrata reovirus.