Effect Of Temperature On The Proliferation Of Mud Crab Dicistrovirus-1 And Reovirus And Their Tissue Distribution

Mud crab(Scylla paramamosain) is an eurycheme and euryhaline economic animal cultured widely in south-east coast of China. Mud crab discistrovirus-1 and mud Crab Reovirus are two important pathogens in crab breeding which result a huge economic losses to crab breeding industry. At present, these two viruses’ research are mostly to established detection methods, there is rarely reported about the influenc factor and tissue distribution of the virus. In this article, we established a MCDV-1 in situ hybridization method, combined with quantitative PCR method to study the distribution of two viruses in mud crab, at the same time to explore that whether the two viruses can be affected by temperature.Above all, We breeding the mud crab in aquarium respectively with temperture 24℃、27℃、31℃ and 35℃, each temperture divided into artifical and natrural infected group. The artificial infected group was injected using extracting solution of MCDV-1 and MCRV, the other group didn’t be injected. Then took five samples at the time of 0 h、1 h、4 h、6 h、12 h、24 h、36 h、48 h、60 h、72 h and 90 h post-injection(hpi) and using real-time PCR to quantitative the MCDV-1 and MCRV copies in the hemolymph, The result showed that: MCDV-1 of all groups increased quickly between 24 h-36 h hpi. In the group of artificial infection, 31℃ was the optimum temperature for MCDV-1 growth, it’s total value can reach to 1×107 copies/ng RNA, while at other temperatures MCDV-1 can only reach 1×105 copies/ng RNA and the order was 35 ℃> 24 ℃> 27 ℃; The proliferative of MCRV changes frequently, over all, the MCRV of 35℃ in two experimental groups would reach two peaks respectively in the 1h and 6h hpi, the viral load reach to 1 × 105 copies / ng RNA, then begin to decline and stabilize; In the group of artificial infection, proliferation laws of 24℃、27℃ and 31℃ was similar and the peak reached in the 48 h hpi, the viral load up to 1×105 copies/ng RNA and the order is 31 ℃> 27 ℃> 24 ℃; while in the group of natural infection, the viral load of 24 ℃、27 ℃ and 31℃ can respectively reach to 1 × 106 copies / ng RNA、1 × 108 copies / ng RNA and 1 × 107 copies / ng RNA. The mud crab of 24℃ and 31℃ in artificial infection group died at 12 h hpi and 27℃’s died at 24 h hpi, the order of mortality between 12 h-72 h hpi is 31℃>24℃>27℃, then the mortality of 27℃ exceeded after 72 h hpi. the crab died quickly at 35℃ doesn’t exclude the impact of stress so this death doesn’t have meanings. The speed order of death in natural infection group is 31℃>27℃>24℃, the mortality of 31℃ was always higher than the other three groups,but the mud crab did’t die at 35℃.To study the tissue distribution of MCDV-1 and MCRV, we sampling gills、hepatopancreas、heart、ganglion、muscle and blood at 0 h、12 h、24 h、36 h、60 h and 90 h hpi respectively from the group of artifical infection and natural infection. Then using the real-time fluorescence quantitative PCR method to detect two virus proliferation in different tissue of mud crab. The result shows that the amount of MCDV-1 in gills was more than the other tissues, the highest levels MCRV was in ganglion. These two virus can finally infect every organization trend to growth with time.The order of MCDV-1 content in different tissues is gill> muscle> ganglion> hepatopancreas> heart> hemolymph and the order of MCRV is heart> liver and pancreas > ganglia> muscle> gill> hemolymph. The MCRV of all organization in natural infection group increased quickly at 12 h hpi and can’t detected MCDV-1 at the same time; the content of MCRV will keep lower after 36 h hpi and then MCDV-1 resume proliferation gradually.We found a relationship between MCDV-1 and MCRV when analysis their tissue distribution and proliferation law. It is fount that When MCDV-1 proliferate rapidly then MCRV proliferation slowly, and vice versa.In the end, we established an in-situ hybridization detection method of MCDV-1 to study the tissue distribution of MCDV-1 and MCRV. Based on the MCDV-1 genome sequences, we designed a pair of primers and obtained a 1314 bp fragment of MCDV-1 gene by RT-PCR. Consequently, probe is prepared using DIG-labeling. The sensitive and specific of the probe were analyzed by dot blot hybridization method. Results showed that this probe can detect a minimum of 8.583107 copies·μL-1 of MCDV-1 and was specific for MCDV-1 detection, without cross-reaction with MCRV(mud crab reovirus)、Ab SV(abalone shriveling syndrome-associated virus)、Ab HV(abalpone herpesvirus)、WSSV(white spot syndrome virus)、TFV(tigerfrogvirus) and KHV(Koi herpesvirus). This probe was used to detect MCDV-1 in the different tissues of the diseased mud crab by in situ hybridization and positive signals of MCDV-1 were mainly observed in hepatopancreas、ganglion、intestines and esophagus.