Genomic and phylogenetic characterization of a bunya-like virus from the freshwater Chinese mitten crab Eriocheir sinensis.

The freshwater Chinese mitten crab (Eriocheir sinensis), an indigenous crustacean in China, has been cultured for more than 30 years. It was reported that the bunya-like virus from Eriocheir sinensis (EsBV) was associated with the tremor disease (TD), which causes high mortality and has a serious impact on production. In this study, full-length genome sequences of EsBV were pursued using next generation sequencing; the genome of EsBV was found to be composed of 6.7 kb L, 3.3 kb M, and 0.8 kb S segments, respectively. PCR detection based genomic sequences showed that the positive rate of EsBV reached 40% in crabs from farming ponds. EsBV had the highest similarity with the Wenling crustacean virus 9, an unassigned, negative sense ssRNA virus. EsBV clustered with the Wenling crustacean virus 9 firstly, and then the branch clustered with Peribunyaviridae clade in every phylogenetic tree - based on L, M and S encoded sequences, respectively, indicating that EsBV can be classified in the family Peribunyaviridae, to which the orthobunyaviruses belongs, but not belonging to any known genera in the family Peribunyaviridae. There were unique complimentary terminal sequences for EsBV, with only partial consensus with members from the orthobunyaviruses. We believe that the findings of this research will be vital for future research about EsBV and will also go a long way in illuminating its relationship with TD. Keywords: Eriocheir sinensis; tremor disease; bunyavirus; EsBV; genome sequences.

Detecting a novel Eriocheir sinensis reovirus by reverse transcription loop-mediated isothermal amplification assay.

The novel Eriocheir sinensis reovirus (EsRV) is a pathogen that causes severe disease and high mortality rates in cultivated crabs. Here, we established a highly sensitive and specific rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that was cheaper and more suitable for field applications in crab aquaculture than those of traditional reverse transcription-polymerase chain reaction (RT-PCR) analysis. The amplification was completed within 45 min under isothermal conditions at 65°C. The RT-LAMP test for EsRV had a detection limit of 15 pg, and sensitivity was 100 times greater than that of conventional RT-PCR. The LAMP primers for EsRV were not amplified by other pathogen strains, indicating good specificity. In addition to detection by electrophoresis, RT-LAMP results were detectable by visual observations of reaction tube turbidity, and calcein was added to visually detect the amplification products. These results indicate that this highly convenient, rapid and sensitive RT-LAMP assay can be used to detect EsRV-infected aquatic organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Tremor disease (TD) is one of the most serious diseases of Eriocheir sinensis. A novel E. sinensis reovirus (EsRV) was identified from E. sinensis afflicted with TD and caused high mortality. We developed a reverse transcription loop-mediated isothermal amplification assay with high specificity, sensitivity and rapidity to detect EsRV, which can be used to diagnose aquatic animal diseases, particularly where expensive diagnostic instruments are not available.