The study on the proliferation of GCRVⅡ in different fish cell lines

Currently, grass carp reovirus genotype Ⅱ(GCRVⅡ) are the leading cause of grass carp haemorrhage disease. To determine the optimum concentration of virus inoculation, various concentrations of HZ08 strain was inoculated with proboscis snout into fibers (PSF) cell of grass carp (PSF) and swim bladder cell of grass carp (GSB). Then we infected 10 kinds of fish cell lines with the optimum inoculation concentration, including liver cell of PSF, GSB, grass carp (L8824), ovary cell of grass carp (CO), etc. After inoculation, we observed the state of the cells with microscope daily, and real-time fluorescence quantitative PCR (qPCR) was used to analyze the proliferation of HZ08 in various cell lines. We also used indirect immunofluorescence to further qualitatively analyze the proliferation of HZ08 in various cell lines five days after infection. The results showed that the optimum inoculation concentration of HZ08 infected cells was 1.0×10⁴ copies/μL. There was no significant cytopathic effect (CPE) observed in 10 kinds of fish cells inoculated with the HZ08 strain. qPCR analysis showed that HZ08 strain can proliferate in GSB, L8824, PSF, CF, CO, CIB, KS, 7 kinds of cells, which proliferated better in GSB, L8824 and PSF cells. The titers can reach 1.14×10⁷, 5.90×10⁶ and 6.30×10⁴ copies/μL respectively. The result of immunofluorescence are coincided with the results of qPCR, there were strong fluorescence signals in GSB, L8824 and PSF cell lines and weak or even no fluorescence signals were observed in other cells. In conclusion, GSB, L8824 and PSF, these 3 kinds of cells are more sensitive cell lines for the proliferation of GCRVⅡ. In conclusion, this research is of great significance to the research of GCRVⅡ and the development of prevention and control product for GCRVⅡ.