Establishment And Application Of A Cell Line From Egg Of Rare Minnow,Gobiocypris Rarus And Its Study On The Proliferation Characteristics Of Grass Carp Reovirus(GCRV)

Grass Carp Reovirus(GCRV),the first aquatic virus isolated in China,which belongs to genus Aquareovirus.GCRV can infect grass carp and black carp resulted in high mortality up tp 80%-100%.Rare minnow,Gobiocypris rarus,is small size laboratory use fish proven to be sensitive to GCRV infection.After being infected,the bodies of rare minnow has obvious symptoms of hemorrhagic disease,and some fishes even die.Epidemiological investigation shows that there are three genotypes of GCRV in China at present,with genotype II as the main epidemic type.However,at present,the research of genotype II GCRV is facing the problems that it can not produce cell lesions and had low viral proliferation.In addition,the common grass carp cells are usually carrying virus.It is urgent to establish more sensitive cell lines to study the pathogenesis of epidemic strains and vaccine development.This research mainly includes two parts:1.The establishment of a cell line from egg of rare minnow: In this study,the eggs of Rare Minnow were cultured in vitro by the trypsin digestion method.And the primary culture and subculture of rare minnow egg cells were successfully carried out by optimizing the medium formulation.The cell was named GrE(Gobiocypris rarus eggs).The optimum culture medium for GrE cells was M199 medium containing 10% fetal bovine serum(FBS),and the cells grew well at a temperature of 28℃.The adherent,growth and division of the cells were fine,and GrE cells were typical epithelioid cells.After successive subcultures,a egg cell line of rare minnow has been successfully established and it has been subcultured to the 70 th generation.The chromosome analysis showed that the GrE’s chromosome mode was 2n=66and the karyotype was 2n=30m+24sm+10t+2st.However,the chromosome number of rare minnow was 2n=50,and the chromosome number of GrE cells has changed,indicating that GrE cells have mutated and become immortalized cell lines.GrE cell line remained its original cell morphology after cryopreservation in liquid nitrogen and resuscitation,and its adherent growth state and proliferation rate were not different from those before cryopreservation.2.The application of GrE cell:(1)Different aquatic animal virus(genotype I and genotyoe II grass carp reovirus(GCRV-Ⅰ,GCRV-Ⅱ),Spring viraemia of carp virus(SVCV),Large mouth bass ranavirus(LMBV),Frog virus3(FV3))with specific concentration were inoculated into the GrE.After inoculation,CPE productions were observed and qualitatively detected by PCR.After inoculation with the five viruses,all GrE cells except those inoculated with GCRV showed obvious cytopathic changes(CPE).And the specific bands of the five viruses were detected by RT-PCR/PCR.These results suggested that the GrE cell lines can be used for the isolation and identification of these viruses mentioned above.Meanwhile,the sensitivity of GrE cells to GCRV-Ⅰ and GCRV-Ⅱ was further verified by indirect immunofluorescence.The titers of GCRV of the two genotypes were 105.59 TCID/m L and 106.47 TCID/m L,respectively.(2)The sensitivities of GrE and CIK to GCRV were further compared.GCRV-Ⅰ and GCRV-Ⅱ with an initial titer of 103 TCID50/ml were inoculated into GrE and CIK cells.GCRV-Ⅰ infection lasted for 7 days and GCRV-Ⅱ infection lasted for 14 days.The virus copies of two different genotypes of GCRV in GrE and CIK cells were calculated.The results demonstrated that the highest copy number of GCRV-Ⅰ in GrE and CIK cells appeared on the4 th day,which was 1.45×105 copies/μL(GrE),1.26×104 copies/μL(CIK),respectively.While the copy numbers of GCRV-Ⅱ both in CIK cells and GrE cells peaked on the 7th day(3.37×104copies/μL)and 12 th day(2.25×105 copies/μL).(3)After 20 passages of GrE cells infected with GCRV-Ⅰ and GCRV-Ⅱ respectively,the GCRV-Ⅰ at generation 1 and 20 could not cause any clinical symptoms of grass carp hemorrhagic disease in rare minnow.there was no significant difference between in the results of histopathological section between those fishes and negative control.Typical clinical symptoms of hemorrhagic disease occurred 3-5 days after two generations of genotype II GCRV injected in healthy rare minnow.There were obvious pathological changes in the fish tissues,including larger intercellular spaces in brain tissues and liver tissues,more yellow hemoglobinophagocytosis cores in spleen and kidney tissues.And in the intestinal tissue,the intestinal capillary hemorrhage was serious.At the initial stage,the intestinal mucosae were slightly necrotic and decayed,and at the later stage,the intestinal villi had decayed and even broke into shapeless.The egg cell line of rare minnow was successfully established in this study,and the optimum growth conditions were determined.The growth characteristics,cell morphologies,chromosome morphologies and the applications to aquatic animal viruses were identified and analyzed.The GrE cells can be used for the isolution and cultre of various common aquaticanimal viruses.GCRV-Ⅰ,GCRV-Ⅱ can proliferate in GrE cells,and GrE cells were more suitable for the proliferation of GCRV-Ⅱ,compared with CIK cells.After the infection with GCRV,the results of clinical symptoms of rare minnows,viral loading and histopathological changes in tissues indicated that the virulence of GCRV-Ⅱ was stable in GrE cells for at least20 generations.The GrE cells established in this study which can be used for epidemiological investigation,pathogenesis and vaccine development of grass carp reovirus epidemic genotypes.