Study On The Tissue And Blood Cell Types Of Grass Carp (Ctenopharyngodon Idella)Infected With GCRV-Ⅱ(Genotype Ⅱ Gress Carp Reovirus)

Grass carp reovirus(GCRV)is the main viral pathogen that endangers the health of grass carp,causing severe viral hemorrhagic disease.Epidemiological studies have shown that GCRV-Ⅱ is currently the most virulent epidemic strain in our country.At present,there have been related studies on the histopathological characteristics,virus distribution and immune-related gene expression of artificially infected GCRV-Ⅱ grass carp.However,the difference between the clinicopathological symptoms and virus distribution of grass carp artificially injection infected and naturally infected with GCRV-Ⅱ virus is still unclear.At the same time,the current research on the target organs and tissues of GCRV infection of grass carp is more in-depth,but there is a lack of studies on the target cells of GCRV infection of grass carp.Studies have shown that GCRV can enter cells through cell endocytosis.The way GCRV enters cells may be related to Clathrin is related to caveolin-mediated endocytosis,but JAMA protein cannot interact with the outer capsid protein of GCRV.Therefore,in the peripheral blood cells,which type of blood cells can be infected by GCRV to perform the function of carrying the virus needs to be studied urgently.Therefore,this study distinguished the pathological symptoms and virus distribution of natural and artificially infected grass carp,and at the same time identified the peripheral blood cell types of GCRV infected grass carp.The main findings are as follows:1.The main target organs of artificially infected GCRV-Ⅱ grass carp and naturally disease grass carp are different.In this study,we compared the clinical symptoms of artificial injection infection and naturally disease fish,and found that the diseased grass carp had obvious spotting bleeding in the orbit,operculum,mouth,abdomen,fin base,intestine,and muscle.Bleeding is more serious than the latter.Observation and comparison of histopathological sections revealed that the infected tissues have different degrees of congestion and red blood cell filling,and the disease of some tissues of artificially infected fish is more serious than that of naturally disease fish.q RT-PCR and Western Blot tests show that GCRV is distributed in different tissues.In the case of artificial infection,the viral load in the intestines,muscles,and liver is higher,and the gill,spleen and mid-kidney viral load is higher in the case of natural onset.And the head kidney had the higher viral load in diseased fish of the two infection methods.Therefore,in the case of artificial inject infection,the intestine may be the primary target organ of GCRV infection,while in the case of natural infection,the gill may be the main target organ of the virus,and all the target organs for virus aggregation are the head kidney,which is the "virus library" of GCRV.2.GCRV-Ⅱ infects leukocytes of grass carp without infecting erythrocytes and thrombocytes.At the same time,in this study,peripheral blood cells were isolated,cultured,and infected separately.Through q RT-PCR,Western Blot,indirect immunofluorescence,flow cytometry,transmission electron microscopy and other methods,a model of GCRV infection of blood cells in vitro was established.PCR and WB results showed that with CIK cells as a positive control,only the infected leukocytes and CIK cells could detect the virus,and the erythrocytes and thrombocytes could not detect the presence of the virus;The results of q RT-PCR showed that the expression of IFN1,IL-1β,MX2 and TNF-α immune-related factors in leukocytes after GCRV infection showed a significant up-regulation trend with the change of infection time,while the expression of IFN1 in erythrocytes after infection did not obvious changes,and thrombocytes show a significant up-regulation trend,indicating that GCRV infection of leukocytes causes related immune responses;transmission electron microscopy results show that virus particles in CIK cells exist in the cytoplasm,either scattered or arranged in a lattice.The diameter of the virus particles is about 70～80nm,and also observed that the density of the irregular virus inclusions(VIBs),virus inclusion body has no obvious membrane structure,including virus particles are forming process.While the virus particles in leukocytes are wrapped by empty vesicles with obvious membrane structure,which was similar to phagocytic vesicles.Meanwhile,CIK cells and leukocytes have severe lesions and loose cell structures,while erythrocytes and thrombocytes have complete structures without obvious lesions,and no virus particles are observed;indirect immunofluorescence and flow cytometry results show that leukocytes and CIK cells are GCRV-positive,and the positive rate increases with the time of infection,while erythrocytes are GCRV-negative after GCRV infection.Therefore,it shows that GCRV can infect leukocytes without infecting erythrocytes and thrombocytes when infecting blood cells in vitro.Leukocytes are target cells infected by GCRV.This study systematically compared the histopathology and virus distribution of artificial infection and naturally occurring grass carp for the first time,and we concluded that the intestines is the main target organ during artificial injection infection,while in the case of natural disease,gill tissue is the main target organ.The conclusions provide detailed information for the pathogenicity of GCRV and the diagnosis of grass carp hemorrhagic disease,and also lays the foundation for further research on virus prevention and control.At the same time,for the first time in this study,the peripheral blood cells of grass carp were isolated and cultured and then artificially infected.It was concluded that GCRV infects the peripheral blood leukocytes of grass carp without infecting erythrocytes and thrombocytes.The leukocytes are the target cells of GCRV infection of grass carp.The research broadened the horizon for the antiviral immunity of GCRV.It provides a theoretical basis for the prevention of grass carp hemorrhagic disease in the grass carp breeding industry,and reduces the economic loss in the process of grass carp breeding.