Study On The Pathogenicity Of Grass Carp Reovirus Co-infected With Aeromonas Hydrophila

Grass carp reovirus and Aeromonas hydrophila are pathogens of grass carp hemorrhagic disease.Both of them can cause inflammation and bleeding in the intestine and other organs of grass carp,resulting in high mortality.Single infection of grass carp reovirus can cause blotchy or massive congestion in various organs and tissues in grass carp,especially in the stage of grass carp fingerling infection.The mortality rate of grass carp reovirus infection is as high as more than 90%,causing huge losses to aquaculture.Aeromonas hydrophila is an opportunistic pathogen that co-infects humans,animals and fish and is widely distributed around the world.Highly pathogenic AA infection can cause systemic symptoms such as bleeding and blood lesions in liver,kidney and other organs of grass carp,which is highly harmful to aquaculture.In aquaculture,it is common to co-infect aquatic products in complex aquatic ecological environments such as bacteria and bacteria,bacteria and viruses,and viruses and viruses.The complicated clinical symptoms caused by co-infection make the diagnosis of aquatic pathogens unclear.At the same time,the pathogenicity and pathogenesis of co-infection in aquatic products are still unclear,and multi-pathogen co-infection also poses new challenges to the prevention and control of aquatic diseases.In this study,we investigated the pathogenicity and potential pathogenic mechanism of GCRV and Ah co-infection in grass carp based on our previous study on the pathogenic mechanism of GCRV and Ah infection in grass carp.The effects of GCRV and Ah infection on their replication were analyzed by plate counting and real-time PCR in vitro.The effect of GCRV and Ah co-infection on the invasion pathway of GCRV and the replication ability of GCRV were analyzed by CPE and western blot combined with drug inhibition assay.Plate counting method was used to analyze the effect of GCRV on the ability of Ah to adhere to and invade cells when co-infected with GCRV and Ah.In order to clarify the pathogenic mechanism of GCRV and Ah alone or co-infection,the ROS levels of CIK cells infected with GCRV and Ah alone and co-infected CIK cells were detected by fluorescent probe method,and the NADPH oxidase activity and mitochondrial membrane potential changes in single and co-infected CIK cells were compared.The main reasons for the changes in ROS levels were explored.The changes in the relative expression levels of cytokines such as IL-8,IL-10,TNF-α,and TGF-β1 in cells infected with GCRV and Ah alone or co-infected were further detected by fluorescence quantitative analysis,and the differences between cell damage under co-infection and single infection were comprehensively analyzed.Based on the in vitro experiment,the grass carp models infected with GCRV and Ah alone and co-infected with GCRV and AH were constructed,and the organ lesions of each group were analyzed by necropsy and histopathological comparison.The degree of inflammation of grass carp tissues and organs in each experimental group was analyzed by measuring the organ index of liver,spleen and other organs.The bacterial load in various tissues and organs of grass carp co-infected with GCRV and Ah was detected by viable count method,and the viral RNA in various organs was detected by fluorescence quantitative PCR to analyze the interaction between the two pathogens in vivo.Furthermore,the effect of co-infection on the pathogenicity of grass carp was analyzed at the animal level by measuring the ROS level and the relative expression of cytokines such as IL-8,IL-10,TNF-α and TGF-β1 in the tissues of experimental grass carp.The results showed that when the cells were infected with Ah before GCRV(Ah+GCRV)or simultaneously infected with GCRV and Ah(GCRV-AH),serious cell damage was caused,which was consistent with the damage caused by Ah infection alone.Under the condition of GCRV infection before Ah infection(GCRV+Ah),the damage of CIK cells was significantly reduced compared with Ah+GCRV group and GCRV-AH group.MTT assay showed that GCRV+Ah group had better cell viability than Ah+GCRV group and GCRV-AH group,and the cell viability was significantly different and could be infected by GCRV.In the analysis of the effect of co-infection on GCRV,it was found that no matter in Ah+GCRV,GCRV+Ah or GCRV-AH groups,there was no significant change in the way of GCRV invading CIK cells and the replication ability of GCRV in CIK cells as long as Ah was present.On the contrary,under the condition of GCRV first infection(GCRV+Ah),the ability of Ah to adhere to and invade CIK cells was significantly decreased compared with Ah infection alone.The level of ROS in CIK cells co-infected with GCRV+Ah group was the highest,which was significantly higher than that in Ah+GCRV,GCRV-AH and GCRV or Ah infection groups.The NADPH oxidase and mitochondrial membrane potential of cells in GCRV+Ah group were significantly higher than those in Ah infection group,and the mitochondrial membrane potential of cells in GCRV+Ah group was also significantly higher than that in AH infection group,which were consistent with those in blank control group.The relative expression of cytokines in the co-infected cells showed that the levels of pro-inflammatory factors were generally decreased,and the levels of anti-inflammatory factors and pro-cell growth factors were increased compared with the Ah infection alone group.The above results were also further verified in the animal model.Compared with Ah infection alone,the clinical symptoms and tissue and organ lesions of grass carp in the early GCRV infection group were significantly reduced after Ah infection.The ROS level of grass carp tissue cells in the GCRV+Ah group was also significantly higher than that of Ah infection alone,which confirmed that GCRV could enhance the killing effect of cells against Ah by increasing the ROS level in the cells infected with GCRV before Ah infection in vivo.Furthermore,by increasing the levels of anti-inflammatory factors and reducing the levels of pro-inflammatory factors in cells,the inflammation induced by Ah can be inhibited,so as to maintain the good vitality of co-infected cells and reduce the influence on the persistent infection of GCRV.These findings provide a new perspective for revealing the pathogenesis of co-infection,and provide theoretical basis and data support for solving this problem.