The Synergistic Pathogenesis Of Avian Leukosis Virus Subgroup J And Reticuloendotheliosis Virus

Multiply virus co-infection is common in nature.viral co-infection usually shows mutual promotion or suppression,and the clinical features,pathological manifestations,infectivity,and clinical indicators appear to change after virus co-infection relative to those of separate infections.Avian leucosis（AL）is a neoplastic disease caused by the Avian leucosis virus（ALV）and Avian sarcoma viruses（ASVs）group of viruses that can cause immunosuppression,growth retardation,and other symptoms.Reticuloendotheliosis disease（RE）is a neoplastic disease caused by Reticuloendotheliosis virus（REV）,which causes immunosuppression,growth retardation and increased mortality in birds.Both viruses are clinically capable of causing severe immunosuppression and growth retardation.Previous survey data show that co-infection of ALV-J and REV is serious in China,and cases of co-infection of ALV-J and REV are frequent in China,with serious effects on chickens after co-infection.Therefore,the study of the pathogenic,infectious and immunosuppressive changes in poultry after co-infection with ALV-J and REV is the key to the subsequent realization of joint purification and control of the spread of these two diseases.To study the synergistic pathogenic effects of ALV-J and REV,400 SPF chicken embryos were randomly divided into 4 groups with an average of 100 embryos per group,and inoculated through the urinary bladder cavity at 6 embryonic ages,with the ALV-J-infected group receiving10^3.0 TCID₅₀ ALV-J-infected cell culture supernatant and the REV-infected group receiving10^3.2 TCID₅₀ REV-infected cell culture supernatant,and the co-infected groups were divided in half The Mock group was inoculated with DMEM.qPCR was performed after incubation to determine whether the virus was successfully inoculated.To clarify the effect of ALV-J and REV co-infection on ALV-J infection.The blood and cloacal swabs samples were collected.It was found that the positive rate of viraemia of the co-infected chickens was higher than the ALV-J-infected chickens at all testing time points.The positive rate of viral shedding of the co-infected chickens was 70%at 7 dpi,with a mean p27OD630 value 0.452.The positive rate of viral shedding in the ALV-J-infected chickens was50%,with a mean p27 OD630 value 0.339.Which indicating that ALV-J and REV co-infection not only significantly increased the positive rate of viremia and increased the level of viremia.In addition,co-infection also delayed production of anti-ALV-J antibodies.The detection of serum samples revealed that 3 chickens in the ALV-J-infected group produced anti-ALV-J antibodies at 6 weeks,while the co-infected group did not produce anti-ALV-J antibodies throughout the detection time period.The indicating that co-infection led to more severe immunosuppression,which delayed production of anti-ALV-J antibodies.Tissue viral load showed that ALV-J loads of the co-infected chickens were significantly higher in spleen,thymus and bursa of fabricius,which suggesting that co-infection enhanced the targeting of ALV-J to immune organs.In order to clarify the effect of co-infection for REV.The positive rate of REV viremia and viral shedding in the ALV-J and REV-and REV-infected chickens were examined.The results showed that positive rate of viremia was no significant difference between the co-infected chickens and REV-infected chickens.The positive rate of viral shedding in the REV-infected chickens peaked at 21 dpi and then started to decrease.The positive rate of viral shedding in the co-infected group maintained an increasing trend and exceeded the mono-infected group at28 dpi,which indicated that ALV-J and REV co-infection increased the positive rate of REV viremia and prolong the time of viral shedding.To clarify the effect of ALV-J and REV co-infected on the immune response to conventional vaccines,30 chickens in each of the four groups were randomly selected at 7 dpi and inoculated intraperitoneally with 3 inactivated vaccines against Newcastle disease（ND）,avian influenza（AI）and fowl adenovirus（FAD）.The blood samples were collected to detect the antibody potency of ND and FAD at 14,28 and 42 dpi.The antibody levels of ND and FAD in the co-infected chickens were significantly lower than the two single-infected chickens at 42dpi,indicating that co-infection significantly reduced the ability of the organism to respond the vaccine.To evaluate the effect of co-infection on the immune system,three chickens in each group were randomly selected and dissected at 14,28 and 42 dpi.The immune organs were taken and examined for immune organ indices,inflammatory factor levels and pathological histological changes.The immune organ indices of thymus and bursa were significantly lower than those of the two mono-infected groups at all time points tested,indicating that co-infection of ALV-J with REV resulted in splenomegaly and atrophy of the thymus and bursa.The expression levels of IL-2,IL-4,IL-6,IL-10,IL-12,IL-13,IFN-γand TNF-αin the spleen were significantly higher in the co-infected chickens than the single-infected chickens at 14,28 and42 dpi.IL-2 and IFN-γare cytokines mediated by Th1 cells.IL-4,IL-10 and IL-13 are cytokines mediated by Th2 cells.IL-6,IL-12 and TNF-αare the main pro-inflammatory factors.The persistent dysregulation of IL-6 and TNF-αexpression can lead to auto-inflammation and tumor.While IL-12 is related to the body’s anti-tumor function,and decreased expression levels lead to a decrease in the body’s anti-tumor capacity.In this study,we demonstrated that co-infection of ALV-J and REV significantly increased the level of ALV-J and REV viremia,enhanced viral shedding and pathogenicity,and delayed the production of specific antibodies;co-infection led to immunosuppression and reduced the body’s ability to respond to vaccines;co-infection increased the damage to immune organs,increased viral load in immune organs,and severe lesions.The co-infection increased damage to immune organs,increased viral load in immune organs,and severe lesions.This study reveals the synergistic effect of ALV-J and REV in vivo,which will provide reliable data to support the co-purification of ALV-J and REV.