| Duck hepatitis A virus 1(DHAV-1)is the most important pathogen causing duck viral hepatitis(DVH)of ducklings which is characterized as an infectious disease with high pathogenicity and mortality.DHAV-1 is epidemic in China with wide distribution and thus seriously endanger the duck industry with huge economic losses.However,it is generally assumed that DHAV-1 is not harmful to mature ducks,due to that there is no typical clinical symptom.But it is lack of evidenced data.Thus,in this study,we utilized virulent DHAV-1 H strain to infect mature ducks by intramuscular injection,to characterize the distributivity and infectivity in adult duck,and the virus-host interactions.The results are as follows:1 The infection of virulent DHAV-1 induces acute and chronic hepatitis in mature ducksDuring the infection course of DHAV-1,there was no clinical symptoms,which was identical to the previous opinion.However,the virus was detectable indeed as soon as 0.5dpi in the liver and blood,and persisted for 252 days and 196 days,respectively,and thus inducing liver injury,coincident with the significantly raised serum biomarkers,including ALT,AST,Tbil,Tria,and globulin,and the decreased albumin.Specific IgG,IgM and IgA1 were produced soon after infection in serum at remarkable levels.However,CD4~+and CD8~+T cells were recruited to the liver at a comparatively low level,which might lead to the persistent infection.The innate immune responses in the liver and blood were induced and activated.In detail,TLR-7 was the dominant sensor due to the highest up-regulated level among the PRRs.Moreover,the transcriptional level of IL-4(Th2cytokine)was higher than IL-2 or IFN-γ(Th1),indicating an incline to Th2 responses.Pearson’s analysis displayed that DHAV-1 RNA titers were negatively correlated with the transcriptional level of IFN-γand IL-6 in the liver and blood,respectively,and the titers of IgG in serum.Thus,our results indicate that virulent DHAV-1 can infect mature ducks showing clinically asymptomatic.But the virus can be persisted in liver and blood to cause acute and chronic hepatitis.Moreover,DHAV-1 can activate the immune system of mature ducks,but has some suppressions on the T cell responses.2 The scattered distribution of virulent DHAV-1 in kidney reveals strategies to escape host immunity leading to the persistent infectionAfter DHAV-1 infection,viral capsids were detected in the mesangial cells and vascular endothelial cells in the kidney at the early phase.Moreover,viral loads were peaked at 1dpi,following with decreases.Meanwhile,viruses were gradually transferred to the scattered renal tubules,and distributed continuously during the later infection course.In addition,DHAV-1 infection caused renal cell lesions around renal tubules,including swelling,degeneration,apoptosis and necrosis,and glomerulonephritis.But,there was no infiltration of neutrophil,nor recruitment of CD4~+or CD8~+T cells in the kidney,indicating loss of cell-mediated immune responses in the kidney.Although the innate immune response was induced,TLR-7 and the downstream antiviral and inflammatory factors,and chemokines,were mainly expressed at the early course of infection.Moreover,there was no significant expression of MHC-1,MHC-II and CCL19,which was probably associated with the deficiency of CD4~+/CD8~+T cell response.In general,the results demonstrated that virulent DHAV-1 could infect the kidney of mature ducks and scatter in the renal tubules,resulting in the limited innate and cellular immune response locally,which was beneficial to viral escape and thus persistent infection.3 The lymphotropism of virulent DHAV-1 to infection lymphoid organsAfter DHAV-1 infection,we could detect the presence of DHAV-1 RNA,capsid and 3D protein in the spleen,Harderian gland,thymus and bursa of Fabricius for a long term,in which the virus was still detectable at 224 dpi.In addition,Pearson’s correlation analysis revealed that viruses produced in the lymphoid organs were positively correlated with the virus in the blood.The detection of immune-related factors showed that the innate immune system of lymphoid organs was activated by DHAV-1 infection,but displayed significant differences between organs.Moreover,Pearson’s correlation analysis showed that IFN-γ,IL-2,IL-4 and IL-6 were negative with DHAV-1 directly,taking part in the viral clearance.In addition,comparing of the expression levels of these factors and the virus clearance in lymphoid organs,it revealed that IFN-γis the most effective anti-DHAV-1 factor.Moreover,it was found that CCL21 was highly expresses persistently in thymus,which affected the emigration of T cells to the periphery,and thus resulted in the loss of T cells in the liver during the medium phase of infection.Therefore,the results indicated that DHAV-1 could infect the lymphoid organs of adult ducks,and that the replication and proliferation of DHAV in lymphoid organs destroyed the function of lymphoid organs,resulting in persistent infections in local and in system.4 Characteristics of virulent DHAV-1 infection in digestive system and the induced mucosal immune responsesTo observe the infection of virulent DHAV-1 in digestive system,we detected the DHAV-1 RNA,which showed that DAHV-1 could infect pancreas,esophagus,duodenum,jejunum,ileum,cecum and rectum as soon as 0.5 dpi,and reached peaks within days 4 post infection,except for the ileum in which the peak titer was at 12 dpi.Moreover,the viral loads were higher in ileum between 10-168 dpi,while at other times in rectum,than other sites.In addition,the second level of virus occurred in esophagus,and the lowest in pancreas which was cleared at 168 dpi that was earlier than others.Specific IgG,IgM and IgA were detected in bile and secretions of esophagus and intestine,which showed higher titers and longer durations of IgA than IgG and IgM.Moreover,IgA~+cells were also detected in esophagus and intestine.However,the level of mucosal immunity was far behind the systemic humoral immunity.In summary,our results indicated that virulent DHAV-1 had infectivity to the digestive system of mature ducks which showed diverse tendency to different parts,with highly infectious to ileum,rectum and esophagus,and weak to pancreas,and that the infection of DHAV-1 could induce the local mucosal immune responses.5 Characteristics of virulent DHAV-1 infection in reproductive system and the induced mucosal immune responsesAt 0.5 dpi,DHAV-1 RNA was detectable in ovary and oviduct of mature ducks,which reached peaks within days 4 after infection,and showed a relative lower level of viral loads in ovary than oviduct.However,although the virus was disappeared between 196-252 dpi in ovary,it was detected again at 280 dpi,distinct from the course of infection in oviduct.The infection courses in magnum and vagina were persisted till to days 280 after infection,while infundibulum,isthmus and uterus showed eliminated DHAV-1 RNA at 280 dpi.Moreover,the viral loads in vagina were higher than in other parts of oviduct.Notably,the vast infection of virulent DHAV-1 in the reproductive system leaded to the appearance of malformed eggs.Specific IgG,IgM and IgA were detected in secretions of ovary and oviduct.In detail,the titers of IgG showed no significant difference at any part,while IgM was higher in magnum and vagina,and IgA was higher in magnum.Additionally,the level of IgA in ovary and oviduct was higher than IgG and IgM,in which IgA~+molecules or cells were presented also.In summary,the results indicated that virulent DHAV-1 could infect and damage the reproductive system of mature ducks,and that the induced mucosal immunity locally was not enough to clear the virus and protect the organs from DHAV-1. |
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