Analysis Of The Prevalence Of AMD In China And Mechanism Research On ADE Of Mink Syk Gene Involved AMDV Infection In Vitro

Aleutian Mink Disease(AMD),also known as plasmacytosis,is an acute or chronic infectious disease caused by Aleutian mink disease virus(AMDV),a member of the parvoviridae family,which mainly damages monocyte-macrophages of mink.AMD is one of the three major epidemics of fur animals and is found in mink farms worldwide,causing serious economic losses.Due to the strong antibody-dependent enhancement(ADE)effect during the process of infection,the large number of antibodies can not neutralize AMDV,but promote the internalization of the virus into macrophages and aggravate the infection,therefore,there is no targeted drug and approved vaccine so far.Spleen tyrosine kinase(Syk)is widely expressed in lymphocytes,hematopoietic cells and fibroblasts,etc.It not only plays a key role in the pathogenesis of certain diseases and cell signal transduction,but also acts as an upstream gene in the macrophage endocytosis pathway mediated by the Fc?R receptor family,and it can also regulate the phagocytosis of macrophages.In order to clarify the prevalence of mink Aleutian in China and to explore the AMD mechanisms in the process of AMDV infection,the following studies were carried out:1.Investigation and Analysis on the prevalence of AMD in ChinaTo further explore the distribution of AMD in China,we conducted a meta-analysis and epidemiological survey of AMD positive rates over the past 40 years(1981-2019):(1)A meta-analysis was applied to systematically evaluate the epidemiological survey articles on domestic AMD published from 1981 to 2017.The results showed that the prevalence of AMDV in China increased from48.0% between 1981 and 2009 to 61.4% between 2010 and 2017,with an overall positive rate of 55.3% between1981 and 2017.Subgroup analysis revealed a regionally diverse distribution of AMD prevalence trends.The prevalence of AMDV in Central China and Northeastern China were 69.8% and 61.3%,respectively.Jiangsu Province had the highest AMDV positive rate,as high as 96.0%,and that in Shanxi Province was the lowest at22.1%.Age subgroup revealed that the prevalence of AMDV in adult minks was higher than that in juvenile minks.The prevalence measured by ELISA method was 76.4%,reaching the highest.The results of metaanalysis indicated that AMDV was seriously prevalent in mink farms in China.(2)Based on the the above meta-analyis results,for further explore the current status of AMD prevalence in parts of Northeast China,a survey was conducted on the prevalence of AMD in parts of Northeastern China(Zuojia town,Jilin Province;Maoershan town,Heilongjiang Province;and Jixian county,Heilongjiang Province)in 2019.The results showed that the overall prevalence of AMDV in 2019 in parts of Northeast China was 46.8%,with significant differences between different regions(P < 0.05).According to the results of multivariate analysis,age of mink and sampling season may be the potential risk factors of AMDV infection(P< 0.05).Combined with the results of meta-analysis,although the current prevalence of AMDV in parts of Northeast China is lower than the total positive rate of AMDV in China,the epidemic situation is still relatively serious.2.Mechanism research on ADE by mink Syk gene regulating AMDV infection in vitroIn order to alleviate the situation of AMDV infection in China,an in-depth study of the ADE mechanism in the course of AMDV infection was conducted with the aim of improving the status of no specific drugs and vaccines for AMDV.(1)An in vitro model of ADE mechanism of AMDV infection in mink was constructed using peripheral blood monocyte-macrophages.In the test group,AMDV-DL125,a strong strain of AMDV,was co-incubated with AMDV-positive sera to form antigen-antibody complexes(AMDV-DL125 PS group).In the control group,AMDV-DL125 was co-incubated with negative sera(AMDV-DL125 NS group).Both groups were infected the isolated and purified mink peripheral blood monocytes-macrophages for 12 h.QPCR was use to detect the replication of AMDV-DL125 during 0-12 h,the results showed that the virus copy number and AMDV VP2 gene expression in AMDV-DL125 PS group cultured for 8-12 h were higher than those in AMDV-DL125 NS group,which showed that the antigen-antibody complex strengthens the replication ability of AMDV-DL125.Indirect immunofluorescence results showed that AMDV–DL125 was more likely to invade monocytemacrophages in the presence of antigen–antibody complexes.The results of q RT-PCR for ADE-related cytokines revealed that the expressions of IFN-?R1,IL-2R?,IL-18,TNF-R1 and TNF-R2 were upregulated while IL-6 was downregulated.ADE related genes were detected by q RT-PCR,and the expressions of SOCS 1,RIG-I,TNFRs and TRAFs were upregulated,while the expressions of JAK-1 and MDA-5 were downregulated.This sresearch model preliminarily confirmed that the ADE effect of AMDV-DL125 was dependent on antigenantibody complex during the infection of mink peripheral blood monocyte-macrophages.(2)The whole transcriptome high-throughput sequencing of the ADE in vitro research model constructed by the above experiment revealed a large number of differentially expressed genes,including 2658 m RNA,2588 lnc RNA,and 1428 mi RNA;554 pairs of genes were found to be mutually regulated.The GO enrichment showed significant enrichment of metabolic process,binding,cytoskeleton and other functions.The KEGG pathway enrichment revealed that Fc?R receptor family mediated phagocytosis,phagosome,endocytosis,NF-?B,MAPK,To LL-like receptor and PI3 K signaling pathway(PI3K-Akt signaling pathway)were significantly enriched.Further studies revealed significant upregulation of Syk expression,a gene located upstream of the Fc?R receptor family mediated macrophage endocytosis pathway,predicting that it may be a key gene regulating the ADE mechanism during infection.In addition,the accuracy of the sequencing results was demonstrated by verifying the relative expression of some differential genes by q RT-PCR technique.(3)To verify the regulatory role of Syk gene on ADE mechanism in the process of AMDV infection,we constructed the overexpression mink Syk gene plasmid Syk-pc DNA3.1 and Syk-si RNA to intervene the expression of Syk gene in peripheral blood mononuclear cells of mink.After adding antigen-antibody complexes to mink peripheral blood mononuclear cellsand incubating them for 12 h,it was found that the copy number of AMDV-DL125 was significantly increased in the overexpressing Syk group and decreased in the suppressing group compared to the control group.The results of indirect immunofluorescenceshowed that monocytes in the overexpression group of Syk were more susceptible to viral invasion.The results of q RT-PCR for ADE-related cytokines showed that IL-10 was upregulated when Syk was overexpressed,further mediating ADE effects.While the expression of antiviral factor receptors(IFN-?R1,TNF-R1 and TNF-R2)was upregulated,indicating that the virus may be in an active state.When Syk expression was inhibited,IL-10 expression was also inhibited and ADE effects were reduced.The expression of antiviral factor receptor was reduced because less virus entered the cell interior.The results showed that Syk played an important role in the regulation of ADE effect during AMDV infection.This study assessed the prevalence of AMDV in China,and preliminarily explored the ADE mechanism that exists in the process of AMDV infection.It provides reasonable suggestions for the prevention and control of AMDV in China,provides research ideas for further exploring the mechanism of AMDV infection,and lays a theoretical foundation for the development of AMDV vaccines.