Cytokines Changes And Viral Distribution In Vivo Of Mink Infection With Mink Enteritis Virus

Mink viral enteritis caused by mink enteritis virus(MEV) is a highly contagious disease, and the symptoms of the infected minks include violent diarrhea,vomiting, and also the morbidity and mortality are high.With the rapid development of fur animal breeding industry, the disease often broke out and caused huge economic losses to the mink industry. So the study of mink enteritis virus pathogenicity and immune-related factors changes plays a significant role in prevent MEV.In this study, the methods of SYBR Green Ⅰ real-time quantitative RT-PCR assay for detection of mink IFN-α, IFN-β and IFN-γ(MiIFN-α, MiIFN-β and MiIFN-γ) mRNA have been established using four pairs of specific primers which designed by the MiIFN-α, MiIFN-β and MiIFN-γ gene sequences,and also with the mink glyceraldehyde-3-phosphate dehydrogenase(MiGAPDH) gene sequences as an internal control. The target genes were amplified and inserted to pMD18-T vector, and then the standard curves of the real-time PCR methods have been established. The results showed that the Ct value of MiIFN-α, MiIFN-β, MiIFN-γ and MiGAPDH mRNA had a good linear relationship(R2=1.00) with the standard from 10copies/μL to 107copies/μL, and the melting curve showed a single peak. The results of sensitivity assay showed that the genetic testing minimum value ware 10 copies/μL. The coefficient variations of inter-assay and intra-assay were both less than 4.5%. The result of clinical test showed that sixth day after the infected of mink enteritis virus, the relative expression of MiIFN-α、MiIFN-β and MiIFN-γ reached the highest level. The expression of MiIFN-α was higher than MiIFN-β and MiIFN-γ,and during the infection period the expression of MiIFN-α was significantly increased.This detection method provided an effective tool for quantitative analysis of mink IFN-α, IFN-β and IFN-γ mRNA.In order to illuminate the dynamic changes of mink cytokines after being infected by different strains of mink enteritis virus(SMPV-11 and MEV-62), the method of fluorescent dye SYBR GreenⅠquantitative RT-PCR has been established and applied to detect the mink cytokines, IFN-α,IFN-β, IL-4 and TNF-α. The results showed that after infection of SMPV-11 and MEV-62 virus the relative expression of IFN-α, IFN-β, INF-γ and IL-4 were gradually increased in 2~6 days, and peaked in 6 days. The expression levels of IFN-α, IFN-β, INF-γ and IL-4 in 10 days and 15 days, SMPV-11 group was higher than MEV-62 group, and with the different the relative expression of TNF-α increased gradually in 0~2 days, peaked in 2 days, reduced to a minimum in 6 days, and then began to increase again. This study of peripheral blood cytokine dynamics provide a theoretical basis for the minks after MEV infection.In order to investigate the distribution of mink enteritis virus in vivo after infected, two experiments have been designed. First, pathological changes, white blood cells changes and virus distribution in different organs have been detected, after the virulent MEV strain SMPV-11 infected by oral. And then after the infection of attenuated MEV vaccine strain MEVB-F61 by muscle injection, the virus distribution in different organs and white blood cells changes have been detected. The results showed that after the infection of SMPV-11, severe clinical symptoms such as fever, loss of appetite andbloody stools appeared, and necropsy show that mesenteric lymph nodes, spleen and liver enlargement have bleeding, intestinal bleeding and wall thinning. After 24 h of infection, MEV viruse can be detected in mink tissues and the high level of MEV concentration at 4~6th days. The clinical symptoms of MEV-F61 was few, and aslo there was no obvious pathology after autopsy. In the brain, kidneys and lungs failed to detect the presence of MEV and low viral load in other tissues. Heart, liver, intestines and other organs have been undetectable MEV in 15 th days. The peak time of detox stage of SMPV-11 group and MEV-F61 group minks is 4~8th days after infected, indicating that this stage is the most critical period for the virus spread outside. And the highest virus load of these two strains were intestinal tract, lymphoid tissue and spleen, suggesting that intestinal tract,mesenteric lymph nodes and spleen are the main target organs for MEV infected. The research of clinical features, pathology and viral distribution in vivo caused by MEV, are conducive not only to the further study of pathogenesis of MEV, but also to the theoretical foundation of MEV attenuated vaccine immune.