Pathogenicity Study Of Porcine Astrovirus Type 1 (PAstV1) GX1 Strain

Astrovirus(AstV)are the single-stranded positive sense RNA virus,non-enveloped,and the diameter of the virus is about 28-30nm.The virus is highly degree of genetic variability and diversity,and has a wide range of hosts.Because of the ability of AstVs to spread across species and adapt to new hosts,it could be a potential zoonotic pathogen.Astrovirus could cause diarrhea in humans and other animals,especially human astrovirus(HAstV),which is considered to be the second largest pathogen of viral diarrhea in infants.Other pathogenic experimental studies have shown that astrovirus infection can cause diarrhea in turkey and mice.In addition,porcine astroviruses(PAstV)are widely spread around the world which had highly infection rate,but the pathogenicity of PAstV in pigs was unknown.Early research of turkey chicken models showed that diarrhea was caused by a decrease in maltase activity which result in osmotic diarrhea because of obstacles in digestion and absorption of sugar in the intestine.At the same time,researchers used human astrovirus to infect CaCo-2 cells and found that the F-actin rearrangement and the permeability of the intestinal barrier was increase,stuggested that the leading to fluid secretion into intestine causing diarrhea.Previous laboratory studies have shown that porcine astrovirus type 1 infection causes mild diarrhea,but its pathogenesis remains unclear,therefore this experiment will further study its pathogenic mechanism on this basis.In this study,twenty-three seven-day-old suckling piglets were randomly assigned to two groups,including twelve in the PAstV-challenged group and eleven in the control group.The porcine astrovirus type 1 strain(PAstV1 strain,TCID50 is 1×104.5/ml)isolated from the laboratory was used for challenge,then carry on observing clinical symptoms,collecting feces,taking body temperature and body weight every day.3 piglets were selected from each of the 2 treatment groups for slaughter at the 2dpi,4dpi,7dpi and 10dpi after the challenge.The pathogenicity of swine astrovirus was studied by real-time RT-PCR and immunohistochemistry.The results showed that PAstV caused transient mild symptoms after infection in suckling piglets.The main clinical symptoms were mild diarrhea,decreased appetite and slow growth;Histopathological changes indicate that the villus of the duodenal,jejunal and ileal mucosal epithelium of infected piglets are degenerated,necrotic and ruptured or irregularly arranged.A large number of lymphocytes and neutrophils infiltrate,different degrees of activation and proliferation of mesenteric lymphocytes occur simultaneously with vacuolar degeneration.Mild inflammatory response such as loss of lymph node structure and its peripheral follicular-like activation hyperplasia or a small amount of peripheral blood vessel expansion and congestion may occur.The Fluorescence quantitative results showed:1)PAstV was detected in the feces of the challenged group compared with the control group,and a large amount of detoxification was found on the 2dpi-6dpi.After the 7dpi,although there was detoxification,the amount of detoxification was significantly reduced;At the 2dpi,4dpi and 7dpi we found the significant difference in the viral load of the intestinal tissue between thechallenged group and the control group(P<0.01),including the ileum,duodenum,jejunum and mesenteric lymph node have higher virus content.However,compare with the challenged group and the control group at 10dpi,only the ileum and colon were significantly different(0.01<P<0.05),and the viral load of the other tissues in the experimental group and the control group was not significantly changed(P>0.05).Overall,the viral load during the entire study of the intestinal tissue in the challengd pigs gradually increased until reaching the peak at 4dpi and then started to fall.IHC results showed that the PAstV antigens were detected in the cytoplasm of the intestinal villi cells in the entire day after the challenge.The antigens could not be observed in the nucleus.The number of immunolabels detected on in the 2nd,4th day were higher,while 7th and 10th day were less,where the antigen-labeled sites were duodenum and jejunum villi and top villi of ileum,cecum,colon and rectum.No immunolabeled PAstV antigen was detected in the control group.Therefore,it confirmed that the porcine astrovirus has a certain pathogenicity to the suckling piglets,and there is still detoxification at the 10dpi after the challenge.Fluorescence quantitative PCR was used to detect cytokine changes in intestinal tissues.It was found that IFN-? and ISG54 appeard up-regulation after challenge,indicating that porcine astrovirus infection stimulates the innate immune system.In addition,we detected of the mRNA expression changesof the barrier permeability of intestinal tissues revealed protein such as the TPJ2 tight junction protein and ZO-1(closed small band protein 1)in intestinal epithelial cells after porcine astrovirus infection by RT-qPCR.The resulted shown the mRNA expression level of TJP1,TJP2 and ZO-1 weredown-regulated,which may lead to intestinal barrier function is reduced and the permeability is increased.Therefore,it is speculated that PAstV may cause an increase in permeability of intestinal epithelial cells after challenge,resulting in secretion of fluid into the intestinal lumen causing diarrhea.This study is the first time to focus the pathogenesis of PAstV,aiming to further understand the pathogenicity and pathogenic mechanism of porcine astrovirus and provide scientific basis for clinical diagnosis,immunization and prevention of astrovirus.