Study On The New Zealand Rabbit For Genotype 4 Swine Hepatitis E Virus Artificial Infection

Genotype 4 swine hepatitis E virus(sHEV-4)has been the important zoonotic pathogen,but animal models for researching the cross-species infection of sHEV-4 are less and mainly infected through the intravenous injection that is not natural infection route and can't objectively show the natural progress.The lack of animal model for cross-species infection of sHEV-4 becomes the bottleneck of researching the pathogenic mechanism further.In this study,we build two kinds of animal models for cross-species infection of sHEV-4 using the New Zealand rabbit as the experimental animal through intraperitoneal injection(i.p.)and intragastric administration(i.g.),and systematically researched the similarities and differences of the viral shedding,pathological change of organs,and viral distribution and replication sites on the two animal models applying histopathology,immunohistochemistry(IHC)and molecular biological methods.Moreover,we initially the function of intestinal mucosal immune during the two infection routes.The results were as follows:1.The acute experiment for rabbits infected by swine HEV-4 through i.p.,i.g.and intestinal injection(i.i.).The HEV RNA could be detected in blood from portal vein and livers of rabbits at 24 and 48 hours post infection(hpi)through i.p..And the viral load was not significant differences at different time points.The HEV RNA could be only detected in intestinal tissues of rabbits post infection through i.g.and i.i..And the viral load in i.i.group was more than it in i.g.group.The lymphocyte activity in sacculus rotundus(SR)significantly elevated in rabbits infected through i.g.and i.i.(P<0.05),and it showed intestinal mucosal immune response was activated.2.Results of rabbits infected with sHEV-4 through i.p.route.The rabbits were infected by sHEV-4 suspension(6.63×107)through i.p.for 7 consecutive days.The positive rate of HEV RNA in feces and throat swabs were 8/8 and 3/8 respectively.And the viral shedding was intermittent.The viral load in feces was from increasing to decreasing.The level of sera aminotransferase were significantly increased at both 28 and 49 dpi(P<0.05).The positive rate of antigen in serum 3/8.And anti-HEV IgG was firstly detected at 42 dpi and positive rate is 1/8.The obvious inflammatory injuries appear in livers and kidneys.HEV RNA could be detected in livers(8/8),intestines(7/8)and other various organs.The viral load was the highest in liver and intestine.HEV ORF2 protein mainly located in the cells from epithelial tissues such as hepatocytes,epithelium of duct in salivary gland and the dendritic cells and macrophages in lymphoid tissues by IHC.The detection of HEV negative-strand RNA and ORF3 protein show that HEV could replicates in the liver,gut,salivary gland,tonsil,lymph node,spleen and so on.The number iIEL and goblet cells(GC)significantly increase(P<0.05).The number of CD3+,CD4+and CD8+T cells in SR and appendix also increased significantly(P<0.05).The number of plasma cells and level of SIgA in intestine increased significantly at 49 dpi(P<0.05).The intestinal mucosal immune was activated.3.Construction of about rabbit model infected by sHEV-4 through i.g..The rabbits were infected with the same viral suspension for 7 consecutive days.The positive rate of HEV RNA in feces and throat swabs were 4/8 and 1/8 respectively.The fecal viral shedding delays,persists short duration and was low load.The level of serum aminotransferase elevated significantly at 49 dpi(P<0.05)and dropped to normal levels at 63 dpi and was lower than it in i.p.infection.The positive rate of serum HEV antigen and antibody were 2/8 and 5/8 respectively.The light inflammatory injuries appear in livers and kidneys.The number of organs with HEV RNA was fewer than infection through i.p..Livers(5/8)and intestines(4/8)were main organs for viral distribution.The detection of HEV negative-strand RNA and ORF3 protein shows that liver was the main replication sites.In part rabbits,intestine,tonsil,salivary gland and spleen were also replication sites of HEV.The number of iIEL significantly increased post infection(P<0.05),and The diameter of white pulp in spleen and lymph nodule enlarged significantly(P<0.05).The number of T cells in duodenum,SR and appendix was significantly increased(P<0.05).At the same time,the number of plasma cells in intestine,spleen and lymph nodes also increased significantly(P<0.05),and level of SIgA was significantly higher than it in i.p.infection.It showed that the the immune response of humoral immune and intestinal mucosal immune in i.g.infection were higher than i.p.infection,and the injury caused by sHEV-4 was lighter in i.g.infection than i.p.infection.The results above show that sHEV-4 can infect New Zealand rabbits through i.p.and i.g.routes and cause the classical viral hepatitis pathological change and injuries of various organs.The positive rate of infection is higher through i.p.than i.g..But it can more objectively imitates the infection progress and injuries of animal in HEV natural infection through i.g.route of infection.HEV can shed in saliva of infected rabbits owing to the viral distribution and replication in salivary gland and tonsil.sHEV-4 can replicate and distribute in various organs but liver and intestine are main sites.It can more effectively activate the humoral immunity of rabbits during i.g.route of infection,and the reason about low positive rate of HEV RNA in feces and tissues through i.g.route of infection may be the early response of intestinal mucosal immune.The results show that rabbit can be used as the animal model for sHEV-4 infection.