Epidemiological Investigation Of Hepatitis E In Kunming Area And Establishment Of Animal Models For Hepatitis E Virus Infection

Hepatitis E (HE) is a zoonotic disease caused by hepatitis E virus (HEV) infection. HE was mainly outbreak in some developing countries with poor sanitary conditions in Asian and African. People are generally susceptible to HEV, in which susceptible populations are mainly older children and young adults. HEV infection can cause miscarriage or death in pregnant women with high mortality of 15% to 30%. The infectious disease report from 2002 to 2004 issued by the Ministry of Health shows that China is a country of high incidence of hepatitis E, a continuing growth for infection rates. HE has become an important public health problem.. More and more researches are evidenced that the HEV can infect pigs, dogs, cows, goats, sheep, deer, primates, chickens, shellfish, rodents and other animals, in which infection rate of swine is the highest, and virus isolated from pigs and human strains also share high homology. Furthermore, HEV was easily degradable and unstable like the other hepatotropic viruses, difficult to isolate a large amount of relatively pure HEV from the feces, bile, etc. and lack of suitable cell culture system, to some extent, limited the further study of HEV. In order to understand HEV infection in Kunming area and try to establish animal models for HEV infection, we carried out serum epidemiological investigation of HE in human, pig, dog in Kunming area, and used gerbils and tree shrews to establish porcine HEV infection model. This study lays a foundation for mechanism study of swine HEV infection, development of effective measures for prevention and control.Specific studies are as follows.1.Investigation of serum epidemiology of hepatitis E in Kunming. To understand the serum epidemiological characteristics of hepatitis E in human, pigs, dogs in Kunming, we used double antigen sandwich ELISA to detect HEV IgG antibodies in human sera (456 samples), swine sera (835 samples), canine sera (285 samples). The results showed that the positive rated for HEV IgG antibodies in human, swine, canine serum samples were 43.20%,55.45% and 20.35%, respectively. In human beings, HEV IgG antibody positive rate in male and female populations were 43.25% and 43.11%, and there is no significant difference between men and women (P> 005.). However, HEV antibody positive rates in groups of 21 to 30 years old (48.82%),31 to 40 years old (50.86%), of 41 to 50 years old (42.86%) were significantly higher than that in groups of 10 to 20 years old (23.08%) and more than 51 years old (32.89%) groups (P<0.05). In swine population, HEV IgG antibody positive rates in groups of piglets (1 month to 2 months of age), fattening pigs (2 months to 6 months of age) and adult pigs (6 months or more) were 46.85%,57.06% and 65.42%, respectively. There was a significant difference (P<0.05) among the different age groups. In dog population, HEV IgG antibody positive rates in Urban stray dogs, family dogs and farm dogs was 59.18%,13.16% and 10.71%, respectively. There was a significant difference between stray dogs and family dogs or farm dogs for HEV IgG antibody positive rates (P<0.05). HEV IgG antibody positive rates in sled dogs, German Shepherd, Kunming dogs were significantly lower than that in other breeds (P<0.05). HEV IgG antibody positive rate in groups of less than 1 year old was significantly higher than that in dog groups of 1 to 5 years old,5 to 10 years old, more than 10 years older (P<0.05). Our studies suggested that HEV infection presented in human, pigs and dogs in Kunming, and HEV IgG positive rates in human, pigs and dogs were age-related. HEV IgG antibody positive rate in human group of 21 to 50 year-olds is relatively higher; HEV IgG positive rate in swine herd gradually increased along with age increase; HEV IgG antibody positive rate in dogs declined along with age increase.2.The molecular epidemiological investigation of swine hepatitis E virus in Kunming and the surrounding counties.To understand the molecular background and epidemiological characteristics of HEV in large-scale pig farms in Kunming, Two pair nested PCR primers was designed and synthesized according to ORF2 conservative region of HEV sequences deposited in GenBank. The nested PCR was applied to 187 fecal samples of piglets, and the samples with 348 bp amplified fragment were regarded as positive. The positive rate was 6.95%. The positive rates of samples collected from Luquan County, Jiangchuan County, Fumin were 4.54%,8.00% and 9.52%, respectively. The amplified fragments were subjected to sequence analysis and phylogenetic analysis. The ucleotide sequences of 348 bp amplified fragments among nine swine HEV shared 87.1%～99.4% nucleotide homology, and shared 77.0%～ 81.9%,52.2%～53.6%,77.0%～88.2% and 77.9% to 96.8% nucleotide homology with those of type Ⅰ,Ⅱ,Ⅲ Ⅳ HEV. Phylogenetic tree analysis showed that the nine swine HEV strains were belonged to genotype IV. Our study indicates that HEV infection is existed in large-scale pig farms in Kunming and the HEV is belonged to genotype Ⅳ.3.The histopathological study of pig liver infected with hepatitis E virus.In order to understand histopathological changes in liver infected with swine hepatitis E virus,176 liver samples were collected from Kunming market and subjected to immunohistochemical staining and hematoxylin-eosin staining. Immunohistochemical staining result showed the positive rate for HEV antigen in liver was 65.34%.51.30% of the liver samples were shown as inflammatory cell infiltration by hematoxylin-eosin staining,28.70% of the liver samples were shown as fibrous connective tissue proliferation,20.87% of the liver samples were shown as liver cell atrophied and 19.13% of the liver samples were shown as liver cell degeneration and necrosis. Our study indicates that HEV infection is prevalent among commercially available pork livers in Kunming and corresponding pathological changes is occurred.4.The experimental study of gerbils infected with swine hepatitis E virus.In order to explore the possibility of establishing swine hepatitis E virus infection in gerbil model, HEV-positive pig fecal supernatant was inoculated into gerbils by intraperitoneal injection. The alanine acid transaminase (ALT) and aspartate aminotransferase (AST) changes in gerbils’ sera were determined periodically, HEV RNAs in livers, feces, blood and small intestines were detected, and histopathological changes and distribution of HEV in the livers were observed. The results showed that the values of ALT and AST in gerbils challenged with HEV were raised on day 7 postinfection, then after began to decrease. But ALT values in challenge group were still higher than that in control group on day 35 postinfection. The ALT values in challenge group were two-fold higher than that in the control group. The AST values in challenge group were decreased to the normal range (based on the negative control) on day 35 postinfection. HEV RNAs can be detected from feces and livers at all time, while HEV RNAs can be detected from blood and intestines occasionally. Liver tissue was shown as lymphocyte infiltration, granular degeneration of liver cells, focal lymphocytic infiltration, liver cell cord disorder and bile duct hyperplasia. Late it presented as multiple lymphocyte infiltration, hepatocellular necrosis, increased Kupffer cells and periportal fibrous connective tissue hyperplasia. There were some pathological changes in small intestine, kidneys, brains, spleens and other tissues. The positive reaction was detected in liver cytoplasm by immunohistochemical staining. Our study indicates that a gerbil model for swine hepatitis E virus infection is established successfully.5.The experimental study of tree shrew infected with swine hepatitis E virus.In order to explore the possibility of establishing swine hepatitis E virus infection in tree shrew model, HEV positive swine fecal supernatant was inoculated in tree shrews by intraperitoneal injection. The ALT and AST changes in tree shrews’ sera were determined periodically, HEV RNAs in livers, feces, blood, small intestines, kidneys and spleens were detected, and histopathological changes.The results showed that the serum ALT and AST values were elevated on day 7 postinfection, then after began to decrease. But the ALT and AST values in challenge group were not reduced to the normal level based on the control group on day 28 postinfection. HEV RNAs can be detected from livers, feces, blood and small intestines of tree shrew on day 7 postinfection, while HEV RNAs can be detected from kidneys and spleens occasionally. Liver tissue was shown as sinus congestion, liver cell degeneration, multiple lymphocytic infiltration, increased Kupffer cells, periportal fibrous connective tissue hyperplasia and other lesions. There were some pathological changes in small intestines, kidneys and other tissues. Our study indicates that a tree shrew model for swine hepatitis E virus infection is established successfully. This study lays a foundation for further study in HEV cross-species transmission and pathogenic mechanisms.