Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits

<div><p>Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV.</p></div

Histopathological analysis of livers and kidneys.

<p>A. There were no gross histopathological lesions in the liver sections from the control group. B. A large number of lymphocytes infiltrated the liver, and the bile ducts proliferated in the portal tracts of the liver in the experimental group. C. No gross lesions were observed in the kidney sections from the control group. D. A large number of lymphocytes infiltrated into the mesenchyme of the renal cortex. H&E staining.</p

Changes in serum HEV antigen and anti-HEV IgG based on ELISA at various days post-inoculation.

<p>A. Serum HEV antigen was first detected in the T5 rabbit at 35 dpi and was elevated until 49 dpi. Serum HEV antigen was also detected in the T6 and T8 rabbits at 49 dpi. B. Anti-HEV IgG appeared in the T5 rabbit at 42 dpi and was elevated until 49 dpi. Other rabbits had no anti-HEV IgG until the end of the experiment. All rabbits in the control group were designated the “control”.</p

HEV ORF2 antigen localization in various tissues from the experimental group detected using immunohistochemical staining.

<p>A. Liver. The positive signal for HEV antigen was detected in hepatocytes and the epithelium of bile ducts. B. Kidney. The positive signal for HEV antigen was detected in the epithelium of the renal tubules. C. Salivary gland. The positive signal for HEV antigen was detected in the duct epithelium of salivary gland. D. Tonsil. The positive signal for HEV antigen was detected in the crypt epithelium and in macrophages in the tonsil. E. Spleen. The positive signal for HEV antigen was detected in macrophages or dendritic cells in the spleen. F. Thymus gland. The positive signal for HEV antigen was detected in thymic epithelial cells in the medulla of the thymus gland. G. Heart. The positive signal for HEV antigen was detected in the myocardium. H. Lung. The positive signal for HEV antigen was detected in the epithelium of the alveoli and bronchioles. I. Pancreas. The positive signal for HEV antigen was detected in the duct epithelium of the pancreas. J. Small intestine. The positive signal for HEV antigen was detected in the mucosal epithelium in the small intestine. K. SR. The positive signal for HEV antigen was detected in the follicle-associated epithelium. L. Appendix. The positive signal for HEV antigen was detected in macrophages or dendritic cells of lymphoid follicles. The positive signal is yellow.</p

HEV ORF2 antigen localization in various tissues from the control group detected using immunohistochemical staining.

<p>No positive signal (yellow) was detected in these tissues of the control group. A-L: liver, kidney, salivary gland, tonsil, spleen, thymus gland, heart, lung, pancreas, small intestine, SR or cecum.</p

Detection of HEV RNA in the feces and throat of the test group.

<p>Detection of HEV RNA in the feces and throat of the test group.</p

Distribution of HEV RNA and negative-stranded RNA in various tissues of rabbits infected with swine HEV at the indicated days post-inoculation.

<p>Distribution of HEV RNA and negative-stranded RNA in various tissues of rabbits infected with swine HEV at the indicated days post-inoculation.</p

ALT and AST levels at 28 and 49 dpi, respectively.

<p>A. The average ALT level was significantly increased in the experimental group compared with the control group (p<0.05) at 28 dpi and 49 dpi. At 49 dpi, the average ALT increased drastically to 96.3 U/L. B. The AST level increased significantly at 28 dpi and 49 dpi (p<0.05). The average AST level reached 102.1 U/L in the experimental group. (*, p<0.05).</p