Vertical transmission of hepatitis E virus in pregnant rhesus macaques

Abstract

Hepatitis E virus (HEV) is the major pathogen of viral hepatitis. HEV causes high mortality in pregnant women. Its infection during pregnancy usually leads to fulminant hepatic failure, spontaneous abortions, premature delivery, or stillbirth. Vertical transmission of HEV has been reported, but the pathogenesis during pregnancy remains largely elusive. Pregnant rhesus macaques were infected with HEV to explore the pathogenesis of genotype 4 HEV infection during pregnancy. Active HEV infections were established with shedding viruses in the feces and blood, and elevated liver enzymes. Notably, higher viral titers and longer durations of HEV infection were found in HEV-infected pregnant rhesus macaques than in non-pregnant macaques. Premature delivery and fetal death occurred in one of the HEV-infected pregnant rhesus macaques. HEV RNA was detected in the liver, spleen, kidneys, and intestines of the dead fetus. This result strongly indicated vertical HEV transmission from mother to fetus. Maternal-transferred antibodies were observed in one of the babies with poor protection. The expressions of interferon-stimulated genes (ISGs) related to HEV infection were completely different between pregnant and non-pregnant rhesus macaques. During pregnancy, impaired innate immune responses, reduced progesterone levels, and shifts in immune states may aggravate HEV infection and result in adverse pregnancy outcomes.

Figure 1

Profile of pregnant rhesus macaques infected with HEV. The copy number of HEV in the serum and stool in pregnant rhesus macaques was quantified by qRT-PCR, and anti-HEV IgG and IgM antibodies were determined by ELISA (A–C) or non-pregnant rhesus macaques (Mean ± SD of three non-pregnant HEV-infected rhesus macaques) (D). Asterisk (*) shown there is a significant difference of viral titers between pregnant rhesus macaques and non-pregnant rhesus macaques in indicated time points. *P < 0.05; **P < 0.01. Arrow indicated the time of delivery. The level/activity of liver enzymes AST (E), ALT (F), ALP (G), and T-BiL (H) was determined at indicated time points.

Figure 2

Vertical transmission of HEV from mother to fetus. The premature dead fetus born from HEV infected pregnant rhesus (06230#) (A). The viral titer of liver, spleen, kidney, and intestine of the dead fetus were quantified by qRT-PCR (B); HEV antigens were detected in the liver, spleen, kidney and intestine of the dead fetus by IHC inoculated with PBS (up panel) or HEV ORF2 specific antibody (down panel) (C). Histopathological analysis of liver, spleen, kidney, and intestine from the dead fetus (D).

Figure 3

HEV infection in neonates born from HEV-infected mothers. Copy number of HEV in the feces (A) or serum (B) of neonates born from HEV-infected mothers quantified by qRT-PCR. Anti-HEV IgM (C) and IgG (D) antibodies determined by ELISA.

Figure 4

Adverse pregnancy outcomes caused by HEV infection. The concentration of progesterone in HEV-infected pregnant rhesus macaques at indicated times points (A). The concentration (mean value) of progesterone during the whole pregnancy (B). The relative expressions of progesterone receptor in HEV-infected pregnant rhesus macaques were normalized with non-pregnant rhesus macaque at indicated time points (C). The folds of progesterone receptor (mean value) during the whole pregnancy (D). The levels of IFN-γ, IL12, IL4 or IL10 were determined in these HEV infected pregnant or non-pregnant rhesus macaques (E–H). The ratio of IL4/IFN-γ using the concentration of IL4 or IFN-γ in each indicated time points (I). The ratio of IL4/IFN-γ using the mean value of IL4 or IFN-γ during the whole pregnancy (J). The ratio of IL12/IL10 using the concentration of IL12 or IL10 in each indicated time points (K). The ratio of IL12/IL10 using the mean value of IL12 or IL10 during the whole pregnancy (L). Arrow indicated the time of delivery.

Figure 5

ISGs expressions in HEV-infected pregnant or non-pregnant rhesus macaques. The gene expression of ISGs in the plasma of HEV-infected pregnant or non-pregnant rhesus macaques. The gene expression of 12 ISGs were quantitated by qRT-PCR and normalized with pre-infection (A–L). The day of farrowing was highlighted by arrow. The relative expression of ISGs in pregnant rhesus macaques was compared with non-pregnant rhesus macaques. Heatmap was generated for the different parameters analyzed at indicated time points (M) or individuals (N).