Fig 1.
Active sub-clinal infection in pigs.
(A) Viremia and fecal viral shedding from pigs inoculated with US-2 HEV. Weekly RNA data are presented. Mock-infected groups remained negative throughout the study. (B) US-2 HEV tissue distribution at 84 days post-inoculation. HEV RNA loads in gall bladder, liver, ileum, and epididymis from US-2 HEV inoculated pigs. Negative control pigs used in the study remained negative. ** = p < 0.01, *** = p < 0.001.
Fig 2.
Hepatitis E virus infects spermatozoa.
(A) Immunohistochemical detection of hepatitis E (red) in the head of spermatozoa obtained from the US-2 infected pigs at day 84 post infection; Hepatitis E virus ORF2 is red; DAPI stain is blue (nucleus). (B) Flow cytometry analysis demonstrating the percentage of sperm cells infected with hepatitis E virus (US-2 strain). (C) Immunodetection of hepatitis E demonstrating infectious sperm cells in HepG2/C3A cells. (D) Flow cytometry analysis demonstrating the percentage of HepG2/C3A cells infected with the sperm cell lysates from mock and US-2 HEV infected pigs. (E) Infectious titer of sperm-derived HEV using HepG2/C3A cells. US-2 HEV RNA loads in culture supernatant (S) and cell lysates (CL) of HepG2/C3A cell cultures after inoculation with the lysed sperm cells. Independent biological experiments, mean ± SD of three replicates, are presented. The dotted line represents the cut-off value demonstrating the background referring to the attachments of the virus to the cell surfaces. (F) HEV RNA loads in sperm cells suspension, seminal fluid, and bile from US-2 HEV inoculated pigs. *** = p < 0.001, **** = p < 0.0001.
Fig 3.
Hepatitis E virus alters spermatozoa motility and morphology.
(A) Light microscopic observation of 200 live spermatozoa harvested from mock or infected pig epididymis. Sperm cells demonstrated decreased progressive motility when infected by HEV US-2. PR–progressive motility of sperm (moving active, either linearly or in a circle, regardless of speed); NP–non-progressive motility (all other patterns of motility with absent progression. IM–immobility. (B) Light microscopic observation of live sperm cells harvested from mock or infected pig epididymis. Sperm from US-2 HEV infected pigs showed a significant increase in spermatozoa with head abnormalities. No significant changes were seen in tail of the sperm cells. * = p < 0.05, ** = p < 0.01. (C) Histogram plot was used to demonstrate the data from flow cytometry. Sperm cells from mock non-infected pigs and from US-2 HEV infected pigs.