Experimental Infection of Pregnant Female Sheep with Zika Virus During Early Gestation

Abstract

Zika virus (ZIKV) is a vertically and sexually transmissible virus resulting in severe congenital malformation. The goal of this study was to develop an ovine model of ZIKV infection. Between 28–35 days gestation (DG), four pregnant animals were infected with two doses of 6 × 106 PFU of ZIKV; four control animals received PBS. Animals were evaluated for 45 days (D) post-infection (PI) and necropsies were performed. Viral RNA was detected in infected ewe peripheral blood mononuclear cells (PBMC) during the first week PI; however, all fluids and tissues were negative upon culture. Anti-ZIKV IgM (1:400) and neutralizing antibodies were detected in all infected animals. Clinical disease, virus, or ZIKV antibodies were not detected in control ewes. After two weeks PI, fetal loss occurred in two infected animals, and at necropsy, three infected animals had placental petechiation and ecchymosis and one had hydramnion. Fetal morphometrics revealed smaller cranial circumference to crown-rump length ratios (p < 0.001) and relative brain weights (p = 0.038) in fetuses of infected animals compared with control fetuses. Immunophenotyping indicated an increase in B cells (p = 0.012) in infected sheep. Additionally, in vitro experiments using both adult and fetal cell lines demonstrated that ovine cells are highly permissive to ZIKV infection. In conclusion, ZIKV infection of pregnant sheep results in a change in fetal growth and gestational outcomes.

Experimental Infection of Mid-Gestation Pregnant Female and Intact Male Sheep with Zika Virus

“…Furthermore, previous research indicates that the viral kinetics of ZIKV in fetal sheep cells has been shown to be similar to that in immortalized Aedes albopictus cells [26]. We have also demonstrated that immortalized adult sheep kidney cells and immortalized fetal sheep testicular cells are susceptible to ZIKV infection and can sustain viral replication for many days [25].ZIKV is also unique in that sexual transmission occurs with infection of the male reproductive tract with possible establishment of prolonged, asymptomatic infection in men [27][28][29][30]. This carrier status in males may make men more likely to sexually transmit ZIKV to females than vice versa [1].…”

“…Furthermore, previous research indicates that the viral kinetics of ZIKV in fetal sheep cells has been shown to be similar to that in immortalized Aedes albopictus cells [26]. We have also demonstrated that immortalized adult sheep kidney cells and immortalized fetal sheep testicular cells are susceptible to ZIKV infection and can sustain viral replication for many days [25].…”

“…An ovine IgM capture ELISA was developed based on previous methods for ZIKV IgM detection and an IgM capture ELISA protocol developed for WNV infection in the equine, as previously described [25,52,54]. Briefly, plates were coated with 10 µg polyclonal rabbit anti-sheep IgM (Bethyl Laboratories, Inc., Montgomery, TX, USA) in carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at 4 • C. After blocking with PBS with 2% equine serum and 0.5% Tween20 for 30 min at RT, samples were diluted at 1:400 in blocking buffer (PBS with 5% milk and 0.5% Tween20) and added in duplicate wells for 1 h at 37 • C. After washing, ZIKV virus or negative cell culture, grown and formalin inactivated as previously described [55], was added to each well and incubated for 1 h at 37 • C. After washing, a 1:50 dilution of anti-flavivirus antibody (D1-4G2-4-15, ATCC) in blocking buffer was added to each well and incubated for 1 h at 37 • C. Following incubation, a 1:100 dilution of sheep anti-mouse IgG antibody conjugated with horseradish peroxidase (Bethyl Laboratories, Inc.) in blocking buffer was added to each well and incubated for 1 h at RT.…”

“…Quantitated cells included CD4+ T helper cells, CD8+ T effector cells, γδ T cells, CD14+ monocytes, and B cells. Cells were stained in duplicate using mouse anti-sheep CD45 (1.11.32, MCA2220PE, BioRad, Hercules, CA, USA), rabbit anti-sheep CD3 (SP7, ab16669, Abcam, Cambridge, MA, USA), goat anti-rabbit IgG (RRID AB_2539814, P-10994, Thermo Fisher Scientific), mouse anti-sheep CD4 (44.38, MCA2213A647, BioRad), mouse anti-sheep CD8 (38.65, MCA2216F, BioRad), mouse anti-human CD14 (TÜK4, MCA1568A700T, BioRad), mouse anti-sheep γδ T cell (WC1-N2, Washington State University Monoclonal Antibody Center, Pullman, WA, USA), goat anti-mouse IgG1 (1070-19, Southern Biotech, Birmingham, AL, USA), mouse anti-ruminant B cell (BAQ44A, S-BOV2064, Washington State University Monoclonal Antibody Center), and rat anti-mouse IgM (RMM-1, 406529, Biolegend, Waltham, MA, USA) as previously described [25].…”

“…The feasibility of sheep as a model for ZIKV infection has been established. We have shown that early gestation ZIKV infection of pregnant sheep results in detectable virus and a robust immune response by the ewes, with femur-sparing patterns of fetal growth retardation and changes in gestational outcomes in the absence of maternal clinical signs of infection [25]. The effects of ZIKV infection seen during the first trimester in sheep are comparable to those observed in humans, as well as fetal outcomes caused by ovine infections; primarily fetal loss [19][20][21][22][23][24].…”