Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas

BACKGROUND: Rift Valley fever virus (RVFV) is an arbovirus of the order Bunyavirales that causes severe disease in ruminants and humans. Outbreaks in sheep herds are characterised by newborn fatalities and abortion storms. The association of RVFV infections with abortions of ovines and other ruminants is well recognized, whereas the pathology resulting in abortion has remained undescribed. Accumulating evidence suggests that RVFV is abortogenic in humans as well, warranting more research on the interaction of RVFV with the ruminant and human placenta. METHODOLOGY/PRINCIPAL FINDINGS: Pregnant ewes were inoculated with a highly virulent strain of RVFV and necropsied at different days post infection. Tissues were collected and analysed by PCR, virus isolation, and immunohistochemistry. The results show that RVFV replicates efficiently in maternal placental epithelial cells before the virus infects foetal trophoblasts. Moreover, the virus was shown to bypass the maternal epithelial cell layer by directly targeting foetal trophoblasts in the haemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with foetal cells. Abortion was associated with widespread necrosis of placental tissues accompanied with severe haemorrhages. Experiments with human placental explants revealed that the same virus strain replicates efficiently in both cyto- and syncytiotrophoblasts. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that RVFV targets the foetal-maternal interface in both ovine and human placentas. The virus was shown to cross the ovine placental barrier via two distinct routes, ultimately resulting in placental and foetal demise followed by abortion. Our finding that RVFV replicates efficiently in human trophoblasts underscores the risk of RVFV infection for human pregnancy.</p

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A novel highly sensitive, rapid and safe Rift Valley fever virus neutralization test

Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization test (VNT). The VNT is superior in sensitivity and specificity and is therefore considered the gold standard serological assay. Classical VNTs make use of virulent RVFV and therefore have to be performed in biosafety level 3 laboratories. Here, we report the development of a novel VNT that is based on an avirulent RVFV expressing the enhanced green fluorescent protein (eGFP), which can be performed safely outside level 3 biocontainment facilities. Evaluation with a broad panel of experimental sera and field sera demonstrated that this novel VNT is faster and more sensitive than the classical VNT.</p

Four-segmented Rift Valley fever virus-based vaccines can be applied safely in ewes during pregnancy

Rift Valley fever virus (RVFV) causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. This mosquito-borne virus, belonging to the genus Phlebovirus of the family Bunyaviridae contains a tri-segmented negative-strand RNA genome. Previously, we developed four-segmented RVFV (RVFV-4s) variants by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins; Gn or Gc. Vaccination/challenge experiments with mice and lambs subsequently showed that RVFV-4s induces protective immunity against wild-type virus infection after a single administration. To demonstrate the unprecedented safety of RVFV-4s, we here report that the virus does not cause encephalitis after intranasal inoculation of mice. A study with pregnant ewes subsequently revealed that RVFV-4s does not cause viremia and does not cross the ovine placental barrier, as evidenced by the absence of teratogenic effects and virus in the blood and organs of the fetuses. Altogether, these results show that the RVFV-4s vaccine virus can be applied safely in pregnant ewes

Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas.

BACKGROUND:Rift Valley fever virus (RVFV) is an arbovirus of the order Bunyavirales that causes severe disease in ruminants and humans. Outbreaks in sheep herds are characterised by newborn fatalities and abortion storms. The association of RVFV infections with abortions of ovines and other ruminants is well recognized, whereas the pathology resulting in abortion has remained undescribed. Accumulating evidence suggests that RVFV is abortogenic in humans as well, warranting more research on the interaction of RVFV with the ruminant and human placenta. METHODOLOGY/PRINCIPAL FINDINGS:Pregnant ewes were inoculated with a highly virulent strain of RVFV and necropsied at different days post infection. Tissues were collected and analysed by PCR, virus isolation, and immunohistochemistry. The results show that RVFV replicates efficiently in maternal placental epithelial cells before the virus infects foetal trophoblasts. Moreover, the virus was shown to bypass the maternal epithelial cell layer by directly targeting foetal trophoblasts in the haemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with foetal cells. Abortion was associated with widespread necrosis of placental tissues accompanied with severe haemorrhages. Experiments with human placental explants revealed that the same virus strain replicates efficiently in both cyto- and syncytiotrophoblasts. CONCLUSIONS/SIGNIFICANCE:This study demonstrates that RVFV targets the foetal-maternal interface in both ovine and human placentas. The virus was shown to cross the ovine placental barrier via two distinct routes, ultimately resulting in placental and foetal demise followed by abortion. Our finding that RVFV replicates efficiently in human trophoblasts underscores the risk of RVFV infection for human pregnancy

Detection of anti-N antibodies by ELISA.

<p>Sera were obtained 7, 14 and 21 days post vaccination (DPV) and at 0, 7, 14 and 21 days post challenge (DPC). Titers are expressed as percentage competition ratio of the optical densities (OD) of the sample and the OD of the negative control (%S/N). All values lower than 40% are considered positive, between 40–50% are considered doubtful and above 50% are considered negative. The 40% and 50% cut-offs are represented by solid and interrupted lines, respectively. Results obtained from analysis of each individual animal from the mock-group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) or high-dose group (H1–H7) are depicted.</p

Rectal temperatures of vaccinated and mock-vaccinated lambs before and after challenge with RVFV.

<p>Fever was defined as a rectal body temperature above 40.5°C (interrupted line). Body temperatures of mock-vaccinated lambs (C1–C8) and lambs vaccinated with a low dose (L1–L8), medium dose (M1–M8) or high dose (H1–H7) of NSR-Gn are depicted individually.</p

Detection of viral RNA in plasma by qRT-PCR.

<p>Plasma samples were collected daily at the first 7 days post challenge (DPC) and subsequently on DPC 9, 11, 14 and 21. Viral RNA copy numbers detected in individual animals of the mock-vaccinated group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) and high-dose group (H1–H7) are depicted.</p

Humoral and cellular immune responses elicited by vaccination of mice with NSR or NSR-Gn.

<p>(A) VNT titers in sera collected from mice vaccinated with NSR or NSR-Gn before vaccination (DPV -1) and 13, 22 and 29 DPV. Bars represent average titers (n = 6) of each group with standard deviation. The detection limit of the assay is depicted by the interrupted line. (B) Detection of IFN-γ-producing splenocytes isolated from mice vaccinated with NSR or NSR-Gn. Splenocytes were isolated and seeded at a density of 5×10⁵ cells/well in triplicate and stimulated for 12 hours with the indicated peptides or the ectodomain of Gn. Bars represent an average number of IFN-γ producing cells (n = 4) per group with standard deviation. The non-parametric Mann-Whitney test was used for statistical analysis and statistical significance between the groups is depicted by asterisks (*p<0.05; **p<0.01).</p