Literature DB >> 31961862

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

Judith Oymans^1,2, Paul J Wichgers Schreur¹, Lucien van Keulen¹, Jet Kant¹, Jeroen Kortekaas^1,2.

Abstract

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.

Entities: CellLine Chemical Disease Gene Species

Year: 2020 PMID： 31961862 PMCID： PMC6994196 DOI： 10.1371/journal.pntd.0007898

Source DB: PubMed Journal: PLoS Negl Trop Dis ISSN： 1935-2727

Introduction

Rift Valley fever virus (RVFV) is a negative-strand RNA virus of the family Phenuiviridae (former family Bunyaviridae), genus Phlebovirus. RVFV is transmitted by mosquitoes, predominantly by species of the genera Aedes and Culex [1]. The virus is pathogenic to domesticated and wild ruminants, of which sheep are the most susceptible. Newborn lambs may succumb within hours after onset of symptoms and seldom survive the infection [2,3]. The most typical pathological feature in lambs is severe necrosis of the liver. Specifically, necropsy of fatal cases reveals a swollen, pale liver with focal to widespread necrosis of hepatocytes. Direct and indirect consequences of liver necrosis include icterus, oedema, and hydrops ascites. Other organs, such as the spleen, heart, kidney and intestines may reveal haemorrhages and congested veins. Fatal cases are generally associated with haemorrhages and signs of shock. Susceptibility decreases with age, although mortality rates in adult sheep may be as high as 60% [4]. The most characteristic feature of RVF outbreaks in sheep herds are abortion storms, in which all pregnant ewes in a herd may abort [5]. Humans can be infected via mosquito bite, although most human cases are attributed to contact with contaminated animal tissues, particularly during the slaughtering of diseased animals [6]. The disease in humans generally follows a transient, febrile course with severe headache and muscle pains. Infection of the eye may induce temporal or permanent loss of vision resulting from retinal damage [7]. In a minority of patients, the infection progresses to encephalitis or haemorrhagic fever, of which the latter is often fatal [4,8,9]. A cross-sectional study of Sudanese women correlated RVFV infection with an elevated miscarriage rate [10]. Notably, experiments with second-trimester human foetal tissue explants recently revealed that human syncytiotrophoblasts are susceptible and permissive to RVFV replication [11]. These reports warrant further studies to determine if RVFV infections pose a perhaps underestimated risk for human pregnancy. The high abortion rates in sheep herds suggest that RVFV crosses the ovine placenta extremely efficiently. The placenta of sheep, and that of other RVFV susceptible ruminant species, is very different from the human placenta. The human placenta has the shape of a single disque whereas the ovine placenta consists of placentomes, varying in number among the different ruminant species [12]. Placentomes are discrete areas of extensive villous interactions between the maternal epithelial cells of the uterus, the caruncle, and the foetal trophoblasts of the allantochorion, the cotelydon, enabling efficient exchange of gases and nutrients between mother and foetus. In sheep and goats, pools of maternal blood are located in the crypts of the foetal villi, directly bordering the foetal trophoblast layer. At these so-called haemophagous zones, maternal erythrocytes are phagocytosed by trophoblasts as a principle source of iron for the developing foetus. Notably, this part of the ovine placenta resembles the haemochorial placenta of humans, in that maternal blood is in direct contact with foetal trophoblasts. However, the blood pools at the haemophagous zones in sheep and goats are filled with stagnant blood and therefore do not contribute significantly to the maternal-foetal exchange of nutrients, whereas in the human haemochorial placenta maternal blood flows along the foetal trophoblasts enabling efficient nutrient exchange. The detrimental consequences of RVFV infection during ovine pregnancy were already reported after the first outbreak of RVF in 1930 and abortion storms have since then become a hallmark of RVF epidemics [2,3,13-15]. Nevertheless, the pathogenesis of RVFV-induced abortion in pregnant ewes was not yet described in literature. In the present study, we experimentally infected pregnant ewes at one third- or at mid-gestation and describe the pathogenic events that result in abortion of the ovine foetus. We furthermore investigated the susceptibility of human placental explants for the same RVFV strain.

Materials and methods

Cells and viruses

Culture media and supplements were obtained from Gibco unless indicated otherwise. Baby Hamster Kidney (BHK-21) cells were maintained in Glasgow minimum essential medium (GMEM) supplemented with 4% tryptose phosphate broth, 1% minimum essential medium nonessential amino acids (MEM NEAA), 1% antibiotic/antimycotic (a/a) and 5% foetal bovine serum (FBS), at 37°C with 5% CO2. Vero-E6 cells were maintained in minimum essential medium (MEM) supplemented with 1% a/a, 5% FBS, 1% glutamine and 1% MEM NEAA, at 37°C with 5% CO2. BHK-21 and Vero-E6 cells were purchased from ATCC. A recombinant version of RVFV strain 35/74, originally isolated from the liver of a sheep during a RVFV outbreak in 1974 in the Free State province of South Africa, was used in the present work as described [16]. The titre was determined on BHK-21 cells as 50% Tissue Culture Infective Dose (TCID50) according to the Spearman-Kärber algorithm [16].

Ethics statements

Animal experiments were conducted in accordance with the Dutch Law on Animal Experiments (Wet op de Dierproeven, ID number BWBR0003081) and the European regulations (EU directive 2010/63/EU) on the protection of animals used for scientific purposes. The procedures were approved by the animal ethics committee of Wageningen Bioveterinary Research (WBVR) and the Dutch Central Authority for Scientific Procedures on Animals (permit number AVD401002017894). Human placentas were obtained after caesarean section of healthy women. This material is regarded as medical waste and therefore does not fall under the scientific medical research law of the Netherlands and does not need approval from an institutional review board. All donors have given written consent and consent forms are stored in accordance with the Dutch privacy law.

Experimental design pregnant ewe trial

At a conventional Dutch sheep farm, 30 Texel-Swifter mix breed ewes that had delivered healthy lambs before, were treated with progesterone sponges to synchronise pregnancies. After removal of the sponges, ewes were naturally mated. Ultrasounds were performed at 6–7 weeks after mating to confirm gestation. Pregnant animals to be enrolled in the studies were subsequently transported to WBVR and allowed to acclimatize for one week before the start of the experimental period under biosafety level 3 (BSL-3) conditions. At 55 days of pregnancy (experiment 1) or 78 days of pregnancy (experiment 2), animals were inoculated via intravenous (IV) route with 105 TCID50 of RVFV in 1 ml medium, or with 1 ml medium (negative control animals). Following challenge, animals were closely monitored for clinical signs, body temperatures were recorded, and EDTA blood samples were collected (Fig 1A). The ewes of experiment 1 were euthanized and necropsied at 6 days post infection and the ewes of experiment 2 were euthanized on day 4 or at abortion (day 7). Ewes and their foetuses were euthanized by intravenous administration of 50 mg/kg sodium pentobarbital (Euthasol, ASTfarma) and subsequent exsanguination. Foetuses were exsanguinated by severing of the umbilical cord after which foetal blood was collected in EDTA tubes. From the ewes, samples were taken from the liver and spleen. From the foetuses, samples were taken from the brain, spleen, leg muscle tissue and liver. Samples were also taken from the amniotic fluid, umbilical cord and the placentomes (3 per foetus). Samples were placed on dry ice and subsequently stored at -80°C. Samples for histology and immunohistochemistry were fixed in 10% phosphate-buffered formalin for at least 48 h followed by routine processing into paraffin blocks.

Fig 1

Experimental design and primary outcome parameters ewes.

Ewes were inoculated intravenously with RVFV or mock inoculated at gestation day (gd) 55 (A) or day 78 (E) and euthanized at 4 (blue cross), 6 (green and purple crosses) or 7 (orange cross) days post inoculation (dpi). Purple numbers represent ewes that were mock-inoculated. Rectal temperatures (B, F), viremia by RT-qPCR (solid line, left y-axis), virus isolations (dotted line, right y-axis) (C, G) and the presence of RVFV in spleen and liver samples of the ewes (D, H) are depicted. Bars represent averages with SDs.

Experimental design and primary outcome parameters ewes.

Infection of human placental explants

Full term placentas were obtained by caesarean section from healthy donors at the Isala hospital in Zwolle, the Netherlands. The placentas were transported on ice and placed in large petridishes with complete medium (40% Dulbecco's Modified Eagle Medium [DMEM], 40% F12 nutrient mixture, 10% FCS supplemented with 1% a/a). Chorionic villi were separated from the placenta and cut in 4x4 mm pieces, after which the samples were washed 3x with PBS + a/a. Placental explants were incubated with 2,5x105 TCID50 of RVFV in 1 ml complete medium, or in 1 ml complete medium (negative controls) in a 24-wells plate. Medium was removed 16 hpi, after which explants were washed 3x with PBS + a/a. Samples were collected at 1, 2 or 4 days post inoculation (dpi). Samples for quantitative reverse-transcription PCR (RT-qPCR) were stored at -80°C. For virus isolation, 200 μl supernatant samples from each timepoint were pooled. For IHC, explants were fixed in 10% phosphate-buffered formalin for 48 h followed by routine processing in paraffin blocks. Each sample was analysed in quadruplicate. The 1 dpi timepoint was taken at 16 hpi after washing the explants. At 3 dpi, 1 ml of fresh medium was added to the 4 dpi samples.

Detection of viral RNA

RNA was extracted from ovine plasma and organ samples. Briefly, organ suspensions were prepared by homogenising 0,3–1 g of tissue in an IKA Ultra Turrax Tube DT-20 containing 7 ml CO2-Independent Medium (CIM) supplemented with 1% a/a. The suspensions were transferred to 15 ml Falcon tubes and cell debris was removed by centrifugation for 15 min at 4952 x g. Fifty μl Proteinase K (5 μg/ml, Sigma) was added to 200 μl of the plasma samples or organ suspensions. Next, 200 μl AL buffer (Qiagen), supplemented with 2 μl polyadenylic acid A (5 mg/ml, Sigma) was added, the samples were thoroughly mixed and incubated at 56°C for 15 min. Subsequently, 250 μl 99% ethanol was added and RNA was isolated using the Qiagen RNeasy kit according to the manufacturer’s protocol. RNA was extracted from human placental explants by first homogenising the explants in 1 ml TRIzol Reagent (Invitrogen) in Lysing Matrix D tubes (MP Biomedicals) using the TeSeE™ Precess 24 bead beater for 2x23 s at 6500 RPM. Supernatant (350 μl) was used for RNA isolation using the Direct-zol RNA miniprep kit (Zymo Research) according the manufacturer’s protocol. Five μl of the RNA was used in a RT-qPCR using the The LightCycler RNA Amplification Kit HybProbe (Roche, Almere, the Netherlands). Primers and probes were purchased from IDT. Forward primer: 5’-AAAGGAACAATGGACTCTGGTCA-3’, reverse primer: 5’-CACTTCTTACTACCATGTCCTCCAAT-3’; Probe: 5’-6FAM-AAAGCTTTGATATCTCTCAGTGCCCCAA-TMR-3’. Cycling conditions were as follows: 45°C for 30 min, 95°C for 5 min, 45 cycles of 5 s at 95°C and 35 s at 57°C, followed by cooling down to 30°C.

Virus isolation

Virus isolations from ovine samples were performed by serial dilution of either plasma in complete CIM supplemented with 3,5 IU/ml heparin, or organ suspension in CIM, followed by incubation with BHK-21 cells. After 1.5 h incubation at RT, the inoculum was replaced by fresh medium and after 5 days of culturing the cells at 37°C cytopathic effects were scored. Virus isolations from the supernatant of the human placenta explants were performed by a 10x serial dilution series of the supernatant in complete medium on Vero cells. At 24 hpi, infection was visualized with an immunoperoxidase monolayer assay (IPMA). Briefly, cells were fixed and permeabilized 24 hpi with 4% paraformaldehyde (10 min) and ice-cold methanol (5 min). After permeabilization the plates were incubated with RVFV specific monoclonal antibody 4-D4, which recognizes the Gn protein [17]. A polyclonal rabbit-α-mouse immunoglobin/HRP antibody (Dako, Denmark) was used as a secondary antibody and 3-Amino-9-ethylcarbazole (AEC; Sigma-Aldrich) was used as a substrate. The titre was expressed as TCID50/ml according to the Spearman-Kärber algorithm [18,19].

Histology and immunohistochemistry

Paraffin embedded tissue was cut into 4 μm sections, collected on silane-coated glass slides and dried for at least 48 h in a 37°C incubator. After deparaffinization and rehydration in graded alcohols, sections were stained routinely with haematoxylin and eosin (H&E) or immunostained for RVFV antigen. For immunostaining, endogenous peroxidase was blocked for 30 min in methanol/H2O2 followed by antigen retrieval through 15 min autoclaving at 121°C in pH 6 citrate buffer (Antigen unmasking solution, Vector Laboratories ). As RVFV Gn-specific primary antibody, monoclonal antibody 4-D4 was used. Specificity of the immunostaining was confirmed with 2 other mAbs directed against different proteins of RVFV. Mouse envision peroxidase (Dako, Denmark) was used as secondary antibody and diaminobenzidine (DAB; Dako, Denmark) as the substrate, according to the manufacturer's instructions. Immunostaining for cytokeratin was performed using a rabbit mAb to cytokeratin 19 (Abcam 52625, USA) followed by α-rabbit-ImmPRESS-AP and Vector Red as substrate (Vector Laboratories, USA). Hematoxylin was used to counterstain the slides.

Results

RVFV infection of pregnant ewes results in abortion within one week

To study the pathogenesis of RVF in pregnant ewes, two experiments were performed. Ewes were infected at either one third of gestation (experiment 1) or at mid gestation (experiment 2). In both experiments, ewes were inoculated via intravenous route with a dose of 105 TCID50 of RVFV, an exposure route and dose that was used in previous studies with sheep [20-22]. In the first experiment, a group of ten ewes was distributed randomly over two groups of five animals of which the first group was inoculated with virus and the second group with medium only (mock) (Fig 1A). As expected, and similar as observed in lambs, all RVFV-inoculated ewes developed fever (Fig 1B), which correlated with viremia as determined by RT-qPCR and virus isolation on plasma samples (Fig 1C). At 4 days post infection (dpi), one of the RVFV inoculated ewes (animal number 1764) acutely succumbed to the infection. Necropsy revealed a swollen, discoloured liver and signs of shock. Analysis of liver and spleen samples by RT-qPCR revealed high levels of viral RNA in both organs (Fig 1D). Extensive haemorrhages were found in the placentas of the two foetuses carried by this ewe and in the uterine wall. The remaining ewes were euthanized and necropsied as scheduled at 6 dpi. All ewes showed a multifocal necrotizing hepatitis which was most severe in the animal that died at 4 dpi. Although no abortions had occurred, all placentas revealed extensive haemorrhages in the placentomes and all foetuses had already died (S1 Fig, S3 Fig and Table 1).

Table 1

Analysis of foetuses collected from RVFV-infected ewes.

One third gestation

DPI

Foetuses

Alive or dead at time of dissection

Placentome

non-cotyledonary allantochorion

Liver

Brain

Amniotic fluid

Umbilical cord

Umbilical blood

PCR

IHC

PCR

IHC

PCR

IHC

PCR

IHC

PCR

IHC

PCR

1764¹-F1

Dead

1764¹-F2

Dead

1760-F1

Dead

+ ²

1761-F1

Dead

1761-F2

Dead

1762-F1

Dead

1762-F2

Dead

1762-F3

Dead

+ ²

1763-F1

Dead

+ ²

1763-F2

Dead

+ ²

Mid-gestation

1841-F1

Alive

1841-F2

Alive

1842-F1

Alive

1842–F2

Alive

1843-F1

Alive

1844-F1

Dead

1844-F2

Dead

1844-F3

Dead

+ ²

1845-F1

Dead

1845-F2

Dead

1846-F1

Alive

+ ²

1846-F2

Dead

+ ²

1846-F3

Dead

+ ²

1Ewe died at 4 dpi

2Blood vessels (blood and endothelium).

Analysis of foetuses collected from RVFV-infected ewes.

Pathological findings in foetuses of necropsied ewes experimentally infected with RVFV at one third (1/3 gestation) or at mid-gestation (1/2 gestation). Ewes were necropsied at 4, 6 or 7 dpi. Viral RNA and proteins were detected by RT-qPCR and IHC, respectively. NT; not tested as foetuses were too autolytic. 1Ewe died at 4 dpi 2Blood vessels (blood and endothelium). To study the pathology at an earlier and later stage than 6 dpi, another group of 6 ewes from the same herd was inoculated with RVFV (experiment 2). Of this group, three ewes were euthanized and necropsied at 4 dpi and the remaining three ewes when abortion was imminent (Fig 1E). Of note, these ewes were at the moment of inoculation at mid gestation. As expected, all ewes of this experiment developed similar rises in rectal temperatures and viremia levels as those of experiment 1 (Fig 1F and 1G). Necropsy at 4 dpi revealed multifocal necrotizing hepatitis in all ewes but no macroscopic abnormalities in placentas and unaffected, live foetuses. In the morning of day 7 post infection, one ewe had aborted 2 foetuses and a second one was is the process of aborting (2 foetuses already expelled, 1 foetus still in the uterus). Necropsy of the third ewe (#1846) revealed three foetuses that were still inside the uterus but with placentomes showing extensive haemorrhages. One of these foetuses was alive, whereas the remaining two foetuses were found dead (Table 1). Analysis of liver and spleen samples revealed very high viral RNA levels in the organs of ewes necropsied at 4 dpi and lower levels in ewes necropsied at 6 dpi or at abortion (Fig 1D and 1H). Viral RNA and viable virus were detected in all placentomes from both experiments (Fig 2). It was striking to observe that viral RNA levels increased in placentomes between days 4 and 7 (Fig 2), whereas viremia and viral RNA levels in spleens and livers declined in the same period (Fig 1D and 1H).

Fig 2

Detection of viral RNA and infectious virus in placentomes.

Viral RNA copies in placentomes as determined by RT-qPCR (black columns; left y-axis) and virus titres in placentomes as determined by virus isolation (grey columns; right y-axis). Results of experiment 1 (A) and 2 (B) represent means and SDs of 3 placentomes per foetus.

Detection of viral RNA and infectious virus in placentomes.

RVFV infects maternal epithelial cells and foetal trophoblasts

The ovine placenta consists of multiple placentomes, in which foetal blood is separated from maternal blood by several cell layers (Fig 3A and 3B). In accordance with literature, we observed an increase in mass of the foetal and maternal villi with concomitant decrease in mesenchyme and expansion of haemophagous zones with progressing pregnancy (Fig 3C and 3D) [23].

Fig 3

The ovine placenta at different stages of gestation.

The ovine placenta at different stages of gestation.

(A) Schematic presentation of an ovine foetus, the cotyledons and their blood supply. Only the foetal parts of the placenta (cotyledons) are displayed, the uterus wall and maternal part of the placenta (caruncles) are not depicted. At the right, a cross section of a placentome is depicted, showing the maternal tissues in shades of pink, and the foetal villi in orange. Haemophagous zones at the base of the foetal villi are depicted in red. (B) A schematic overview of the cotyledon (center) with the different cell layers of the synepitheliochorial placenta at the left and the haemophagous zone at the right. In the synepitheliochorial placenta, the foetal blood is separated from maternal blood by several maternal and foetal cell layers. In the haemophagous zone maternal blood is in direct contact with the foetal trophoblasts. (C, D) HE staining of placentomes, the haemophagous zones and synepitheliochorial placenta at 1/3 gestation (C) and 1/2 gestation (D). Red interrupted lines indicate the boundaries between maternal and foetal tissues. F = foetal villus, M = maternal villus, MB = maternal blood. Notice the increase in the foetal/maternal villous interface in the synepitheliochorial placenta and the increase in size and erythrophagous activity of the trophoblasts of the haemophagous zone between 1/3 and 1/2 of the gestation period. Necropsy of the ewe of experiment 1 that died at 4 dpi (#1764) revealed macroscopic abnormalities: extensive haemorrhages in the uterine wall, at the base of the placentomes and within the placentomes. Necropsy of ewes that were euthanized at 4 dpi in experiment 2 revealed placentomes that did not show any macroscopic abnormalities. Immunohistochemistry showed the presence of RVFV antigen as small foci spread throughout the placentomes collected at 4 dpi in experiments 1 and 2 (Fig 4A). These foci consisted of a cluster of strongly stained epithelial/syncytial cells of the maternal villus with occasionally a single positive trophoblast in the epithelial lining of the opposite foetal villus (Fig 4B). Clusters of RVFV-positive cells were also found in the trophoblast epithelium lining the haemophagous zone (Fig 4C).

Fig 4

RVFV antigen in placentomes.

RVFV antigen in placentomes.

Immunohistochemical (IHC) detection of RVFV antigen in cross sections of the placentomes at 4 dpi (experiment 2) and 6 dpi (experiment 1). At 4 dpi (A) only small foci of antigen positive cells are visible throughout the placentome while at 6 dpi (F) almost the entire placentome stains positive for RVFV antigen. Higher magnification of the synepitheliochorial placenta (SP) at 4 dpi (B) shows strong labelling of the maternal epithelial cells with only an individual positively stained foetal trophoblast (black arrowhead). At 6 dpi both maternal and foetal cell layers are strongly stained (G). In the haemophagous zone (HZ) at 4 dpi (C) only small clusters of foetal trophoblasts stain positive for RVFV antigen while at 6 dpi (H) the entire foetal trophoblast lining of the haemophagous zone stains positive. Panels D, E, I and J represent cross sections of uninfected placentomes corresponding to B, C, G and H, respectively, showing absence of background IHC staining. Red interrupted lines indicate the boundaries between maternal and foetal tissues. F; foetal villus, M; maternal villus, MB; maternal blood. Bar = 5000 μm (A, F) or 100 μm (B-J). At 6 dpi, extensive haemorrhages were observed in the placentomes of the RVFV infected ewes (S1 Fig). In addition, in some placentomes maternal and foetal villi were starting to separate. Both maternal epithelial cells (viable and necrotic) and foetal trophoblasts were strongly positive for RVFV antigen throughout the entire placentome (Fig 4F). Compared to the placentomes at 4 dpi, the area of RVFV positive maternal epithelial cells and foetal trophoblasts was greatly increased covering almost the entire maternal and foetal epithelial lining of the placentome including the haemophagous zone (Fig 4G and 4H). H&E staining revealed haemorrhages in the maternal villi with extensive necrosis of the maternal epithelium but only limited areas of necrosis of foetal trophoblasts (Fig 5A). In addition, a heavy influx of neutrophils was present mainly in the stratum compactum of the lamina propria and at the base of the maternal villi (Fig 5B).

Fig 5

Histopathology of the placentomes at imminent abortion.

Histopathology of the placentomes at imminent abortion.

HE staining of placentomes at 6 dpi. (A) Haemorrhages (black asterisk) and necrosis of maternal epithelial cells (arrowheads). Notice the relatively intact foetal epithelium. (B) Influx of neutrophils (red asterisk) and necrosis of maternal epithelium (arrowheads). F = foetal villus, M = maternal villus, Bar = 100 μm. At imminent abortion at 7 dpi, placentomes showed extensive haemorrhages with varying degrees of separation of maternal and foetal parts. Histology and immunohistochemistry revealed similar results as those obtained from analysis of the 6 dpi group with haemorrhages and a neutrophilic inflammatory response in maternal villi, necrosis of maternal epithelium and strong positive staining for RVFV in maternal epithelial cells and foetal trophoblasts. In placentomes where foetal villi had already separated from the maternal caruncle, large areas of denuded maternal villi were seen. In addition in some maternal villi, blood vessels were found that stained positively for RVFV in the endothelium and/or smooth muscle cells in the blood vessel wall (S2 Fig). Notably, endothelial cells did not reveal signs of apoptosis or necrosis.

Detection of RVFV in foetal tissues

Ewe 1764 that acutely succumbed to the infection in experiment 1 carried two foetuses. RVFV RT-qPCR revealed viral RNA in the livers of both foetuses (Table 1 and Fig 6A). The foetuses of the ewes necropsied at 4 dpi in experiment 2 were all alive and appeared to be normal at the moment of necropsy (S3 Fig). However, RT-qPCR showed the presence of viral RNA in the blood of all foetuses and in the livers of 3 out of 5 foetuses (Fig 6B). These results show that RVFV is able to reach the foetus within 4 days.

Fig 6

Detection of viral RNA and viral antigen in foetal organs.

Detection of viral RNA and viral antigen in foetal organs.

Detection of RVFV RNA by RT-qPCR in of organ suspensions of foetal organs collected in experiment 1 (A) and experiment 2 (B). Bars represent averages with SDs. Staining of RVFV antigen in samples collected from liver (C), brain (D) umbilical cord (E) and leg muscle (F). Notice the strong staining of endothelial cells in the blood vessels within the various organs. Bar = 500 μm (D, E, F), 100 μm (C) or 20 μm (inset D). All 8 foetuses of the ewes necropsied at 6 dpi were found dead within the uterus (Table 1). The livers of these foetuses were also positive for viral RNA and in 4 out of 8 foetuses viral antigen was detected by immunohistochemistry. In these 4 foetuses, the liver showed massive necrosis with only a few viable hepatocytes left (Fig 6C). Brain samples collected from 5 of the 8 foetuses were also found to contain viral RNA. Immunohistochemical staining of brain tissues from these foetuses revealed viral antigen in the blood and the endothelium of the blood vessels throughout the brain and arachnoidea (Fig 6D). No viral antigen was detected in neurons or glial cells. RVFV antigen was also detected in endothelial cells of blood vessels of the umbilical cord (Fig 6E) and muscle tissues (Fig 6F). Most of the aborted foetuses in experiment 2 were severely autolytic and unsuited for further analysis (S3 Fig). The foetuses that were suitable for further analysis presented with severely necrotic livers and contained high levels of viral RNA in liver and brain samples. Hepatocytes and endothelial cells were strongly positive for RVFV antigen.

RVFV strain 35/74 infects cytotrophoblasts and syncytiotrophoblasts in human placental explants

To study if RVFV strain 35/74, which was originally isolated from sheep, replicates in human placentas, explants of human term placentas were inoculated with the virus. The results show that the virus replicates efficiently in cytotrophoblasts and syncytiotrophoblasts (Fig 7), as revealed by RT-qPCR, virus isolation and IHC. In some areas of the placenta, viral antigen was detected in cytotrophoblasts but appeared to be absent in syncytiotrophoblasts, suggesting that the former are more permissive to RVFV replication (Fig 7C). The differences and similarities between the ovine and human placenta are depicted in S4 Fig.

Fig 7

RVFV in human placental explants.

Detection of viral RNA (A) and infectious virus (B) in human full term placental explants at different timepoints post infection. Viral RNA was detected by RT-qPCR and infectious virus by virus isolation. (C, D, E) Immunohistochemical staining of RVFV with mAb 4-D4, counterstained with haematoxylin. (C) Single villus with syncytiotrophoblasts (black arrowheads) staining negative for RVFV antigen with cytotrophoblasts (brown staining, red arrowhead) staining positive. (D) Single villus in which no cytotrophoblasts are present, with positive staining of syncytiotrophoblasts (black arrowheads). (E) Single villus showing absence of background IHC staining in non-infected control placental explant. Syncytiotrophoblasts are indicated with black arrowheads. (F) Immunohistochemical staining of epithelial cells in a non-infected placental villus with a mAb to cytokeratin showing both the syncytiotrophoblast layer (black arrowheads) and the cytotrophoblast layer (red arrowheads). Bar = 20 μm (C, D, E) or 50 μm (F).

RVFV in human placental explants.

Discussion

The most characteristic feature of RVFV epizootics are abortion storms in sheep herds. During these events, all pregnant ewes in an affected herd may abort their foetuses. Although the detrimental effects of RVFV infection on ruminant pregnancy are well-recognized, the route that the virus uses to cross the placenta and the pathogenic events that result in abortion have remained undescribed. Here, we report that maternal and foetal epithelial cells in the ovine placenta are highly susceptible and permissive for RVFV and that abortion results from severe pathology of the placenta. In some ewes, the rapid progression of placental demise caused foetal mortality before foetuses could be infected. During gestation, the progesterone hormone keeps the uterus and myometrium in a quiescent state to allow successful foetal development until parturition [24]. During the first trimester of gestation in sheep, progesterone is produced by the corpus luteum which resides in the ovaries. During the second and third trimester, the developing placenta becomes an additional source of progesterone. Placental progesterone is produced by binucleate trophoblasts that migrate to the maternal epithelium to fuse with uterine epithelial cells to form so-called syncytial cells. At about 90 days of pregnancy, half of the progesterone in the pregnant ewe is produced by the corpus luteum and half by the placenta. Our study has revealed that both trophoblasts and (syncytial) epithelial cells are major target cells of RVFV. Widespread necrosis of these cells likely results in a drop of systemic progesterone levels. In addition, virus-induced necrosis of placental cells leads to the production of pro-inflammatory chemokines and cytokines like TNF-α, IFN-γ and IL-8, which stimulate prostaglandin (E2/F2α) excretion by the placental epithelium. Prostaglandins bring the corpus luteum in regression, resulting in a further drop in progesterone levels. In the absence of the inhibitory effect of progesterone, prostaglandins induce uterine contractions and cervical effacement resulting in abortion. Inflammatory cytokines, most notably TNF-α, are also known to reduce vascular integrity. In the face of lowered coagulation factors in maternal blood resulting from liver necrosis, this could explain the observed placental haemorrhages. Our study has revealed that RVFV uses at least two routes to cross the ovine placenta. The first route involves the haemophagous zones of the placenta, where foetal trophoblasts are in direct contact with maternal blood. Trophoblast cells in the haemophagous zone are specialised in phagocytosis of erythrocytes from the blood to provide the foetus with iron. The presence of RVFV in maternal pools of blood in the haemophagous zone may therefore result in direct infection of trophoblasts or via co-uptake of the virus during erythrophagy. The second route initiates with infection of maternal epithelial cells of the placenta. Virus released from these cells exposes foetal trophoblasts. Progeny virus produced by infected trophoblasts is subsequently released into the foetal mesenchyme exposing endothelial cells of foetal bloodvessels, which were also identified as target cells of RVFV. The finding that foetal endothelial cells and endothelial cells in the maternal caruncle are target cells of RVFV is notable, as most of our previous studies with juvenile and adult sheep did not reveal endothelial cell infection. However, in one of our studies, RVFV-positive endothelial cells were detected in lymphoid organs of a lamb that peracutely succumbed after developing exceptionally high viremia [25]. In another study, endothelial cells of the skin became infected after feeding of mosquitoes on a viremic lamb [26]. These findings, together with the haemorrhagic manifestations that were seen in RVFV infected placentomes in this study, calls for further research into the interaction of RVFV with endothelial cells. In a recently published study by McMillen and co-workers, RVFV infection of human placental explants was shown for the first time, using a strain that was originally isolated from humans [11]. Our experiments with human placental explants corroborate this research and highlight the potential risks of RVFV infection during human pregnancy. In the same study, the pathogenesis of RVFV infection in pregnant Sprague-Dawley rats was described. This work demonstrated that vertical transmission in these rats occurs through direct placental infection and that viral loads in the placenta were higher than in the liver and other maternal organs, similar as observed in the present study. In our study, the ovine placenta was not only found to contain the highest viral loads but was also the only organ still containing high levels of infectious virus at the moment of necropsy. These findings suggest that RVFV efficiently counteracts innate immune responses in placental cells. RVFV counteracts host innate immunity through several functions of the nonstructural NSs protein [27-32]. One of the major functions of NSs is the downregulation of type I interferon (IFN) responses, which occurs through downregulation of general host gene transcription and the direct inhibition of IFN-β mRNA production [27,28,32]. Whereas in most cell types, type I IFNs play a major role in innate immunity, in cells of epithelial origin, such as the cells of the placenta, innate immunity is regulated by type III IFNs, referred to as IFN-λ [33]. Importantly, both type I and type III IFNs trigger the JAK/STAT pathway, which was shown to be targeted by NSs [34]. We therefore hypothesize that RVFV NSs facilitates replication in placental tissues by downregulating JAK/STAT signalling. On the other hand, previous studies have demonstrated that RVFV can also cross the ovine placental barrier without NSs [35]. These infections did not result in abortions but instead in stillbirths and congenital malformations, including arthrogryposis and hydranencephaly, resembling pathology in ovine foetuses infected by members of the genus Orthobunyavirus, such as Schmallenberg virus. In conclusion, the present work has revealed how RVFV crosses the ovine placental barrier and has provided novel insights into the pathology that results in abortion in the most susceptible target species. The sheep isolate that was used was shown to replicate efficiently in human placental explants as well, calling for further research on the risk of RVFV infection during human pregnancy.

RVFV infection results in extensive placental haemorrhages.

Placenta from a healthy ewe (A) and from an ewe inoculated with RVFV, necropsied six days after inoculation (B). Placentas were collected during experiment 1 at one third of gestation. (TIF) Click here for additional data file.

Replication of RVFV in caruncles is associated with bleeding and infection of maternal endothelial cells.

(A) Micrograph of HE-stained caruncle tissue of ewe 1845 euthanized at 7 dpi (experiment 2). Notice the extensive haemorrhages at the base of the caruncle (arrowhead and inset). (B) Immunohistochemical staining of RVFV antigen. Positive staining is only seen in those areas where necrotic maternal epithelium is still present. Some maternal blood vessels are also stained (arrowhead and inset) and show the presence of RVFV antigen in the endothelial cells and the smooth muscle cells of the tunica media (inset). Bar = 1000 μm (A, B), 100 μm (inset A), or 50 μm (inset B). LP; lamina propria, C; caruncle. (TIF) Click here for additional data file.

Pathological manifestations in foetuses collected from RVFV infected ewes.

Healthy foetuses collected from ewes necropsied at one third (A) or at mid-gestation (B). (C) Foetuses carried by ewe 1764 that succumbed 4 days after inoculation with RVFV in experiment 1. (D) Live foetus collected from an ewe that was necropsied at 4 dpi in experiment 2. (E) Autolytic foetus collected from an ewe necropsied at 6 dpi in experiment 1. (F) Two aborted foetuses from experiment 2 (left) and one foetus (right) that was still inside the uterus at the moment of necropsy. (TIF) Click here for additional data file.

Schematic presentation of the ovine and human placenta.

A human placenta consists of a single discoid plaque whereas an ovine placenta consists of placentomes (A). A cross section of both placentas is depicted, showing the maternal tissues in shades of pink, and the foetal villi in orange. Blood and arteries are depicted in red, veins are depicted in blue (B). In the synepitheliochorial placenta (C, left panel), the foetal blood is separated from maternal blood by several maternal and foetal cell layers. In the haemophagous zone (C, middle panel) maternal blood is in direct contact with the foetal trophoblasts, which is similar to the human haemochorial placenta (C, right panel). (TIF) Click here for additional data file. 27 Aug 2019 Dear Oymans: Thank you very much for submitting your manuscript "Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas" (#PNTD-D-19-01103) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time. We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer. When you are ready to resubmit, please be prepared to upload the following: (1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. (2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file); the other a clean version (uploaded as the article file). (3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type. Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images). (4) If applicable, we encourage you to add a list of accession numbers/ID numbers for genes and proteins mentioned in the text (these should be listed as a paragraph at the end of the manuscript). You can supply accession numbers for any database, so long as the database is publicly accessible and stable. Examples include LocusLink and SwissProt. (5) To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-methods Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. We hope to receive your revised manuscript by Oct 26 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email. To submit a revision, go to https://www.editorialmanager.com/pntd/ and log in as an Author. You will see a menu item call Submission Needing Revision. You will find your submission record there. Sincerely, Abdallah M. Samy, PhD Guest Editor PLOS Neglected Tropical Diseases Paulo Pimenta Deputy Editor PLOS Neglected Tropical Diseases *********************** Reviewer's Responses to Questions Key Review Criteria Required for Acceptance? As you describe the new analyses required for acceptance, please consider the following: Methods -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? -Is the study design appropriate to address the stated objectives? -Is the population clearly described and appropriate for the hypothesis being tested? -Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? -Were correct statistical analysis used to support conclusions? -Are there concerns about ethical or regulatory requirements being met? Reviewer #1: (No Response) Reviewer #2: Yes -------------------- Results -Does the analysis presented match the analysis plan? -Are the results clearly and completely presented? -Are the figures (Tables, Images) of sufficient quality for clarity? Reviewer #1: Rift Valley fever virus (RVFV) is an arbovirus of the order Bunyavirales that causes severe disease in ruminants and humans. Surprisingly, pathology resulting in abortion remains poorly described. In the present work, pregnant ewes were inoculated intravenously with a highly virulent strain of RVFV and necropsied at different time points post inoculation. Tissues were collected and analyzed by real-time qPCR, virus isolation, and immunohistochemistry. Results reveal that maternal epithelial cells are the first placental cells to be infected, followed by fetal trophoblasts. In addition, RVFV can bypass the maternal epithelial cell layer by directly targeting fetal trophoblasts in the hemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with fetal cells. Abortion was associated with widespread necrosis of placental tissues and severe hemorrhage. Experiments with human placental explants revealed efficient infection and replication in both cyto- and syncytiotrophoblasts. Results demonstrate that RVFV targets the fetal-maternal interface in both ovine and human placentas. The virus crossed the ovine placental barrier via two distinct routes, ultimately resulting in placental and fetal demise followed by abortion. Finding show that the same RVFV strain replicates efficiently in human trophoblasts and underscores the risk of RVFV infection during human pregnancy. This is an excellently executed and well-illustrated study. Authors have nicely analyzed and compared ovine and human placental tissue with respect to RVFV infection. It remains unclear how closely human placental explants mimic the in situ situation. In order to infect an ovine fetus a pathogen has to overcome several tissue layers compared to human placental tissue. This could indicate that human fetal infection should occur very frequently. Authors report ….. “In addition, RVFV can bypass the maternal epithelial cell layer by directly targeting fetal trophoblasts in the hemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with fetal cells.” Does this represents a back-door for any kind of placental infection in ruminants? Conditions of RVFV PCR including used primers should be provided in more detail. Table 1 should be accompanied by a more informative legend explaining the different colors, NT, abbreviations, mentioned internal case numbers, meaning of 1/3 and ½ gestation etc. Table should be readable without reading the remaining manuscript. Numbering of figures starting at line 253 should occur in numeric/alphabetic order (e.g. please start with Figure 1a and not 1e). It would be interesting to know whether alive fetuses of experiment 2 would develop long-term alterations in any other organ? How would the authors explain the difference with respect to placental/fetal susceptibility between experiment 1 and 2? Line 272: it is stated: “…..observed an increase in mass of the fetal and maternal villi with concomitant decrease in mesenchyme (meaning mesenchymal tissue?)…….”. How was this statement substantiated (e.g. quantitative or semi-quantitative analysis?)? Furthermore, how would authors explain this tissue enlargement and decrease in mesenchymal tissue within such a short time period, especially in experiment 1? What about placental tissue of experiment 2 without macroscopic lesions? The question remains whether the extensive hemorrhage in placental tissue is solely due to a direct virus effect or represents a secondary event, at least in part, mediated by (maternal?) liver damage. Overall, I miss a comment on the pathology found in the ewes. It would be important to know whether animals suffered from severe liver damage, which could complicate or contribute to placental lesions. Furthermore, is there a preferential infection of placentomes during pregnancy or did animals suffer predominantly from liver necrosis and placental lesions represent a concurrent but less sever organ manifestation? It remains unclear whether abortion is due to placental necrosis, drop in systemic progesterone levels or systemic effects of RVFV infection in the ewe. I miss a comment on the vasculature in the affected placentas (e.g. necrosis, vasculitis?). It is stated that…”blood vessels were found that stained positively for RVFV in the endothelium and/or smooth muscle cells in the blood vessel wall…”.in line 303/304. In addition, the prominent endothelial staining in figure 6 raises the question whether RVFV infection triggered morphologically recognizable or functional cell damage. Did these vessels also display necrosis (caspase 3, TUNEL-positivity?)? Line 320: what is meant by …endothelial staining…(e.g. immunohistochemistry for endothelial cells or RVFV?)? Minor points: Figure 4: Please indicate (mark) more clearly the fetal and maternal part of the placentome. Findings in figure 7 should be depicted at higher magnification for c and d, in addition. Blood-tingled number tags should be removed in the supplementary file figure 1 and 2. Supplementary file figure 1 and 2: Please provide a more explanatory, descriptive legend. Supplementary file figure 3b: Positive maternal vessels are not recognizable at the tip of the arrow. Mesenchyme is spelled differently in figure 3 and the text (line 273 and 374). Reviewer #2: See my specific comments below regarding figures -------------------- Conclusions -Are the conclusions supported by the data presented? -Are the limitations of analysis clearly described? -Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? -Is public health relevance addressed? Reviewer #1: (No Response) Reviewer #2: See my comments regarding speculation with regards to the conclusions -------------------- Editorial and Data Presentation Modifications? Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: (No Response) Reviewer #2: Comments regarding figure and data clarity are below -------------------- Summary and General Comments Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed. Reviewer #1: (No Response) Reviewer #2: Livestock are highly susceptible to infection with Rift Valley fever virus (RVFV). Pregnant ewes suffer fetal abortions when infected in nature, and this massive fetal infection has been known since the time that RVF was first described. However, the mechanism by which RVFV infects the ovine placental has not been thoroughly documented in either a natural or experimental setting, and this manuscript addresses this important gap in the field. The authors perform experimental inoculation of pregnant sheep at 2 time points during gestation. The ewes and their fetuses are monitored for RVFV infection and pathology. The authors are to be commended for completion of a difficult study. Given the limitations of a large animal study with a high-containment agricultural pathogen, the data presented here contribute significantly to our understanding of livestock placental infection by RVFV. Comments are mostly minor for clarity of text and data presentation. This reviewer would caution the authors to avoid unnecessary speculation in the discussion. While the histology was limited to H&E and basic IHC, this reviewer feels the data largely support the conclusions. Specific comments: 1. Line 54 – Please clarify that the severe outcome of RVFV during LIVESTOCK pregnancy is well documented. The effect in human pregnancy is not as clear. 2. Paragraph beginning at line 89 and ending at 105 describing the ovine placental structure should reference the appropriate citations. 3. Line 143-44: Please clarify what “right upfront the national breeding season” means. 4. Line 171: Please clarify what 1% a/a means. 5. Throughout the manuscript, the term “virus isolation” is used when it appears to refer to infectious virus quantification by TCID50. 6. Results section beginning on line 235: Please provide information on how the inoculum dose and the gestation time points were chosen. 7. Line 257: “dpi 7” should be “7 dpi” 8. Line 275-277: The authors refer to hemorrhage of the uterine wall and placentomes of ewe 1764– does this refer to macroscopic hemorrhage (as seen in Supp Fig 1) or microscopic hemorrhage visible by histology? 9. Line 307 – remove the comma after Experiment 1. 10. Line 313-315 – Do the authors feel that the 4/8 dead fetuses without vRNA in the liver were not directly infected but rather died due to destruction of the associated placenta? 11. Lines 348-365 – Description of sheep pregnancy development needs the addition of references. 12. Line 356-365 – Since neither progesterone nor cytokine levels were measured in this study, the conclusions from this paragraph are speculative. 13. Lines 366-376 – Description of two routes that RVFV uses to cross the placenta is also largely speculative. The data in Fig 4 clearly show a lot of viral antigen within the placenta at 6 dpi, but without a true serial sacrifice experiment, it is difficult to definitively say what ‘route’ is used. Comments regarding figures: 1. Fig. 1 – please show rectal temperatures of the control group 2. Fig. 4 – What counterstain is used for the IHC staining? How certain are you of the trophoblast cell identification without cell-specific markers or a counterstain? It would be helpful to have a square or box in (a) and (d) indicating the zoomed-in areas shown in b, c, e, and f. It would also be helpful to have uninfected control placentomes stained for IHC visualize any background. In addition, labeling of the structures in Fig 4 (similar to the labeling in Fig 3c,d) would help to orient the reader since most readers are not intimately familiar with placental histology. There does not seem to be an arrow in 4b that is referred to in the legend. 3. Fig. 5 – There is no black asterisk that is referred to in the legend. 4. Fig. 7 – How many human placental donors were tested? How many tissue explants were tested per donor per time point? Is the data in this figure a compilation of replication from different donors or a single donor? What is the counterstain for the IHC? In c and d, are these tissue explants the cross section of a single villus? Please clarify what it is that the reader is looking at. In such a small piece of tissue, how confident are you of the cytotrophoblasts and syncytiotrophoblasts without a specific cell marker? Addition of arrows to indicate the different cell types would be helpful. Is there any background IHC staining in uninfected human placental explants? Control staining images would be helpful. 5. Supplemental Fig 1 – are the negative control placenta and the RVFV-infected placenta from the same day of gestation? 6. Supplemental Fig 2 – same question for the control animals. Are they the same gestational age as the fetuses in c, d or e, f? -------------------- PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No 10 Oct 2019 Submitted filename: Response to Reviewers.docx Click here for additional data file. 1 Nov 2019 Dear Miss Oymans, We are pleased to inform you that your manuscript, "Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas", has been editorially accepted for publication at PLOS Neglected Tropical Diseases. Before your manuscript can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please note: your manuscript will not be scheduled for publication until you have made the required changes. IMPORTANT NOTES * Copyediting and Author Proofs: To ensure prompt publication, your manuscript will NOT be subject to detailed copyediting and you will NOT receive a typeset proof for review. The corresponding author will have one final opportunity to correct any errors when sent the requests mentioned above. Please review this version of your manuscript for any errors. * If you or your institution will be preparing press materials for this manuscript, please inform our press team in advance at plosntds@plos.org. If you need to know your paper's publication date for media purposes, you must coordinate with our press team, and your manuscript will remain under a strict press embargo until the publication date and time. PLOS NTDs may choose to issue a press release for your article. If there is anything that the journal should know, please get in touch. *Now that your manuscript has been provisionally accepted, please log into EM and update your profile. Go to http://www.editorialmanager.com/pntd, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process. *Note to LaTeX users only - Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines [http://www.plosntds.org/static/latexGuidelines.action] in the meantime. Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases. Best regards, Abdallah M. Samy, PhD Guest Editor PLOS Neglected Tropical Diseases Paulo Pimenta Deputy Editor PLOS Neglected Tropical Diseases 13 Jan 2020 Dear Miss Oymans, We are delighted to inform you that your manuscript, "Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas," has been formally accepted for publication in PLOS Neglected Tropical Diseases. We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication. The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly. Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers. Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases. Best regards, Serap Aksoy Editor-in-Chief PLOS Neglected Tropical Diseases Shaden Kamhawi Editor-in-Chief PLOS Neglected Tropical Diseases

26 in total

1. Four-segmented Rift Valley fever virus induces sterile immunity in sheep after a single vaccination.

Authors: Paul J Wichgers Schreur; Jet Kant; Lucien van Keulen; Rob J M Moormann; Jeroen Kortekaas
Journal: Vaccine Date: 2015-02-07 Impact factor: 3.641

2. Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Authors: M Bouloy; C Janzen; P Vialat; H Khun; J Pavlovic; M Huerre; O Haller
Journal: J Virol Date: 2001-02 Impact factor: 5.103

3. Efficacy of three candidate Rift Valley fever vaccines in sheep.

Authors: J Kortekaas; A F G Antonis; J Kant; R P M Vloet; A Vogel; N Oreshkova; S M de Boer; B J Bosch; R J M Moormann
Journal: Vaccine Date: 2012-03-24 Impact factor: 3.641

4. NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription.

Authors: Agnès Billecocq; Martin Spiegel; Pierre Vialat; Alain Kohl; Friedemann Weber; Michèle Bouloy; Otto Haller
Journal: J Virol Date: 2004-09 Impact factor: 5.103

5. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

Authors: Matthias Habjan; Andreas Pichlmair; Richard M Elliott; Anna K Overby; Timo Glatter; Matthias Gstaiger; Giulio Superti-Furga; Hermann Unger; Friedemann Weber
Journal: J Virol Date: 2009-02-11 Impact factor: 5.103

6. Co-housing of Rift Valley Fever Virus Infected Lambs with Immunocompetent or Immunosuppressed Lambs Does Not Result in Virus Transmission.

Authors: Paul J Wichgers Schreur; Lucien van Keulen; Jet Kant; Nadia Oreshkova; Rob J M Moormann; Jeroen Kortekaas
Journal: Front Microbiol Date: 2016-03-07 Impact factor: 5.640

7. Transmission of Rift Valley fever virus from European-breed lambs to Culex pipiens mosquitoes.

Authors: Rianka P M Vloet; Chantal B F Vogels; Constantianus J M Koenraadt; Gorben P Pijlman; Martin Eiden; Jose L Gonzales; Lucien J M van Keulen; Paul J Wichgers Schreur; Jeroen Kortekaas
Journal: PLoS Negl Trop Dis Date: 2017-12-27

8. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

Authors: Tetsuro Ikegami; Krishna Narayanan; Sungyong Won; Wataru Kamitani; C J Peters; Shinji Makino
Journal: PLoS Pathog Date: 2009-02-06 Impact factor: 6.823

9. A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells.

Authors: Nicolas Le May; Zeyni Mansuroglu; Psylvia Léger; Thibaut Josse; Guillaume Blot; Agnès Billecocq; Ramon Flick; Yves Jacob; Eliette Bonnefoy; Michèle Bouloy
Journal: PLoS Pathog Date: 2008-01 Impact factor: 6.823

10. Rift Valley Fever Vaccine Virus Clone 13 Is Able to Cross the Ovine Placental Barrier Associated with Foetal Infections, Malformations, and Stillbirths.

Authors: Birgit Makoschey; Emma van Kilsdonk; Willem R Hubers; Mieke P Vrijenhoek; Marianne Smit; Paul J Wichgers Schreur; Jeroen Kortekaas; Véronique Moulin
Journal: PLoS Negl Trop Dis Date: 2016-03-31

14 in total

1. Reverse Genetics System for Shuni Virus, an Emerging Orthobunyavirus with Zoonotic Potential.

Authors: Judith Oymans; Paul J Wichgers Schreur; Sophie van Oort; Rianka Vloet; Marietjie Venter; Gorben P Pijlman; Monique M van Oers; Jeroen Kortekaas
Journal: Viruses Date: 2020-04-17 Impact factor: 5.048

Review 2. Livestock Challenge Models of Rift Valley Fever for Agricultural Vaccine Testing.

Authors: Andrea Louise Kroeker; Shawn Babiuk; Bradley S Pickering; Juergen A Richt; William C Wilson
Journal: Front Vet Sci Date: 2020-05-27

3. Shuni Virus Replicates at the Maternal-Fetal Interface of the Ovine and Human Placenta.

Authors: Judith Oymans; Lucien van Keulen; Guus M Vermeulen; Paul J Wichgers Schreur; Jeroen Kortekaas
Journal: Pathogens Date: 2020-12-29

4. Reproducing the Rift Valley fever virus mosquito-lamb-mosquito transmission cycle.

Authors: Paul J Wichgers Schreur; Rianka P M Vloet; Jet Kant; Lucien van Keulen; Jose L Gonzales; Tessa M Visser; Constantianus J M Koenraadt; Chantal B F Vogels; Jeroen Kortekaas
Journal: Sci Rep Date: 2021-01-14 Impact factor: 4.379

Review 5. Insights into the Pathogenesis of Viral Haemorrhagic Fever Based on Virus Tropism and Tissue Lesions of Natural Rift Valley Fever.

Authors: Lieza Odendaal; A Sally Davis; Estelle H Venter
Journal: Viruses Date: 2021-04-20 Impact factor: 5.048

Review 6. Infections at the maternal-fetal interface: an overview of pathogenesis and defence.

Authors: Christina J Megli; Carolyn B Coyne
Journal: Nat Rev Microbiol Date: 2021-08-25 Impact factor: 60.633

7. Rift Valley fever during the COVID-19 pandemic in Africa: A double burden for Africa's healthcare system.

Authors: Olivier Uwishema; Elie Chalhoub; Tania Torbati; Success Chekwube David; Carlo Khoury; Lucas Loiola Ponte Albuquerque Ribeiro; Yves Nasrallah; Bezawit Kassahun Bekele; Helen Onyeaka
Journal: Health Sci Rep Date: 2022-01-06

8. Time-Resolved Analysis of N-RNA Interactions during RVFV Infection Shows Qualitative and Quantitative Shifts in RNA Encapsidation and Packaging.

Authors: Miyuki Hayashi; Eric P Schultz; Jean-Marc Lanchy; J Stephen Lodmell
Journal: Viruses Date: 2021-12-02 Impact factor: 5.048

9. Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice.

Authors: Kaori Terasaki; Birte Kalveram; Kendra N Johnson; Terry Juelich; Jennifer K Smith; Lihong Zhang; Alexander N Freiberg; Shinji Makino
Journal: PLoS Negl Trop Dis Date: 2021-09-13

10. Early Pathogenesis of Wesselsbron Disease in Pregnant Ewes.

Authors: Judith Oymans; Lucien van Keulen; Paul J Wichgers Schreur; Jeroen Kortekaas
Journal: Pathogens Date: 2020-05-13