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.
The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3′ terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.IMPORTANCE Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.
The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp “snatches” capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism but show that it functions efficiently only for primers that are short or cannot stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently.IMPORTANCE Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.
BackgroundShuni virus (SHUV) is an orthobunyavirus that belongs to the Simbu serogroup. SHUV was isolated from diverse species of domesticated animals and wildlife, and is associated with neurological disease, abortions, and congenital malformations. Recently, SHUV caused outbreaks among ruminants in Israel, representing the first incursions outside the African continent. The isolation of SHUV from a febrile child in Nigeria and seroprevalence among veterinarians in South Africa suggests that the virus may have zoonotic potential as well. The high pathogenicity, extremely broad tropism, potential transmission via both biting midges and mosquitoes, and zoonotic features warrants prioritization of SHUV for further research. Additional knowledge is essential to accurately determine the risk for animal and human health, and to assess the risk of future epizootics and epidemics. To gain first insights into the potential involvement of arthropod vectors in SHUV transmission, we have investigated the ability of SHUV to infect and disseminate in laboratory-reared biting midges and mosquitoes.Methodology/Principal findingsCulicoides nubeculosus, C. sonorensis, Culex pipiens pipiens, and Aedes aegypti were orally exposed to SHUV by providing an infectious blood meal. Biting midges showed high infection rates of approximately 40–60%, whereas infection rates of mosquitoes were lower than 2%. SHUV successfully disseminated in both species of biting midges, but no evidence of transmission in orally exposed mosquitoes was found.Conclusions/SignificanceThe results of this study show that different species of Culicoides biting midges are susceptible to infection and dissemination of SHUV, whereas the two mosquito species tested were found not to be susceptible.
The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.
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