Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1.

Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

The Orthobunyavirus Germiston Enters Host Cells from Late Endosomes.

With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.

The transmission ecology of Tahyna orthobunyavirus in Austria as revealed by longitudinal mosquito sampling and blood meal analysis in floodplain habitats.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is a mosquito-borne virus that may cause mild flu-like symptoms or neurological symptoms in humans. It is historically associated with floodplain habitats in Central Europe, and the mammalophilic floodwater mosquito, Aedes vexans, is thought to be the principal vector. There are few contemporary reports of TAHV transmission ecology within mosquitoes or their vertebrate hosts, and virus infections are rarely reported (and probably seldom diagnosed). The objectives of this study were to survey the mosquito population for TAHV in three floodwater habitats and describe host usage by the predominant floodwater mosquito species to potentially define TAHV transmission at these foci. METHODS: We performed longitudinal mosquito sampling along three major rivers in eastern Austria to characterize the mosquito community in floodplain habitats, and tested for the presence of TAHV in pools of mosquitoes. We characterized TAHV rescued from mosquito pool homogenate by sequencing. We surveyed mosquito host selection by analyzing mosquito blood meals. RESULTS: We identified TAHV in two pools of Ae. vexans captured along the Leitha River. This mosquito, and other floodwater mosquitoes, used large mammals (red deer, roe deer, wild boar) as their hosts. The sequence of the rescued virus was remarkably similar to other TAHV isolates from the region, dating back to the first isolate of TAHV in 1958. CONCLUSIONS: In general, we confirmed that TAHV is most likely being transmitted by Ae. vexans, although the precise contribution of vertebrate-amplifying hosts to the ecological maintenance of the virus is unclear. The pattern of host selection matches the estimated exposure of the same large mammal species in the region to TAHV based on a recent serosurvey, but hares were also hosts at the site where TAHV was detected. We also confirm humans as hosts of two floodwater mosquito species, providing a potential mechanism for spillover of TAHV or other mosquito-borne viruses.

Orthobunyaviruses: From Virus Binding to Penetration into Mammalian Host Cells.

With over 80 members worldwide, Orthobunyavirus is the largest genus in the Peribunyaviridae family. Orthobunyaviruses (OBVs) are arthropod-borne viruses that are structurally simple, with a trisegmented, negative-sense RNA genome and only four structural proteins. OBVs are potential agents of emerging and re-emerging diseases and overall represent a global threat to both public and veterinary health. The focus of this review is on the very first steps of OBV infection in mammalian hosts, from virus binding to penetration and release of the viral genome into the cytosol. Here, we address the most current knowledge and advances regarding OBV receptors, endocytosis, and fusion.

Evaluation of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Mosquitoes Competence to Oropouche virus Infection.

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.

High dispersal capacity of Culicoides obsoletus (Diptera: Ceratopogonidae), vector of bluetongue and Schmallenberg viruses, revealed by landscape genetic analyses.

BACKGROUND: In the last two decades, recurrent epizootics of bluetongue virus and Schmallenberg virus have been reported in the western Palearctic region. These viruses affect domestic cattle, sheep, goats and wild ruminants and are transmitted by native hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae). Culicoides dispersal is known to be stratified, i.e. due to a combination of dispersal processes occurring actively at short distances and passively or semi-actively at long distances, allowing individuals to jump hundreds of kilometers. METHODS: Here, we aim to identify the environmental factors that promote or limit gene flow of Culicoides obsoletus, an abundant and widespread vector species in Europe, using an innovative framework integrating spatial, population genetics and statistical approaches. A total of 348 individuals were sampled in 46 sites in France and were genotyped using 13 newly designed microsatellite markers. RESULTS: We found low genetic differentiation and a weak population structure for C. obsoletus across the country. Using three complementary inter-individual genetic distances, we did not detect any significant isolation by distance, but did detect significant anisotropic isolation by distance on a north-south axis. We employed a multiple regression on distance matrices approach to investigate the correlation between genetic and environmental distances. Among all the environmental factors that were tested, only cattle density seems to have an impact on C. obsoletus gene flow. CONCLUSIONS: The high dispersal capacity of C. obsoletus over land found in the present study calls for a re-evaluation of the impact of Culicoides on virus dispersal, and highlights the urgent need to better integrate molecular, spatial and statistical information to guide vector-borne disease control.

Infection, Dissemination, and Transmission Potential of North American Culex quinquefasciatus, Culex tarsalis, and Culicoides sonorensis for Oropouche Virus.

Oropouche virus (OROV), a vector-borne Orthobunyavirus circulating in South and Central America, causes a febrile illness with high rates of morbidity but with no documented fatalities. Oropouche virus is transmitted by numerous vectors, including multiple genera of mosquitoes and Culicoides biting midges in South America. This study investigated the vector competence of three North American vectors, Culex tarsalis, Culex quinquefasciatus, and Culicoides sonorensis, for OROV. Cohorts of each species were fed an infectious blood meal containing 6.5 log10 PFU/mL OROV and incubated for 10 or 14 days. Culex tarsalis demonstrated infection (3.13%) but not dissemination or transmission potential at 10 days post infection (DPI). At 10 and 14 DPI, Cx. quinquefasciatus demonstrated 9.71% and 19.3% infection, 2.91% and 1.23% dissemination, and 0.97% and 0.82% transmission potential, respectively. Culicoides sonorensis demonstrated 86.63% infection, 83.14% dissemination, and 19.77% transmission potential at 14 DPI. Based on these data, Cx. tarsalis is unlikely to be a competent vector for OROV. Culex quinquefasciatus demonstrated infection, dissemination, and transmission potential, although at relatively low rates. Culicoides sonorensis demonstrated high infection and dissemination but may have a salivary gland barrier to the virus. These data have implications for the spread of OROV in the event of a North American introduction.

First report of antiviral activity of nordihydroguaiaretic acid against Fort Sherman virus (Orthobunyavirus).

The genus Orthobunyavirus are a group of viruses within arbovirus, with a zoonotic cycle, some of which could lead to human infection. A characteristic of these viruses is their lack of antiviral treatment or vaccine for its prevention. The objective of this work was to study the in vitro antiviral activity of nordihydroguaiaretic acid (NDGA), the most important active compound of Larrea divaricata Cav. (Zigophyllaceae), against Fort Sherman virus (FSV) as a model of Orthobunyavirus genus. At the same time, the effect of NDGA as a lipolytic agent on the cell cycle of this viral model was assessed. The method of reducing plaque forming units on LLC-MK2 cells was used to detect the action of NDGA on CbaAr426 and SFCrEq231 isolates of FSV. NDGA did not show virucidal effect, but it had antiviral activity with a similar inhibition in both isolates, which was dose dependent. It was established that the NDGA has a better inhibition 1-h post-internalization (p.i.), showing a different behavior in each isolate, which was dependent upon the time p.i. Since virus multiplication is dependent on host cell lipid metabolism, the antiviral effect of NDGA has been previously related to its ability to disturb the lipid metabolism, probably by interfering with the 5-lipoxigenase (5-LOX) and the sterol regulatory element-binding proteins (SREBP) pathway. We determined by using caffeic acid, a 5-LOX inhibitor, that the inhibition of this enzyme negatively affected the FSV replication; and by means of resveratrol, a SREBP1 inhibitor, it was showed that the negative regulation of this pathway only had action on the SFCrEq231 reduction. In addition, it was proved that the NDGA acts intracellularly, since it showed the ability to incorporate into LLC-MK2 cells. The information provided in this work converts the NDGA into a compound with antiviral activity in vitro against FSV (Orthobunyavirus), which can be subjected to structural modifications in the future to improve the activity.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

Shuni virus-induced meningoencephalitis after experimental infection of cattle.

Shuni virus (SHUV), an insect-transmitted orthobunyavirus of the Simbu serogroup within the family Peribunyaviridae, may induce severe congenital malformations when naïve ruminants are infected during gestation. Only recently, another clinical presentation in cattle, namely neurological disease after postnatal infection, was reported. To characterize the course of the disease under experimental conditions and to confirm a causal relationship between the virus and the neurological disorders observed in the field, six calves each were experimentally inoculated (subcutaneously) with two different SHUV strains from both clinical presentations, that is encephalitis and congenital malformation, respectively. Subsequently, the animals were monitored clinically, virologically and serologically for three weeks. All animals inoculated with the 'encephalitis strain' SHUV 2162/16 developed viremia for three to four consecutive days, seroconverted, and five out of six animals showed elevated body temperature for up to three days. No further clinical signs such as neurological symptoms were observed in any of these animals. However, four out of six animals developed a non-suppurative meningoencephalitis, characterized by perivascular cuffing and glial nodule formation. Moreover, SHUV genome could be visualized in brain tissues of the infected animals by in situ hybridization. In contrast to the 'encephalitis SHUV strain', in animals subcutaneously inoculated with the strain isolated from a malformed newborn (SHUV 2504/3/14), which expressed a truncated non-structural protein NSs, a major virulence factor, no viremia or seroconversion, was observed, demonstrating an expected severe replication defect of this strain in vivo. The lack of viremia further indicates that virus variants evolving in malformed foetuses may represent attenuated artefacts as has been described for closely related viruses. As the neuropathogenicity of SHUV could be demonstrated under experimental conditions, this virus should be included in differential diagnosis for encephalitis in ruminants, and cattle represent a suitable animal model to study the pathogenesis of SHUV.

Emerging orthobunyaviruses associated with CNS disease.

The Orthobunyavirus genus comprises a wide range of arthropod-borne viruses which are prevalent worldwide and commonly associated with central nervous system (CNS) disease in humans and other vertebrates. Several orthobunyaviruses have recently emerged and increasingly more will likely do so in the future. Despite this large number, an overview of these viruses is currently lacking, making it challenging to determine importance from a One Health perspective. Causality is a key feature of determining importance, yet classical tools are unfit to evaluate the causality of orthobunyaviral CNS disease. Therefore, we aimed to provide an overview of orthobunyaviral CNS disease in vertebrates and objectify the causality strength of each virus. In total, we identified 27 orthobunyaviruses described in literature to be associated with CNS disease. Ten were associated with disease in multiple host species of which seven included humans. Seven viruses were associated with both congenital and postnatal CNS disease. CNS disease-associated orthobunyaviruses were spread across all known Orthobunyavirus serogroups by phylogenetic analyses. Taken together, these results indicate that orthobunyaviruses may have a common tendency to infect the CNS of vertebrates. Next, we developed six tailor-made causality indicators and evaluated the causality strength of each of the identified orthobunyaviruses. Nine viruses had a 'strong' causality score and were deemed causal. Eight had a 'moderate' and ten a 'weak' causality score. Notably, there was a lack of case-control studies, which was only available for one virus. We, therefore, stress the importance of proper case-control studies as a fundamental aspect of proving causality. This comprehensible overview can be used to identify orthobunyaviruses which may be considered causal, reveal research gaps for viruses with moderate to low causality scores, and provide a framework to evaluate the causality of orthobunyaviruses that may newly emerge in the future.

Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays.

Oropouche orthobunyavirus (OROV) is an emerging arbovirus with a high potential of dissemination in America. Little is known about the role of peripheral blood mononuclear cells (PBMC) response during OROV infection in humans. Thus, to evaluate human leukocytes susceptibility, permissiveness and immune response during OROV infection, we applied RNA hybridization, qRT-PCR and cell-based assays to quantify viral antigens, genome, antigenome and gene expression in different cells. First, we observed OROV replication in human leukocytes lineages as THP-1 monocytes, Jeko-1 B cells and Jurkat T cells. Interestingly, cell viability and viral particle detection are maintained in these cells, even after successive passages. PBMCs from healthy donors were susceptible but the infection was not productive, since neither antigenome nor infectious particle was found in the supernatant of infected PBMCs. In fact, only viral antigens and small quantities of OROV genome were detected at 24 hpi in lymphocytes, monocytes and CD11c+ cells. Finally, activation of the Interferon (IFN) response was essential to restrict OROV replication in human PBMCs. Increased expression of type I/III IFNs, ISGs and inflammatory cytokines was detected in the first 24 hpi and viral replication was re-established after blocking IFNAR or treating cells with glucocorticoid. Thus, in short, our results show OROV is able to infect and remain in low titers in human T cells, monocytes, DCs and B cells as a consequence of an effective IFN response after infection, indicating the possibility of leukocytes serving as a trojan horse in specific microenvironments during immunosuppression.

A Genome-Wide CRISPR-Cas9 Screen Reveals the Requirement of Host Cell Sulfation for Schmallenberg Virus Infection.

Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses.IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.

Impact of Gut Bacteria on the Infection and Transmission of Pathogenic Arboviruses by Biting Midges and Mosquitoes.

Tripartite interactions among insect vectors, midgut bacteria, and viruses may determine the ability of insects to transmit pathogenic arboviruses. Here, we investigated the impact of gut bacteria on the susceptibility of Culicoides nubeculosus and Culicoides sonorensis biting midges for Schmallenberg virus, and of Aedes aegypti mosquitoes for Zika and chikungunya viruses. Gut bacteria were manipulated by treating the adult insects with antibiotics. The gut bacterial communities were investigated using Illumina MiSeq sequencing of 16S rRNA, and susceptibility to arbovirus infection was tested by feeding insects with an infectious blood meal. Antibiotic treatment led to changes in gut bacteria for all insects. Interestingly, the gut bacterial composition of untreated Ae. aegypti and C. nubeculosus showed Asaia as the dominant genus, which was drastically reduced after antibiotic treatment. Furthermore, antibiotic treatment resulted in relatively more Delftia bacteria in both biting midge species, but not in mosquitoes. Antibiotic treatment and subsequent changes in gut bacterial communities were associated with a significant, 1.8-fold increased infection rate of C. nubeculosus with Schmallenberg virus, but not for C. sonorensis. We did not find any changes in infection rates for Ae. aegypti mosquitoes with Zika or chikungunya virus. We conclude that resident gut bacteria may dampen arbovirus transmission in biting midges, but not so in mosquitoes. Use of antimicrobial compounds at livestock farms might therefore have an unexpected contradictory effect on the health of animals, by increasing the transmission of viral pathogens by biting midges.

Low incidence of Schmallenberg virus infection in natural cases of abortion in domestic ruminants in Hungary.

An epizootic caused by a new orthobunyavirus called Schmallenberg virus (SBV) was recognised in European ruminants in 2011 and 2012. The re-emergence of the infection was reported in several countries in the subsequent years. Although the main clinical sign of SBV infection is abortion, the impact of SBV in natural cases of abortion in domestic ruminants had not been systematically examined before this study. The aim of the study was to investigate the role of SBV infection and to compare it to the importance of other causes of abortion by examining 537 natural cases of abortion that had occurred between 2011 and 2017 in Hungary. The cause of abortion was determined in 165 (31%) cases. An infectious cause was proved in 88 (16%) cases. SBV infection was found only in a total of four cases (0.8%) using real-time polymerase chain reaction. Three of them proved to be inapparent SBV infection, and one case was attributed to SBV-induced abortion by detecting non-purulent encephalitis and SBV nucleoprotein by immunohistochemistry in a brain tissue sample. According to the results, SBV played a minor role in natural cases of domestic ruminant abortion in Hungary during the 7-year period following the first SBV outbreak in 2011.

The Cap-Snatching Mechanism of Bunyaviruses.

In common with all segmented negative-sense RNA viruses, bunyavirus transcripts contain heterologous sequences at their 5' termini originating from capped host cell RNAs. These heterologous sequences are acquired by a so-called cap-snatching mechanism. Whereas for nuclear replicating influenza virus the source of capped primers as well as the cap-binding and endonuclease activities of the viral polymerase needed for cap snatching have been functionally and structurally well characterized, our knowledge on the expected counterparts of cytoplasmic replicating bunyaviruses is still limited and controversial. This review focuses on the cap-snatching mechanism of bunyaviruses in the light of recent structural and functional data.

Immunogenicity and efficacy of Schmallenberg virus envelope glycoprotein subunit vaccines.

The Schmallenberg virus (SBV) is an orthobunyavirus that causes abortions, stillbirths, and congenital defects in pregnant sheep and cattle. Inactivated or live attenuated vaccines have been developed in endemic countries, but there is still interest in the development of SBV vaccines that would allow Differentiating Infected from Vaccinated Animals (DIVA). Therefore, an attempt was made to develop novel DIVA-compatible SBV vaccines using SBV glycoproteins expressed in baculovirus. All vaccines and phosphate buffered saline (PBS) controls were prepared with adjuvant and administered subcutaneously to cattle at 6 month of age. The first trial included 2 groups of animals vaccinated with either carboxyl-terminus glycoprotein (Gc) or PBS and boosted after 2 weeks. In the second trial, 3 groups of cattle were administered either Gc, Gc and amino-terminus glycoprotein (Gn), or PBS with a booster vaccination after 3 weeks. The animals were challenged with SBV 9 days after the booster vaccination in the first study, and 3 weeks after the booster vaccination in the second study. Using a SBV Gc-specific enzyme-linked immunosorbent assay, antibodies were first detected in serum samples 14 days after the first vaccination in both trials, and peaked on days 7 and 9 after the booster in the first and second trials, respectively. Low titers of neutralizing antibodies were detected in serum from only 3/6 and 2/4 animals in the first and second trial, respectively, at 14 days after the first vaccination. The titers increased 2 to 3-fold after the booster vaccination. SBV-specific RNA was detected in the serum and selective tissues in all animals after SBV challenge independent of vaccination status. The SBV candidate vaccines neither prevented viremia nor conferred protection against SBV infection.

Schmallenberg Disease-A Newly Emerged Culicoides-borne Viral Disease of Ruminants.

First appearing in 2011 in Northern Europe, Schmallenberg virus (SBV), an Orthobunyavirus of the Simbu serogroup, is associated with clinical disease mainly in ruminants such as cattle, sheep and goats. The clinical signs are characterized by abortion and congenital deformities in newborns. The virus is transmitted by Culicoides midges of the Obsoletus complex. SBV infection induces a solid protective immunity that persists for at least 4 or 6 years in sheep and cattle, respectively. SBV infection can be diagnosed directly by real-time RT-qPCR and virus isolation or indirectly by serological assays. Three vaccines are commercially available in Europe. This article provides a comprehensive literature review on this emerging disease regarding pathogenesis, transmission, diagnosis, control and prevention. This review also highlights that although much has been learned since SBV's first emergence, there are still areas that require further study to devise better mitigation strategies.

Reliable and Standardized Animal Models to Study the Pathogenesis of Bluetongue and Schmallenberg Viruses in Ruminant Natural Host Species with Special Emphasis on Placental Crossing.

Starting in 2006, bluetongue virus serotype 8 (BTV8) was responsible for a major epizootic in Western and Northern Europe. The magnitude and spread of the disease were surprisingly high and the control of BTV improved significantly with the marketing of BTV8 inactivated vaccines in 2008. During late summer of 2011, a first cluster of reduced milk yield, fever, and diarrhoea was reported in the Netherlands. Congenital malformations appeared in March 2012 and Schmallenberg virus (SBV) was identified, becoming one of the very few orthobunyaviruses distributed in Europe. At the start of both epizootics, little was known about the pathogenesis and epidemiology of these viruses in the European context and most assumptions were extrapolated based on other related viruses and/or other regions of the World. Standardized and repeatable models potentially mimicking clinical signs observed in the field are required to study the pathogenesis of these infections, and to clarify their ability to cross the placental barrier. This review presents some of the latest experimental designs for infectious disease challenges with BTV or SBV. Infectious doses, routes of infection, inoculum preparation, and origin are discussed. Particular emphasis is given to the placental crossing associated with these two viruses.

Changing surveillance objectives during the different phases of an emerging vector-borne disease outbreak: The Schmallenberg virus example.

In the late summer of 2011, a sudden rise in incidence of fever, drop in milk production and diarrhoea was observed in dairy cows in the eastern region of the Netherlands and in north-western Germany. In the autumn of 2011, a novel orthobunyavirus was identified by metagenomic analyses in samples from acutely diseased cows on a farm near the German city of Schmallenberg, and was thereafter named Schmallenberg virus (SBV). Due to the novelty of the virus, there was an immediate need for knowledge regarding the epidemiological characteristics of SBV-infections to inform surveillance and control strategies. A rapid assessment of the spread and impact of an emerging disease supports decision-makers on allocation of resources. This paper reviews the disease mitigation activities during and after the SBV epidemic in the Netherlands, to illustrate the phases in surveillance when a new (vector-borne) pathogen emerges in a country or region. Immediate and short-term disease mitigation activities that were initiated after SBV was identified are discussed in detail, as well as ways to enhance future surveillance (e.g. by syndromic surveillance) and preparedness for similar disease outbreaks. By doing so, lessons learnt from the SBV epidemic will also improve surveillance for other emerging diseases in cattle.