Pigs in southern Italy are exposed to three ruminant pathogens: an analysis of seroprevalence and risk factors analysis study.

BACKGROUND: Pigs are susceptible to several ruminant pathogens, including Coxiella burnetti, Schmallenberg virus (SBV) and bovine viral diarrhea virus (BVDV). These pathogens have already been described in the pig population, although the dynamics of the infection and the impact on pig farms are currently unclear. The aim of this work was to evaluate the presence of these infections in the pig population of the Campania region, southern Italy, and to evaluate the risk factors associated with a greater risk of exposure. RESULTS: A total of 414 serum samples belonging to 32 herds were tested for the presence of antibodies against SBV, Coxiella, and BVD using commercial multispecies ELISA kits. SBV (5.3%) was the most prevalent pathogen, followed by Coxiella (4.1%) and BVD (3%). The risk factors included in the study (age, sex, province, farming system, ruminant density and major ruminant species) had no influence on the probability of being exposed to BVD and Coxiella, except for the location, in fact more pigs seropositive to Coxiella were found in the province of Caserta. However, the univariate analysis highlighted the influence of age, location, and sex on exposure to SBV. The subsequent multivariate analysis statistically confirmed the importance of these factors. The presence of neutralizing antibodies for SBV and BVDV, or antibodies directed towards a specific phase of infection for Coxiella was further confirmed with virus-neutralization assays and phase-specific ELISAs in a large proportion of positive samples. The presence of high neutralizing antibody titers (especially for SBV) could indicate recent exposures. Twelve of the 17 positive samples tested positive for antibodies against Coxiella phase I or II antigens, indicating the presence of both acute and chronic infections (one animal tested positive for both phases antibodies). CONCLUSIONS: Our study indicates a non-negligible exposure of pigs from southern Italy to the above pathogens. Further studies are necessary to fully understand the dynamics of these infections in pigs, the impact on productivity, and the public health consequences in the case of Coxiella.

[Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody].

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

Innate immune response against vector-borne bunyavirus infection and viral countermeasures.

Bunyaviruses are a large group of important viral pathogens that cause significant diseases in humans and animals worldwide. Bunyaviruses are enveloped, single-stranded, negative-sense RNA viruses that infect a wide range of hosts. Upon entry into host cells, the components of viruses are recognized by host innate immune system, leading to the activation of downstream signaling cascades to induce interferons (IFNs) and other proinflammatory cytokines. IFNs bind to their receptors and upregulate the expression of hundreds of interferon-stimulated genes (ISGs). Many ISGs have antiviral activities and confer an antiviral state to host cells. For efficient replication and spread, viruses have evolved different strategies to antagonize IFN-mediated restriction. Here, we discuss recent advances in our understanding of the interactions between bunyaviruses and host innate immune response.

First evidence of Schmallenberg virus infection in domestic ruminants in Kosovo and Albania.

Schmallenberg virus (SBV), a novel Orthobunyavirus, emerged in European domestic ruminants in 2011 causing abortions and malformations in newborns and none or mild clinical symptoms in adult animals. Here, a total of 364 bovine, ovine and caprine serum samples were collected in Kosovo and Albania between May 2014 and August 2016 and analyzed for the presence of anti­SBV antibodies. Sera were tested using an enzyme­linked immunosorbent assay (ELISA), and 48 ELISA­positive samples were subsequently analyzed by serum neutralization test (SNT). The overall percentage of ELISA positive results was 17.9%; 23.1% (53/229) was the prevalence observed in Kosovo (cattle 45.5%, sheep 19.2% and goat 6.8%), while 8.9% (12/135) was that observed in Albania (cattle 11.1%, sheep 0% and goat 20.0%). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested. This is the first study reporting SBV circulation in domestic ruminants in Kosovo and Albania, with indication that this virus has been present in Kosovo and Albania at least since 2014 without being detected.

Arbovirus serosurvey (Orthobunyavirus, Flavivirus, and Alphavirus) in a draft horse population from Santa Fe, Argentina (2013-2016).

In the present study, we serosurveyed the exposure of 222 draft horses to different arboviruses in the city of Santa Fe, Argentina. Plaque reduction neutralization tests confirmed exposure to Fort Sherman virus (FSV), Saint Louis encephalitis virus (SLEV), West Nile virus (WNV), and Río Negro virus (RNV). Apparently, Western and Eastern equine encephalitis viruses did not circulate in the population tested. The confirmation of five seroconversions for WNV, FSV, and SLEV and the association between prevalence and age are indicative of recent circulation. These results highlight the importance of considering draft horses in arboviral surveillance in urban and rural areas of developing countries.

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.

Epidemiological surveillance of Schmallenberg virus in small ruminants in southern Spain.

Schmallenberg virus (SBV) is an emerging Culicoides-borne Orthobunyavirus that affects ruminant species. Between 2011 and 2013, it was responsible for a large-scale epidemic in Europe. In the present study, we aimed to determine the seroprevalence, spatial distribution and risk factors associated with SBV exposure in sheep and goats in the region where the first Schmallenberg disease outbreak in Spain was reported. Blood samples from 1,796 small ruminants from 120 farms were collected in Andalusia (southern Spain) between 2015 and 2017. Antibodies against SBV were detected in 536 of 1,796 animals (29.8%; 95%CI: 27.7-32.0) using a commercial blocking ELISA. The individual seroprevalence according to species was 31.1% (280/900; 95%CI: 28.1-34.1) in sheep and 28.6% (256/896; 95%CI: 25.6-31.5) in goats. The farm prevalence was 76.7% (95%CI: 69.1-84.2). Seropositivity to SBV was confirmed in both sheep and goats in all provinces by virus neutralization test. Two significant (p < .001) spatial clusters of high seroprevalence were identified. The generalized estimating equation analysis showed that management system (extensive), temperature (>14ºC) and altitude (<400 metres above sea level) were risk factors associated with SBV exposure in small ruminants. Our results highlight widespread but not homogeneous circulation of SBV in small ruminant populations in Spain.

Serosurveillance of Schmallenberg virus in wild ruminants in Spain.

Schmallenberg disease (SBD) is an emerging vector-borne disease that affects domestic and wild ruminants. A long-term serosurvey was conducted to assess exposure to Schmallenberg virus (SBV) in all the wild ruminant species present in mainland Spain. Between 2010 and 2016, sera from 1,216 animals were tested for antibodies against SBV using a commercial blocking ELISA. The overall prevalence of antibodies was 27.1% (95%CI: 24.7-29.7). Statistically significant differences among species were observed, with significantly higher seropositivity found in fallow deer (Dama dama) (45.6%; 99/217), red deer (Cervus elaphus) (31.6%; 97/307) and mouflon (Ovis aries musimon) (28.0%; 33/118) compared to Barbary sheep (Ammotragus lervia) (22.2%; 8/36), Iberian wild goat (Capra pyrenaica) (19.9%; 49/246), roe deer (Capreolus capreolus) (17.5%; 34/194) and Southern chamois (Rupicapra pyrenaica) (10.2%; 10/98). Seropositive animals were detected in 81.4% (57/70; 95%CI: 70.8-88.8) of the sampled populations. SBV seroprevalence ranged from 18.8% (48/256) in bioregion (BR)2 (north-central, Mediterranean) to 32.3% (31/96) in BR1 (northeastern or Atlantic, Eurosiberian). Anti-SBV antibodies were not found before 2012, when the first outbreak of SBD was reported in Spain. In contrast, seropositivity was detected uninterruptedly during the period 2012-2016 and anti-SBV antibodies were found in yearling animals in each of these years. Our results provide evidence of widespread endemic circulation of SBV among wild ruminant populations in mainland Spain in recent years. Surveillance in these species could be a useful tool for monitoring SBV in Europe, particularly in areas where wild ruminants share habitats with livestock.

Zoo animals as sentinels for Schmallenberg virus monitoring in Spain.

Schmallenberg virus (SBV) is a newly emerged vector-borne pathogen that affects many domestic and wild animal species. A serosurvey was carried out to assess SBV exposure in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals. Between 2002 and 2019, sera from 278 animals belonging to 73 different species were collected from five zoos (A-E). Thirty-one of these animals were longitudinally sampled at three of these zoo parks during the study period. Seropositivity was detected in 28 (10.1 %) of 278 animals analyzed by blocking ELISA. Specific anti-SBV antibodies were confirmed in 20 (7.2 %; 95 %CI: 4.2-10.3) animals of six different species using virus neutralization test (VNT). The multiple logistic regression model showed that "order" (Artiodactyla) and "zoo provenance" (zoo B; southern Spain) were risk factors potentially associated with SBV exposure. Two (8.7 %) of the 31 longitudinally-sampled individuals showed specific antibodies against SBV at all samplings whereas seroconversion was detected in one mouflon (Ovis aries musimon) and one Asian elephant (Elephas maximus) in 2016 and 2019, respectively. To the best of the author's knowledge, this is the first surveillance conducted on SBV in zoos in Spain. The results confirm SBV exposure in zoo animals in this country and indicate circulation of the virus before the first Schmallenberg disease outbreak was reported in Spain. Surveillance in zoological parks could be a complementary approach to monitoring SBV activity. Further studies are warranted to assess the impact of this virus on the health status of susceptible zoo animals.

A novel Schmallenberg virus subunit vaccine candidate protects IFNAR-/- mice against virulent SBV challenge.

Schmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR-/- mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.

Clinical, MRI, and histopathological findings of congenital focal diplomyelia at the level of L4 in a female crossbred calf.

BACKGROUND: This case report describes the clinical signs of a calf with focal diplomyelia at the level of the fourth lumbar vertebra. Magnetic resonance imaging (MRI) images and histological findings of the affected spinal cord are included in this case report. This case differs from previously reported cases in terms of localization and minimal extent of the congenital anomaly, clinical symptoms and findings during further examinations. CASE PRESENTATION: The calf was presented to the Farm Animal Health clinic, Faculty of Veterinary Medicine, Utrecht University, with an abnormal, stiff, 'bunny-hop' gait of the pelvic limbs. Prominent clinical findings included general proprioceptive ataxia with paraparesis, pathological spinal reflexes of the pelvic limbs and pollakiuria. MRI revealed a focal dilated central canal, and mid-sagittal T2 hyperintense band in the dorsal part of the spinal cord at the level of the third to fourth lumbar vertebra. By means of histology, the calf was diagnosed with focal diplomyelia at the level of the fourth lumbar vertebra, a rare congenital malformation of the spinal cord. The calf tested positive for Schmallenberg virus antibodies, however this is not considered to be part of the pathogenesis of the diplomyelia. CONCLUSIONS: This case report adds value to future clinical practice, as it provides a clear description of focal diplomyelia as a previously unreported lesion and details its diagnosis using advanced imaging and histology. This type of lesion should be included in the differential diagnoses when a calf is presented with a general proprioceptive ataxia of the hind limbs. In particular, a 'bunny-hop' gait of the pelvic limbs is thought to be a specific clinical symptom of diplomyelia. This case report is of clinical and scientific importance as it demonstrates the possibility of a focal microscopic diplomyelia, which would not be evident by gross examination alone, as a cause of hind-limb ataxia. The aetiology of diplomyelia in calves remains unclear.

Reverse genetics approaches for the development of bunyavirus vaccines.

The Bunyavirales order is the largest group of RNA viruses, which includes important human and animal pathogens, that cause serious diseases. Licensed vaccines are often not available for many of these pathogens. The establishment of bunyavirus reverse genetics systems has facilitated the generation of recombinant infectious viruses, which have been employed as powerful tools for understanding bunyavirus biology and identifying important virulence factors. Technological advances in this area have enabled the development of novel strategies, including codon-deoptimization, viral genome rearrangement and single-cycle replicable viruses, for the generation of live-attenuated vaccine candidates. In this review, we have summarized the current knowledge of the bunyavirus reverse genetics approaches for the generation of live-attenuated vaccine candidates and their evaluation in animal models.

Pan-viral protection against arboviruses by activating skin macrophages at the inoculation site.

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.

Post-epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia.

Schmallenberg virus (SBV) is a vector-borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA-positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re-emergence of SBV in Europe is predicted to occur when population-level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV-positive wild ruminants suggest that these species might be possible sources for the re-emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017-2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real-time RT-PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.

Schmallenberg virus neutralising antibody responses in sheep.

BACKGROUND: Schmallenberg virus (SBV) is a midge borne virus of cattle and sheep. Infection is typically asymptomatic in adult sheep but fetal infection during pregnancy can result in abortion, stillbirth, neurological disorders and malformations of variable severity in newborn animals. It was first identified in Germany and the Netherlands in 2011 and then circulated throughout Europe in 2012 and 2013. Circulation in subsequent years was low or non-existent until summer and autumn 2016, leading to an increased incidence of deformed newborn lambs and calves in 2016-17. This study reports SBV circulation in October 2016 within a group of 24 ewes and 13 rams. The ewes were monitored at 3 times points over an 11 week period (September to December 2016). RESULTS: Most ewes displayed an increase in SBV VNT with antibody titre increases greater in older, previously exposed ewes. Two ewes had SBV RNA detectable by RT-qPCR, one on 30/09/16 and one on 04/11/16. Of these ewes, one had detectable serum SBV RNA (indicating viraemia) despite pre-existing antibody. The rams had been previously vaccinated with a commercial inactivated SBV vaccine, they showed minimal neutralising antibody titres against SBV 8 months post-vaccination and all displayed increased titre in October 2016. CONCLUSION: This data suggests that SBV circulated for a minimum period of 5 weeks in September to October 2016 in central England. Ewes previously exposed to virus showed an enhanced antibody response compared to naïve animals. Pre-existing antibody titre did not prevent re-infection in at least one animal, implying immunity to SBV upon natural exposure may not be life-long. In addition, data suggests that immunity provided by killed adjuvanted SBV vaccines only provides short term protection (< 8 months) from virus.

Comparative evaluation of two commercial ELISA kits for detection of antibodies against Akabane virus in cattle serum.

BACKGROUND: Akabane disease (AD), a barrier to international trade for endemic areas with far economic impact on the countries, is caused by Akabane virus (AKAV). Commercial enzyme-linked immunosorbent assay (ELISA) is a commonly used diagnostic technique for AKAV infection, including the IDEXX and IDVET ELISA kits. However, the comparative evaluation of the IDEXX and IDVET ELISA kits has not been published. The object of this study was to evaluate the test performance of the two commercial ELISA kits in detecting serum anti-AKAV antibodies in cattle. RESULTS: With virus neutralization test (VNT) as the "relative gold standard", the diagnostic sensitivity (DSe) was 80.39% (123/153) and 93.46% (143/153) for the IDEXX and IDVET ELISA kit, when suspect samples were included. The diagnostic specificity (DSp) for the IDEXX and IDVET ELISA kit was 93.48% (502/537) and 82.31% (442/537), respectively. CONCLUSION: Both of the tested ELISA kits could be applied to detect antibodies against AKAV in cattle serum. The IDVET ELISA kit had a higher DSe. The IDEXX ELISA kit possessed the higher DSp. These results have important implications if the kits are used to screen herds or individual cattle in surveillance programs, or at border crossings for import-export inspection and quarantine.

Description of the first Schmallenberg disease outbreak in Spain and subsequent virus spreading in domestic ruminants.

Schmallenberg disease (SBD) is an emerging disease transmitted mainly among ruminant species by biting midges of the genus Culicoides. Since the Schmallenberg virus (SBV) was first identified in Germany in late 2011, it rapidly spread to other European countries. The aims of the present study were to describe the first SBD outbreak in Spain and to assess the spread and risk factors associated with SBV infection in domestic ruminants from nearby farms during the following year. In March 2012, one malformed stillborn lamb from a sheep farm located in Cordoba province (Southern Spain) was subjected to necropsy. Pathological compatible lesions and molecular analyses confirmed the first SBV infection in Spain. Afterwards, serum samples from 505 extensively reared domestic ruminants from 29 farms were analysed using both blocking ELISA and virus neutralization test against SBV. The overall seroprevalence was 54.4% (CI95%: 50.0-58.7). Antibodies were detected in 70.6%, 46.0% and 34.8% of cattle, sheep and goats, respectively. A generalized estimating equation model indicated that the main risk factors associated with SBV infection were: species (cattle), age (adult), and absence of animal insecticide treatment. Pathological and molecular results confirmed the presence of SBV in Spain few months after it was firstly identified in Germany. The seroprevalence detected indicates a widespread circulation of SBV in nearby domestic ruminant farms one year after this first outbreak was reported in Spain. Further studies are warranted to determine the spatio-temporal trend of SBV in domestic ruminants in this country.

International proficiency trial demonstrates reliable Schmallenberg virus infection diagnosis in endemic and non-affected countries.

Schmallenberg virus (SBV), an orthobunyavirus infecting ruminants, emerged in 2011 in Central Europe, spread very rapidly throughout the continent and established an endemic status, thereby representing a constant threat not only to the European livestock population, but also to neighboring countries. Hence, in endemically infected regions, the maintenance and regular verification of diagnostics is needed and in not yet affected regions, suitable diagnostic systems should be established to be prepared for a potential introduction of the disease. In addition, also for the trade of animals into free regions, highly reliable and sensitive diagnostics are of utmost importance. Therefore, a laboratory proficiency trial was initiated to allow for performance evaluations of test systems available for SBV-diagnostics, but also for evaluation of veterinary diagnostic laboratories performing those tests. Ten serum samples (six seropositive, four seronegative) were provided for serological analysis, four of the seropositive samples were provided undiluted, while the remaining samples represented 1/2 and 1/4 dilutions of one of the aforementioned samples in negative serum. Ten further sera (five virus-positive, five negative) were sent to the participants to be analyzed by SBV genome detection methods. A total of 48 diagnostic laboratories from 15 countries of three continents (Europe, Asia, North America) and three kit manufacturers participated in the SBV proficiency test, thereby generating 131 result sets, corresponding to 1310 individual results. The sample panel aimed for serological analysis was tested 72 times; the applied diagnostic methods comprised different commercial ELISAs and standard micro-neutralization tests. The sample set aimed for genome detection was analyzed in 59 approaches by various commercial or in-house (real-time) RT-PCR protocols. Antibody or genome positive samples were correctly identified in every case, independent of the applied diagnostic test system. For seronegative samples, three incorrect, false-positive test results were produced. Virus-negative samples tested false-positive in two cases. Thus, a very high diagnostic accuracy of 99.58% and 99.66% was achieved by the serological and virological methods, respectively. Hence, this ring trial demonstrated that reliable and robust SBV-diagnostics has been established in veterinary diagnostic laboratories in affected and non-affected countries.

Orthobunyavirus spike architecture and recognition by neutralizing antibodies.

Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.

Characterization of Triniti virus supports its reclassification in the family Peribunyaviridae.

Triniti virus (TNTV) has been isolated in Trinidad and Tobago and in Brazil. To date little is known about this virus, which is classified as an ungrouped virus within the family Togaviridae. Here, three isolates of TNTV were characterized both genetically and antigenically. The genome was shown to contain three RNA segments: small (S), medium (M) and large (L). Genome organization, protein sizes and protein motifs were similar to those of viruses in the genus Orthobunyavirus, family Peribunyaviridae. Antigenic reactivity revealed the three TNTV isolates to be closely related, but no serologic cross-reaction with other orthobunyaviruses. Morphological observation by transmission electron microscopy indicated that virus size and symmetry were compatible with those of viruses in the family Peribunyaviridae. Our serological, morphological and molecular results support the taxonomic reclassification of TNTV as a member of the genus Orthobunyavirus, family Peribunyaviridae.