Infection dynamics and persistence of hepatitis E virus on pig farms - a review.

BACKGROUND: Hepatitis E virus (HEV) genotype 3 and 4 is a zoonosis that causes hepatitis in humans. Humans can become infected by consumption of pork or contact with pigs. Pigs are the main reservoir of the virus worldwide and the virus is present on most pig farms. MAIN BODY: Though HEV is present on most farms, the proportion of infected pigs at slaughter and thus the level of exposure to consumers differs between farms and countries. Understanding the cause of that difference is necessary to install effective measures to lower HEV in pigs at slaughter. Here, HEV studies are reviewed that include infection dynamics of HEV in pigs and on farms, risk factors for HEV farm prevalence, and that describe mechanisms and sources that could generate persistence on farms. Most pigs become infected after maternal immunity has waned, at the end of the nursing or beginning of the fattening phase. Risk factors increasing the likelihood of a high farm prevalence or proportion of actively infected slaughter pigs comprise of factors such as farm demographics, internal and external biosecurity and immunomodulating coinfections. On-farm persistence of HEV is plausible, because of a high transmission rate and a constant influx of susceptible pigs. Environmental sources of HEV that enhance persistence are contaminated manure storages, water and fomites. CONCLUSION: As HEV is persistently present on most pig farms, current risk mitigation should focus on lowering transmission within farms, especially between farm compartments. Yet, one should be aware of the paradox of increasing the proportion of actively infected pigs at slaughter by reducing transmission insufficiently. Vaccination of pigs may aid HEV control in the future.

Study on inactivation of porcine epidemic diarrhoea virus, porcine sapelovirus 1 and adenovirus in the production and storage of laboratory spray-dried porcine plasma.

AIM: Evaluation of the thermal and physical conditions for inactivation of adenovirus (AdV), porcine sapelovirus 1 (PSV1) and the economically important viruses porcine epidemic diarrhoea virus (PEDV) and porcine circovirus 2 (PCV2) in the production of spray-dried porcine plasma (SDPP). METHODS AND RESULTS: Citrate-treated porcine plasma of pH 7·5, 9·8 and 10·2 (8·5% dry-matter) was spiked with PEDV, PSV1, PCV2 and AdV and incubated at 3°C for maximum 24 h, and at 44 or 48°C for maximum 10 min (Experiment 1). Spiked citrate-treated concentrated plasma of pH 7·5 and 9·8 (24% dry-matter) was spray dried in a laboratory scale apparatus (Experiment 2). Aliquots of SDPP were stored over a period of 0-10 weeks at 11 and 20°C (Experiment 3). Reverse transcription(RT)-quantitative PCR detected no notable reduction in viral genomes in treated plasma and SDPP samples. No infectious PSV1 was re-isolated from plasma and SDPP samples in cell culture. At pH 10·2 and 3°C, infectivity of PEDV in plasma was reduced with a reduction factor of 4·2 log 10 (LRF) at 10 h contact time, whereas heating to 44°C for at least 1 min at alkali pH was needed to achieve a LRF of 4·2 for AdV. Spray drying at an outlet temperature of 80°C reduced AdV infectivity effectively (LRF = 5·2) and PEDV infectivity for 95% (LRF = 1·4). After storage at 20°C for 2 weeks no infectious PEDV was re-isolated from SDPP anymore (LRF ≥4·0). Due to growth of antibiotic-resistant bacteria from plasma in cell cultures used for PCV2 isolation, no data regarding inactivation of PCV2 were obtained. CONCLUSIONS: Five percent of PEDV stayed infectious after our spray drying conditions. Spray drying in combination with storage for ≥2 weeks at 20°C eliminated infectivity of PEDV effectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The conditions for inactivation of virus in plasma and SDPP determined are important for producers to inactivate PEDV during production of SDPP.

Knowledge gaps and research priorities in the prevention and control of hepatitis E virus infection.

Hepatitis E virus (HEV), family Hepeviridae, is a main cause of epidemic hepatitis in developing countries and sporadic and cluster cases of hepatitis in industrialized countries. There are an increasing number of reported cases in humans especially in industrialized countries, and there is a high potential for transboundary spread of zoonotic genotypes of the virus through the transport of pigs, pig products and by-products. Bloodborne transmission of the virus has been reported with a significant medical concern. To better coordinate HEV research and design better control measures of HEV infections in animals, a group of HEV experts reviewed the current knowledge on the disease and considered the existing disease control tools. It was concluded that there is a lack of in-depth information about the spread of the virus from pigs to humans. The role of animals other than pigs in the zoonotic transmission of the virus to humans and the extent of foodborne transmission are poorly understood. Factors involved in development of clinical disease such as infectious dose, susceptibility and virulence of virus strains need to be studied more extensively. However, such studies are greatly hindered by the absence of a broadly applicable, efficient and sensitive in vitro cell culture system for HEV. Diagnostic tools for HEV are available but need to be further validated, harmonized and standardized. Commercially available HEV vaccines for the control of HEV infection in animal populations are needed as such vaccines can minimize the zoonotic risk for humans. Anti-HEV drugs for treatment of HEV-infected patients need to be studied more extensively. The detailed expert review can be downloaded from the project website at http://www.discontools.eu/.

ICTV Virus Taxonomy Profile: Hepeviridae.

The family Hepeviridae includes enterically transmitted small non-enveloped positive-sense RNA viruses. It includes the genera Piscihepevirus, whose members infect fish, and Orthohepevirus, whose members infect mammals and birds. Members of the genus Orthohepevirus include hepatitis E virus, which is responsible for self-limiting acute hepatitis in humans and several mammalian species; the infection may become chronic in immunocompromised individuals. Extrahepatic manifestations of Guillain-Barré syndrome, neuralgic amyotrophy, glomerulonephritis and pancreatitis have been described in humans. Avian hepatitis E virus causes hepatitis-splenomegaly syndrome in chickens. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Hepeviridae, which is available at www.ictv.global/report/hepeviridae.

High-resolution phylogeny providing insights towards the epidemiology, zoonotic aspects and taxonomy of sapoviruses.

The evolution, epidemiology and zoonotic aspects of Sapoviruses (SaV) are still not well explored. In this study, we applied high-resolution phylogeny to investigate the epidemiological and zoonotic origins as well as taxonomic classification of animal and human SaV. Bayesian framework analyses showed an increase in porcine SaV (PoSaV) population dynamics and genetic diversity between 1975 and 1982, resulting in a SaV gene flow and generation of new strains among porcine and human populations. Our results also show the contribution of different animal populations involved in SaV epidemiology and highlight zoonotic aspects, as exemplified by the crucial role that swine, dogs, mink and humans play in SaV spread. Additionally, phylogenetic analysis suggests that bats may play key role in SaV epidemiology. According to our hypothesis, these animals may act as reservoirs or intermediate host species, contributing to viral spread in zoonotic and other epidemiological scenarios and facilitating the generation of new SaV genogroups and genotypes through recombination events. Data from large-scale phylogeny partition based on patristic distance, did not show a correlation between transmission clusters on generation of SaV genogroups, nevertheless we present both important findings about SaV taxonomy and important considerations useful for further taxonomical studies.

Outbreak of Porcine Epidemic Diarrhea Virus in Portugal, 2015.

An outbreak of porcine epidemic diarrhea virus (PEDV) in the South of Portugal in January 2015 and the spread of PEDV northwards in the territory are described. Comparative analysis of the amplified sequences showed a very high (99.0%) identity with the PEDV variant most recently reported in the United States and also show complete (100%) identity to the strains recently reported in Germany, supporting the hypothesis that a unique strain is currently circulating in Europe. The origin of this PEDV variant still needs to be elucidated and further studies in the remaining European countries may contribute to the knowledge.

Detection of serum neutralizing antibodies to Simbu sero-group viruses in cattle in Tanzania.

BACKGROUND: Orthobunyaviruses belonging to the Simbu sero-group occur worldwide, including the newly recognized Schmallenberg virus (SBV) in Europe. These viruses cause congenital malformations and reproductive losses in ruminants. Information on the presence of these viruses in Africa is scarce and the origin of SBV is unknown. The aim of this study was to investigate the presence of antibodies against SBV and closely related viruses in cattle in Tanzania, and their possible association with reproductive disorders. RESULTS: In a cross-sectional study, serum from 659 cattle from 202 herds collected in 2012/2013 were analyzed using a commercial kit for SBV ELISA, and 61 % were positive. Univariable logistic regression revealed significant association between ELISA seropositivity and reproductive disorders (OR = 1.9). Sera from the same area collected in 2008/2009, before the SBV epidemic in Europe, were also tested and 71 (54.6 %) of 130 were positive. To interpret the ELISA results, SBV virus neutralization test (VNT) was performed on 110 sera collected in 2012/2013, of which 51 % were positive. Of 71 sera from 2008/2009, 21 % were positive. To investigate potential cross reactivity with related viruses, 45 sera from 2012/2013 that were positive in SBV ELISA were analyzed in VNTs for Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda, Simbu and Tinaroo viruses. All 45 sera were positive for one or more of these viruses. Twenty-nine sera (64.4 %) were positive for SBV, and one had the highest titer for this virus. CONCLUSIONS: This is the first indication that Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda and Tinaroo viruses circulate and cause negative effect on reproductive performance in cattle in Tanzania. SBV or a closely related virus was present before the European epidemic. However, potential cross reactivity complicates the interpretation of serological studies in areas where several related viruses may circulate. Virus isolation and molecular characterization in cattle and/or vectors is recommended to further identify the viruses circulating in this region. However, isolation in cattle is difficult due to short viremic period of 2 to 6 days, and isolation in vectors does not necessarily reflect the situation in cattle.

Schmallenberg Virus in Culicoides Biting Midges in the Netherlands in 2012.

A total of 130 pools of Culicoides biting midges collected between May and September 2012 in the Netherlands were assayed for Schmallenberg virus (SBV). The Culicoides midges were caught in the same area as where in 2011 a high proportion of Culicoides pools tested positive for SBV, in majority with a high viral load (Ct values between 20 and 30). Two of a total of 42 pools comprising 50 midges/pool of the Obsoletus complex from the 2012 collection tested weak positive (Ct values: 34.96 and 37.66), indicating a relatively low viral load. On an individual midge level, the proportion of SBV-infected Culicoides of the Obsoletus complex caught in the same area and in a comparable period of the year was significantly lower in 2012 (0.1% = 1 per 1050 tested) compared with 2011 (0.56% = 13 per 2300 tested).

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Limited interlaboratory comparison of Schmallenberg virus antibody detection in serum samples.

Eight veterinary institutes in seven different countries in Europe participated in a limited interlaboratory comparison trial to evaluate laboratory performances of Schmallenberg virus (SBV) antibody detection in serum. Seven different sheep sera and three different cattle sera were circulated, and all participating institutes were asked to test these sera using SBV antibody detection assay(s) in place in their laboratories. All laboratories within the trial performed a virus neutralisation test (VNT) as well as one or two ELISAs on all samples, and swiftly detected SBV antibodies using these assays. VNT was more sensitive in detecting SBV antibodies than several of the used ELISA assays. Based on the test results, one cattle and one sheep SBV antibody-positive serum were selected to serve as reference sera, which now can be supplied to other laboratories on request.

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine.

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Schmallenberg virus detection in bovine semen after experimental infection of bulls.

To study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 106 TCID50). After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested for SBV by qRT-PCR assay. At 24 days post-inoculation both animals were subjected to necropsy and the genital organs and lymph nodes draining these organs were also tested for SBV RNA (qRT-PCR). After SBV infection both animals in the study showed viraemia (qRT-PCR) with fever and diarrhoea. SBV RNA could be detected in semen from both animals. The highest SBV RNA concentrations in semen were found in the first week (days 4-7 post-inoculation) but concentrations were relatively low (Ct values 30-39). Viable SBV was only isolated from blood samples and not from semen or genital tissues.

Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants.

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

Schmallenberg virus outbreak in the Netherlands: routine diagnostics and test results.

At the end of 2011, a new Orthobunyavirus was discovered in Germany and named Schmallenberg virus (SBV). In the Netherlands malformations in new-born ruminants were made notifiable from the 20th of December 2011. After a notification, malformed new-borns were necropsied and brain tissue was sampled for reverse transcription-polymerase chain reaction (RT-PCR). In addition, blood samples from mothers of affected new-borns were tested for antibodies in a virus neutralization test (VNT). The aim of this study was to summarize and evaluate the diagnostic data obtained and to gain insight into the possible regional differences. In total 2166 brains were tested: 800 from lambs, 1301 from calves and 65 from goat kids. Furthermore 1394 blood samples were tested: 458 from ewes, 899 from cows and 37 from goats. Results showed that 29% of the lamb brains, 14% of the calf brains, and 9% of the goat kid brains were RT-PCR positive. The number of malformed and RT-PCR positive lambs decreased over time while the number of malformed and RT-PCR positive calves increased. In the VNT 92% of the ewes, 96% of the cows and 43% of the goats tested positive. Combining RT-PCR and VNT results, 18% of all farms tested positive in both the RT-PCR and VNT. The relative sensitivity and specificity of the RT-PCR are 19% and 97% respectively, and of the VNT 99% and 6%. The results show a widespread exposure to SBV and the regional evaluation seems to indicate an introduction of SBV in the central/eastern part.

Molecular epidemiology of bat lyssaviruses in Europe.

Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2). Between 1977 and 2011, 961 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV-1. There have been 25 suspected cases of EBLV-2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in south-west Russia in 2002 (West Caucasian bat virus) and in Germany in 2010 (Bokeloh bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV-1 and EBLV-2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV-2 cases for which viral sequences (N gene, 400 nucleotides) are available (n = 21). Oropharyngeal swabs collected from two healthy Myotis daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV-2). Despite the relatively low number of EBLV-2 cases, a surprisingly large amount of anomalous data has been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV-1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV-1 isolates from Spain and the EBLV-2 isolates to provide support for the existence of sublineages.

'Schmallenberg virus'--a novel orthobunyavirus emerging in Europe.

In 2011, a novel orthobunyavirus of the Simbu serogroup, the Schmallenberg virus (SBV), was discovered using a metagenomic approach. SBV caused a large epidemic in Europe in ruminants. As with related viruses such as Akabane virus, it appears to be transmitted by biting midges. Transplacental infection often results in the birth of malformed calves, lambs and goat kids. In more than 5000 farms in Germany, The Netherlands, Belgium, France, UK, Italy, Spain, Luxembourg, Denmark and Switzerland acute infections of adult ruminants or malformed SBV-positive offspring were detected, and high seroprevalences were seen in adult ruminants in the core regions in The Netherlands, Germany and Belgium. The discovery of SBV, the spread of the epidemic, the role of vectors, the impact on livestock, public health issues, SBV diagnosis and measures taken are described in this review. Lessons to be learned from the Schmallenberg virus epidemic and the consequences for future outbreaks are discussed.

Replication of hepatitis E virus in three-dimensional cell culture.

Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

Transmission dynamics of hepatitis E virus in pigs: estimation from field data and effect of vaccination.

Hepatitis E is a viral disease that causes serious concerns for public health. Hepatitis E virus (HEV) genotype 3 is endemic in commercial pig farms worldwide that act as a reservoir. Pig-to-human transmission may occur when infectious animals enter the food chain at slaughter, through consumption of contaminated meat, direct exposure or use of by-products. To reduce the fraction of infectious animals at slaughter age and thus the risk for public health, it is important to understand the transmission dynamics of HEV in pig populations. In this study, we estimate the transmission rate parameter and mean infectious period of HEV in pigs from field data, using a Bayesian analysis. The data were collected in ten commercial pig herds that are each divided into three different age groups. Two transmission models were compared, assuming that animals are infected either locally by their group mates or globally by any infectious animal regardless of its group. For local and global transmission, the transmission rate parameters were 0.11 (posterior median with 95% credible interval: 0.092-0.14 day(-1)) and 0.16 (0.082-0.29 day(-1)), the mean infectious periods were 24 (18-33) days and 27 (20-39) days and the reproduction numbers were 2.7 (2.2-3.6) and 4.3 (2.8-6.9). Based on these results, global transmission is considered to be the more conservative model. Three effects of vaccination were explored separately. When vaccination is not sufficient to eliminate the virus, a shorter mean infectious period decreases the fraction of infectious animals at slaughter age, whereas a reduced transmission rate parameter adversely increases it. With a reduced susceptibility, vaccination of animals at a later age can be a better strategy than early vaccination. These effects should be taken into account in vaccine development.

Detection and characterization of hepatitis E virus in domestic pigs of different ages in Portugal.

This study represents the primary hepatitis E virus (HEV) surveillance in domestic pigs in Portugal, five pig farms were investigated in 5 different Portuguese regions, ten faecal samples were collected at four different stages of the production. All faecal samples were tested for hepatitis E virus by real-time RT-PCR. At least one sample from each farms of all age groups tested positive for HEV. The prevalence in the pig herds varied from 10% to 30% and the mean prevalence was 32% in weaners, 20% in growers, 32% in fatteners and 4% in adult dry sows. Phylogenetic analysis of the detected HEV sequences indicated that the circulating virus strains belong under the genotype 3.

Epizootic of ovine congenital malformations associated with Schmallenberg virus infection.

Epizootic outbreaks of congenital malformations in sheep are rare and have, to the best of our knowledge, never been reported before in Europe. This paper describes relevant preliminary findings from the first epizootic outbreak of ovine congenital malformations in the Netherlands. Between 25 November and 20 December 2011, congenital malformations in newborn lambs on sheep farms throughout the country were reported to the Animal Health Service in Deventer. Subsequently, small ruminant veterinary specialists visited these farms and collected relevant information from farmers by means of questionnaires. The deformities varied from mild to severe, and ewes were reported to have given birth to both normal and deformed lambs; both male and female lambs were affected. Most of the affected lambs were delivered at term. Besides malformed and normal lambs, dummy lambs, unable to suckle, were born also on these farms. None of the ewes had shown clinical signs during gestation or at parturition. Dystocia was common, because of the lambs' deformities. Lambs were submitted for post-mortem examination, and samples of brain tissue were collected for virus detection. The main macroscopic findings included arthrogryposis, torticollis, scoliosis and kyphosis, brachygnathia inferior, and mild-to-marked hypoplasia of the cerebrum, cerebellum and spinal cord. Preliminary data from the first ten affected farms suggest that nutritional deficiencies, intoxication, and genetic factors are not likely to have caused the malformations. Preliminary diagnostic analyses of precolostral serum samples excluded border disease virus, bovine viral diarrhoea virus, and bluetongue virus. In December 2011, samples of brain tissue from 54 lambs were sent to the Central Veterinary Institute of Wageningen University Research, Lelystad. Real-time PCR detected the presence of a virus, provisionally named the Schmallenberg virus, in brain tissue from 22 of the 54 lambs, which originated from seven of eight farms that had submitted lambs for post-mortem examination. This Schmallenberg virus was first reported in Germany and seems to be related to the Shamonda, Aino, and Akabane viruses, all of which belong to the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. These preliminary findings suggest that the Schmallenberg virus is the most likely cause of this epizootic of ovine congenital malformations, which is the first such outbreak reported in Europe.