Kerstin Wernike

Novel orthobunyavirus in Cattle, Europe, 2011.

In 2011, an unidentified disease in cattle was reported in Germany and the Netherlands. Clinical signs included fever, decreased milk production, and diarrhea. Metagenomic analysis identified a novel orthobunyavirus, which subsequently was isolated from blood of affected animals. Surveillance was initiated to test malformed newborn animals in the affected region.

Oral exposure, reinfection and cellular immunity to Schmallenberg virus in cattle

Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Germany in 2011. In adult ruminants SBV causes mild transient disease, but foetal infection can lead to severe malformations. Owing to its recent discovery, the knowledge about the pathogenesis is limited. In this study, two heifers seroconverted after a previous SBV infection and five SBV antibody-negative calves were subcutaneously inoculated, another two animals received SBV orally and three were kept as controls. In naïve cattle infected subcutaneously viral RNA was detected in serum and blood samples for several days. Seropositive or orally inoculated animals as well as the uninfected controls remained negative throughout the study. Seroconversion was observed only after subcutaneous exposure of the naïve animals to SBV. In lymphocytes from peripheral blood SBV genome was not detected, but the lymphocyte homeostasis in blood was influenced.

Schmallenberg virus challenge models in cattle: infectious serum or culture-grown virus?

Schmallenberg virus (SBV), discovered in Europe in 2011, causes mild transient disease in adult ruminants, but fetal infection can lead to severe malformation in cattle, sheep and goats.To elucidate the pathogenesis of this novel orthobunyavirus, considerable efforts are required. A reliable and standardized infection model is essential for in vivo studies. In the present study, two groups of four cattle were inoculated with either serum passaged in cattle only or cell culture-grown virus. The replication of culture-grown SBV in cattle was reduced compared to virus inoculated via infectious serum. In a second experiment, the infectious serum was titrated in calves; the tested batch contained 102.83 infectious doses per mL. Hence, serum-borne virus that was only passaged in the natural host is a suitable option for a standardized SBV infection model.

Schmallenberg virus-two years of experiences.

In autumn 2011, a novel species of the genus Orthobunyavirus of the Simbu serogroup was discovered close to the German/Dutch border and named Schmallenberg virus (SBV). Since then, SBV has caused a large epidemic in European livestock. Like other viruses of the Simbu serogroup, SBV is transmitted by insect vectors. Adult ruminants may show a mild transient disease, while an infection during a critical period of pregnancy can lead to severe congenital malformation, premature birth or stillbirth. The current knowledge about the virus, its diagnosis, the spread of the epidemic, the impact and the possibilities for preventing infections with SBV is described and discussed.

Schmallenberg virus experimental infection of sheep.

Since late 2011, a novel orthobunyavirus, named Schmallenberg virus (SBV), has been implicated in many cases of severely malformed bovine and ovine offspring in Europe. In adult cattle, SBV is known to cause a mild transient disease; clinical signs include short febrile episodes, decreased milk production and diarrhoea for a few days. However, the knowledge about clinical signs and pathogenesis in adult sheep is limited. In the present study, adult sheep of European domestic breeds were inoculated with SBV either as cell culture grown virus or as virus with no history of passage in cell cultures. Various experimental set-ups were used. Sampling included blood collection at different time points during the experimental period and selected organ material at autopsy. Data from this study showed, that the RNAemic period in sheep was as short as reported for cattle; viral genome was detectable for about 3-5 days by real-time RT-PCR. In total, 13 out of 30 inoculated sheep became RNAemic, with the highest viral load in animals inoculated with virus from low cell culture passaged or the animal passaged material. Contact animals remained negative throughout the study. One RNAemic sheep showed diarrhoea for several days, but fever was not recorded in any of the animals. Antibodies were first detectable 10-14 days post inoculation. Viral RNA was detectable in spleen and lymph nodes up to day 44 post inoculation. In conclusion, as described for cattle, SBV-infection in adult sheep predominantly results in subclinical infection, transient RNAemia and a specific antibody response. Maintenance of viral RNA in the lymphoreticular system is observed for an extended period.

Schmallenberg Virus Recurrence, Germany, 2014.

Schmallenberg virus (SBV) emerged in Germany in 2011, spread rapidly across Europe, and almost disappeared in 2013. However, since late summer 2014, new cases have occurred in adult cattle. Full-genome analysis revealed some amino acid substitution differences from the first SBV sample. Viremia developed in experimentally infected sheep and cattle for 4–6 days.

Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus: Protection against Virulent Virus Challenge in Cattle and Sheep

ABSTRACT Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Porcine reproductive and respiratory syndrome virus Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

Detection and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus by Multiplex Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV) are classified into the two distinct genotypes “North American (NA, type 2)” and “European (EU, type 1)”. In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV), characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

Schmallenberg virus infection of adult type I interferon receptor knock-out mice.

Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Europe in late 2011. It causes mild and transient disease in adult ruminants, but fetal infection can lead to abortion or severe malformations. There is considerable demand for SBV research, but in vivo studies in large animals are complicated by their long gestation periods and the cost of high containment housing. The goal of this study was to investigate whether type I interferon receptor knock-out (IFNAR−/−) mice are a suitable small animal model for SBV. Twenty IFNAR−/− mice were inoculated with SBV, four were kept as controls. After inoculation, all were observed and weighed daily; two mice per day were sacrificed and blood, brain, lungs, liver, spleen, and intestine were harvested. All but one inoculated mouse lost weight, and two mice died spontaneously at the end of the first week, while another two had to be euthanized. Real-time RT-PCR detected large amounts of SBV RNA in all dead or sick mice; the controls were healthy and PCR-negative. IFNAR−/− mice are susceptible to SBV infection and can develop fatal disease, making them a handy and versatile tool for SBV vaccine research.