Patricia König

Efficacy of three inactivated vaccines against bluetongue virus serotype 8 in sheep.

Bluetongue has become a major animal health issue in the European Union. The member states and Switzerland have agreed on a vaccination strategy. Three different inactivated monovalent vaccines against bluetongue virus serotype 8 were selected for the compulsory vaccination program carried out in Germany in 2008. The efficacy of these vaccines was evaluated in a pilot study in sheep immunised under field conditions by clinical, virological and serological examination before and after experimental challenge infection with a BTV-8 field isolate. Antibody levels prior to challenge infection differed between the vaccinated groups, but all seroconverted animals were fully protected against clinical disease and virus replication. Only one vaccinated animal was very weakly positive in the real-time RT-PCR at day 10 after challenge infection, and one seronegative sheep in one of the vaccine groups was not protected.

Evaluation of humoral response and protective efficacy of three inactivated vaccines against bluetongue virus serotype 8 one year after vaccination of sheep and cattle

The long-term efficacy of three commercially available inactivated vaccines against bluetongue virus serotype 8 (BTV-8) (BLUEVAC) 8, Zulvac 8, and BTVPUR AlSap 8) was evaluated in a seroprevalence study and challenge experiments. Seroprevalences 1 year after vaccination ranged from 75% to 100%. In two infection experiments, groups of vaccinated sheep and cattle selected either randomly or for low antibody levels were challenged with a European BTV-8 strain 12 months after vaccination. With two exceptions, all animals, including those with low antibody levels prior to challenge, were protected from viral replication and clinical disease even at low initial antibody levels. Vaccination of susceptible ruminants in yearly intervals is thus considered an adequate scheme for BTV-8 control in Europe.

Emergence of bluetongue virus serotype 6 in Europe—German field data and experimental infection of cattle

In late 2008, bluetongue virus (BTV) serotype 6 (BTV-6), which had never occurred in Europe before, was first detected in the Netherlands and Germany. While the origin of the virus remains unknown, the prevalence of infections in cattle was investigated in a virological (N=28,658) and serological (N=2075) field survey in Lower Saxony, where 45 cases confined to the district Grafschaft Bentheim were found. Blood from affected animals was used for the experimental infection of three cattle with different BTV antibody status, leading to sustained viraemia in one animal naïve for BTV. Of two animals that had detectable antibodies against BTV serotype 8, one became transiently infected and seroconverted for BTV-6 while the other did not react. In conclusion, while only a very limited spread of BTV-6 could be observed in the field, experimental infection of cattle did not show substantial differences of the course of infection in comparison to other BTV serotypes.

Glycoprotein M of bovine herpesvirus 1 (BHV-1) is nonessential for replication in cell culture and is involved in inhibition of bovine respiratory syncytial virus F protein induced syncytium formation in recombinant BHV-1 infected cells

Cell cultures infected with BHV-1/F(syn), a recombinant bovine herpesvirus 1 (BHV-1) which expresses a synthetic open reading frame encoding the fusion (F) protein of the bovine respiratory syncytial virus (BRSV), showed a cytopathic effect (CPE) indistinguishable from that induced by wildtype BHV-1 although transient transfection experiments demonstrated that expression of the F protein leads to formation of large syncytia. Since it has been shown that glycoprotein M (gM) of pseudorabies virus inhibits BRSV F-induced syncytium formation in transient plasmid transfection experiments [Pseudorbies virus glycoprotein M inhibits membrane fusion. J. Virol. 74 (2000) 6760], the gM ORF of wtBHV-1 and BHV-1/F(syn) was interrupted. Infection of cell cultures with the resulting gM(-) mutant of BHV-1/F(syn) led to formation of syncytia, whereas the CPE in gM(-)BHV-1 infected cells was comparable to the CPE in wtBHV-1 infected cultures. Our results demonstrate that gM is not essential for BHV-1 replication in cell culture and that gM is involved in inhibition of the cell fusion activity of the BHV-1 expressed BRSV F protein.

Deletion Mutants of Schmallenberg Virus Are Avirulent and Protect from Virus Challenge

ABSTRACT Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were tested in vitro and in vivo in type I interferon receptor knockout mice (IFNAR−/−) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR−/− mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses. IMPORTANCE SBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

Infectious blood or culture-grown virus: A comparison of bluetongue virus challenge models

The World Organisation for Animal Health (OIE) currently recommends using infectious ruminant blood as challenge inoculum in bluetongue virus (BTV) vaccination and challenge experiments. The use of virus grown in cultured cells is discouraged because culture passages can lead to changes in virus phenotype, including reduced replication efficiency and virulence in the host, while the OIE considers clinical disease in control animals indispensable evidence of successful infection. In the present study, two groups of five sheep were inoculated with either infectious calf blood lysate or culture-grown bluetongue virus of serotype 8 (BTV-8) (2 × 10(4)TCID(50) and 5 × 10(5)TCID(50), respectively). No pronounced difference in the induction and progression of viraemia as determined by real-time RT-PCR, which is the most objective parameter in the evaluation of vaccine efficacy, was observed. In a second experiment, the virulence of both inocula was confirmed by fatal infection of interferon receptor-deficient mice. The recent availability of highly sensitive molecular methods for the detection of BTV can finally shift the focus away from clinical disease. For the sake of objective and repeatable BTV challenge experiments, the OIE should reconsider its policy on culture-grown virus.

Innocuousness and safety of classical swine fever marker vaccine candidate CP7_E2alf in non-target and target species

Chimeric pestivirus CP7_E2alf is a promising live marker vaccine candidate against classical swine fever. Prior to a possible application in the field, several safety aspects have to be addressed. Due to the fact that CP7_E2alf is based on a bovine viral diarrhea virus backbone, its behavior in ruminants is of particular interest. In the framework of this study, its innocuousness in non-target species was addressed by inoculation of calves, young goats, lambs, and rabbits. To this means, high titres of CP7_E2alf were applied orally to three animals of each species. Additional animals were left as unvaccinated contact controls. During the study, all animals remained clinically healthy, and neither fever nor leukopenia were observed. Virus could not be isolated from purified white blood cells or from nasal or faecal excretions. Moreover, none of the animals (inoculated or contact control) seroconverted. In the target species, innocuousness, shedding and transmission of vaccine virus was addressed in different animal trials that were carried out primarily for the purpose of efficacy, potency or duration of immunity studies. In all experiments, CP7_E2alf proved to be completely safe for the vaccinees and unvaccinated contact controls. Furthermore, no shedding or transmission was detected in any of the experiments. Even after parental vaccination, vaccine virus genome was barely detectable in blood or organ samples of vaccinated animals. Thus, CP7_E2alf can be regarded as completely safe for both target and non-target species.

Experimental infection of South American camelids with bluetongue virus serotype 8

Bluetongue (BT) is an infectious, non-contagious disease of wild and domestic ruminants. It is caused by bluetongue virus (BTV) and transmitted by Culicoides biting midges. Since 1998, BT has been emerging throughout Europe, threatening not only the naïve ruminant population. Historically, South American camelids (SAC) were considered to be resistant to BT disease. However, recent fatalities related to BTV in captive SAC have raised questions about their role in BTV epidemiology. Data on the susceptibility of SAC to experimental infection with BTV serotype 8 (BTV-8) were collected in an animal experiment. Three alpacas (Vicugna pacos) and three llamas (Lama glama) were experimentally infected with BTV-8. They displayed very mild clinical signs. Seroconversion was first measured 6-8 days after infection (dpi) by ELISA, and neutralising antibodies appeared 10-13 dpi. BTV-8 RNA levels in blood were very low, and quickly cleared after seroconversion. However, spleens collected post-mortem were still positive for BTV RNA, over 71 days after the last detection in blood samples. Virus isolation was only possible from blood samples of two alpacas by inoculation of highly sensitive interferon alpha/beta receptor-deficient (IFNAR(-/-)) mice. An in vitro experiment demonstrated that significantly lower amounts of BTV-8 adsorb to SAC blood cells than to bovine blood cells. Although this experiment showed that SAC are generally susceptible to a BTV-8 infection, it indicates that these species play a negligible role in BTV epidemiology.

Equid herpesvirus 5-associated dermatitis in a horse—Resembling herpes-associated erythema multiforme

An equid herpesvirus 5 (EHV-5) infection was detected in lesioned skin from a nine-year-old Holsteiner stallion in the south of Germany. Macroscopically, the animal displayed a non-pruritic, multifocal, pustular dermatitis around both eyes, nostrils and the muzzle, which had been ongoing for one year. Histopathologically, skin lesions were characterized by orthokeratotic to parakeratotic hyperkeratosis, pustular dermatitis, epidermal hyperplasia, apoptotic keratinocytes, a lympho-plasmahistiocytic interface dermatitis with hydropic degeneration of keratinocytes, and perivascular to diffuse, lympho-histiocytic infiltrations. The stratum granulosum and the upper part of the stratum spinosum contained multiple amphophilic, intranuclear inclusion bodies. By in situ hybridization and immunohistochemistry herpesvirus DNA and protein, respectively, were detected within keratinocytes containing inclusion bodies. Sequencing of the PCR-product revealed the presence of EHV-5 DNA. This is the first description of a dermatitis associated with EHV-5 in a horse, indicating that EHV-5 should be considered as an etiology of lymphohistiocytic interface dermatitis with intranuclear inclusion bodies in horses and is similar to herpes-associated erythema multiforme in humans.

Characterization of C-strain “Riems” TAV-epitope escape variants obtained through selective antibody pressure in cell culture

Classical swine fever virus (CSFV) C-strain “Riems” escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.