Bovine viral diarrhoea: a playground for virologists and a target for eradication.

197 adverse reactions of Swissmedic-authorized veterinary medicinal products were reported during the year 2012 (2011: 167). Species and drug classes remain unchanged over the years: most of the reports related to reactions following the use of antiparasitic products (37.6 %), antiinfectives (15.7 %) or non-steroidal antiinflammatory drugs (11.7 %) in companion animals (94 dogs and 53 cats) followed by cattle/calves (29). Additionally, 45 cases transmitted by the Swiss Toxicological Information Centre in Zürich were processed. We discuss a paradoxical reaction under the potential influence of acepromazine as well as a modified protocol for treating permethrin intoxication in cats. Finally, the vaccinovigilance program received 95 declarations following the application of various vaccines, mainly to dogs or cats.

Epizootiologic Investigations of Selected Abortive Agents in Free-Ranging Alpine Ibex (Capra Ibex Ibex) in Switzerland

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems clue to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella. spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0-11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

Epidemiological study of pestiviruses in South American camelids in Switzerland

BACKGROUND: In the context of the ongoing eradication campaign for bovine viral diarrhea virus (BVDV) in cattle in Switzerland, the role of South American camelids (SAC) as a possible virus reservoir needed to be evaluated. OBJECTIVE: To assess and characterize the prevalence of pestivirus infections in SAC in Switzerland. ANIMALS: Serum samples collected from 348 animals (40 herds) in 2008 and from 248 animals (39 herds) in 2000 were examined for antibodies against pestiviruses and for the presence of BVDV viral RNA. METHODS: Cross-sectional study using stratified, representative herd sampling. An indirect BVDV-ELISA was used to analyze serum samples for pestivirus antibodies, and positive samples underwent a serum neutralization test (SNT). Real-time RT-PCR to detect pestiviral RNA was carried out in all animals from herds with at least 1 seropositive animal. RESULTS: In 2008, the overall prevalence of animals positive for antibodies (ELISA) and pestiviral RNA or was 5.75 and 0%, respectively. In 2000, the corresponding prevalences were 3.63 and 0%, respectively. The seroprevalences (SNT) for BVDV, border disease virus or undetermined pestiviruses were estimated to be 0, 1.73, and 4.02% in 2008, and 0.40, 1.21, and 2.02% in 2000, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: At the present time, SAC appear to represent a negligible risk of re-infection for the BVDV eradication program in cattle in Switzerland.

Nonstructural proteins NS2-3 and NS4A of classical swine fever virus: essential features for infectious particle formation

The nonstructural protein NS2-3 of pestiviruses undergoes tightly regulated processing. For bovine viral diarrhea virus it was shown that uncleaved NS2-3 is required for infectious particle formation while cleaved NS3 is essential for genome replication. To further investigate the functions of NS2-3 and NS4A in the pestivirus life cycle, we established T7 RNA polymerase-dependent trans-complementation for p7-NS2-3-4A of classical swine fever virus (CSFV). Expression of NS2-3 and NS4A in trans restored the production of infectious particles from genomes lacking NS2-3 expression. Co-expression of cleaved NS4A was essential. None of the enzymatic activities harbored by NS2-3 were required for infectious particle formation. Importantly, expression of uncleavable NS2-3 together with NS4A rescued infectious particles from a genome lacking NS2, demonstrating that cleaved NS2 per se has no additional essential function. These data indicate that NS2-3 and NS3, each in association with NS4A, have independent functions in the CSFV life cycle.

BVDV: a pestivirus inducing tolerance of the innate immune response

Animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) retain a strain-specific B- and T-cell immunotolerance. Pestiviral RNA triggers interferon (IFN) synthesis, and the viral RNase E(rns) inhibits IFN expression induced by extracellular viral RNA. In addition, N(pro) promotes the degradation of the transcription factor IRF-3, which effectively blocks IFN expression in BVDV-infected cells. As not all the potential target cells are infected in PI animals, these are 'chimeric' with respect to BVDV. This suggests that N(pro) and E(rns) are non-redundant IFN antagonists that act in infected and non-infected cells, respectively. Moreover, E(rns) may take a paradoxical function, both as virulence as well as "attenuation" factor: The former by preventing the activation of the innate and, consequently, of the adaptive immune system, the latter by minimizing the detrimental effects of systemic IFN production. Thus, BVDV maintains "self-tolerance" by avoiding the induction of IFN while itself being largely resistant to it without, however, interfering with the IFN action against unrelated viruses ('nonself'). This unique extension of 'self' to a virus suggests that the host's own RNases may have evolved as a guard against inadvertent activation of the innate immune system by host RNA, thus establishing a state of "innate tolerance".

Sheep persistently infected with Border disease readily transmit virus to calves seronegative to BVD virus.

Bovine viral diarrhea- and Border disease viruses of sheep belong to the highly diverse genus pestivirus of the Flaviviridae. Ruminant pestiviruses may infect a wide range of domestic and wild cloven-hooved mammals (artiodactyla). Due to its economic importance, programs to eradicate bovine viral diarrhea are a high priority in the cattle industry. By contrast, Border disease is not a target of eradication, although the Border disease virus is known to be capable of also infecting cattle. In this work, we compared single dose experimental inoculation of calves with Border disease virus with co-mingling of calves with sheep persistently infected with this virus. As indicated by seroconversion, infection was achieved only in one out of seven calves with a dose of Border disease virus that was previously shown to be successful in calves inoculated with BVD virus. By contrast, all calves kept together with persistently infected sheep readily became infected with Border disease virus. The ease of viral transmission from sheep to cattle and the antigenic similarity of bovine and ovine pestiviruses may become a problem for demonstrating freedom of BVD by serology in the cattle population.

Pestiviruses.

Pestiviruses cause economically important diseases among domestic ruminants and pigs, but they may also infect a wide spectrum of wild species of even-toed ungulates (Artiodactyla). Bovine viral diarrhea virus (BVDV) and Border disease virus of sheep infect their hosts either transiently or persistently. Cellular and humoral immunotolerance to the infecting strain is a unique feature of persistent infection (PI) by ruminant pestiviruses. Persistence, caused by transplacental infection early in fetal development, depends on virally encoded interferon antagonists that inactivate the host's innate immune response to the virus without globally interfering with its function against other viruses. At epidemiological equilibrium, approximately 1-2% of animals are PI. Successful BVDV control programs show that removal of PI animals results in viral extinction in the host population. The nucleotide sequences of ruminant pestiviruses change little during persistent infection. Nevertheless, they display large heterogeneity, pointing to a long history of virus-host coevolution in which avirulent strains are more successful.

Prolonged activity of the pestiviral RNase Erns as an interferon antagonist after uptake by clathrin-mediated endocytosis

The RNase activity of the envelope glycoprotein E(rns) of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E(rns) is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E(rns). Here, we show that the activity of soluble E(rns) as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E(rns) protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E(rns) mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E(rns) remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E(rns) acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCE The pestiviral RNase E(rns) was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E(rns) at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E(rns) is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E(rns) remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.

Pestiviral E(rns) blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

Expert Opinion on the Perceived Effectiveness and Importance of On-Farm Biosecurity Measures for Cattle and Swine Farms in Switzerland.

Biosecurity is crucial for safeguarding livestock from infectious diseases. Despite the plethora of biosecurity recommendations, published scientific evidence on the effectiveness of individual biosecurity measures is limited. The objective of this study was to assess the perception of Swiss experts about the effectiveness and importance of individual on-farm biosecurity measures for cattle and swine farms (31 and 30 measures, respectively). Using a modified Delphi method, 16 Swiss livestock disease specialists (8 for each species) were interviewed. The experts were asked to rank biosecurity measures that were written on cards, by allocating a score from 0 (lowest) to 5 (highest). Experts ranked biosecurity measures based on their importance related to Swiss legislation, feasibility, as well as the effort required for implementation and the benefit of each biosecurity measure. The experts also ranked biosecurity measures based on their effectiveness in preventing an infectious agent from entering and spreading on a farm, solely based on transmission characteristics of specific pathogens. The pathogens considered by cattle experts were those causing Bluetongue (BT), Bovine Viral Diarrhea (BVD), Foot and Mouth Disease (FMD) and Infectious Bovine Rhinotracheitis (IBR). Swine experts expressed their opinion on the pathogens causing African Swine Fever (ASF), Enzootic Pneumonia (EP), Porcine Reproductive and Respiratory Syndrome (PRRS), as well as FMD. For cattle farms, biosecurity measures that improve disease awareness of farmers were ranked as both most important and most effective. For swine farms, the most important and effective measures identified were those related to animal movements. Among all single measures evaluated, education of farmers was perceived by the experts to be the most important and effective for protecting both Swiss cattle and swine farms from disease. The findings of this study provide an important basis for recommendation to farmers and policy makers.

The viral RNase E(rns) prevents IFN type-I triggering by pestiviral single- and double-stranded RNAs

Interferon (IFN) type-I is of utmost importance in the innate antiviral defence of eukaryotic cells. The cells express intra- and extracellular receptors that monitor their surroundings for the presence of viral genomes. Bovine viral diarrhoea virus (BVDV), a Pestivirus of the family Flaviviridae, is able to prevent IFN synthesis induced by poly(IC), a synthetic dsRNA. The evasion of innate immunity might be a decisive ability of BVDV to establish persistent infection in its host. We report that ds- as well as ssRNA fragments of viral origin are able to trigger IFN synthesis, and that the viral envelope glycoprotein E(rns), that is also secreted from infected cells, is able to inhibit IFN expression induced by these extracellular viral RNAs. The RNase activity of E(rns) is required for this inhibition, and E(rns) degrades ds- and ssRNA at neutral pH. In addition, cells infected with a cytopathogenic strain of BVDV contain more dsRNA than cells infected with the homologous non-cytopathogenic strain, and the intracellular viral RNA was able to excite the IFN system in a 5'-triphosphate-, i.e. RIG-I-, independent manner. Functionally, E(rns) might represent a decoy receptor that binds and enzymatically degrades viral RNA that otherwise might activate the IFN defence by binding to Toll-like receptors of uninfected cells. Thus, the pestiviral RNase efficiently manipulates the host's self-nonself discrimination to successfully establish and maintain persistence and immunotolerance.

Investigation of border disease and bovine virus diarrhoea in sheep from 76 mixed cattle and sheep farms in eastern Switzerland

The purpose of this study was to examine the occurrence of sheep persistently infected with Border disease virus (BDV) on 76 mixed cattle and sheep farms and whether seroconversion to BDV infection occurred in cattle of these farms. Seroprevalence of BDV and bovine viral disease virus (BVDV) infection in sheep was also investigated. Quantitative RT-PCR for pestivirus detection and an ELISA to detect pestivirus antibodies were used in 2'384 and 2'291 ovine blood samples, respectively. Another 27 seropositive sheep from ten flocks underwent serum neutralization testing to differentiate between BDV and BVDV antibodies. A BDV titre that was at least four times higher than the BVDV titre was interpreted as the result of BDV infection. Titres against BVDV were interpreted in an analogous fashion. All examined sheep were pestivirus-negative, 310 sheep were seropositive, 119 had an indeterminate titre and 1'862 were seronegative. The flock seroprevalence ranged from 0.0 to 73.9 %. Three of the 27 flocks that underwent serum neutralization testing were interpreted as BDV-infected because of 6 sheep with higher BDV titres, and 6 flocks were interpreted as BVDV-infected because of 14 sheep with higher BVDV titres.

Seroprevalence and characterization of pestivirus infections in small ruminants and new world camelids in Switzerland

The seroprevalence of pestivirus infections in small ruminants and new world camelids in Switzerland was determined. In 5'059 sera of sheep from 382 herds, 503 sera of goats from 54 herds and 109 sera of alpacas and lamas from 53 herds, population prevalences of 16.1% (sheep), 25.4% (goats) and 4.6% (new world camelids), respectively, were found. In order to determine the source of infection, the serological reactions were further characterized by cross-neutralization against two pestiviruses representing the genotypes BVDV (Bovine Virus Diarrhea Virus)-1 and BDV (Border Disease Virus)-1. Based on the ratio of respective antibody titres, 56.1% of the infections in sheep were induced by a BDV-1, 12.9% by a BVDV-1 and 31.0% by an unresolved pestivirus. In goats, the corresponding proportions were 23.4%, 10.2% and 66.4%, respectively. In Alpacas and Lamas, the source of infection of 1 animal was BDV-1 and that of 4 seropositive animals remained unresolved. In view of the phylogenetic relationship between pestiviruses, the unresolved source of infection is most probably attributable to other pestivirus genotypes circulating in small ruminants and new world camelids. Due to the predominance of pestiviral genotypes other than BVDV-1, the risk of transmission of BVDV from persistently infected small ruminants and new world camelids to cattle appears to be moderate, apart from close direct contact in mixed animal husbandry, communal pasturing and grazing in the Alps.

Evaluation of farm-level parameters derived from animal movements for use in risk-based surveillance programmes of cattle in Switzerland

BACKGROUND: This study focused on the descriptive analysis of cattle movements and farm-level parameters derived from cattle movements, which are considered to be generically suitable for risk-based surveillance systems in Switzerland for diseases where animal movements constitute an important risk pathway. METHODS: A framework was developed to select farms for surveillance based on a risk score summarizing 5 parameters. The proposed framework was validated using data from the bovine viral diarrhoea (BVD) surveillance programme in 2013. RESULTS: A cumulative score was calculated per farm, including the following parameters; the maximum monthly ingoing contact chain (in 2012), the average number of animals per incoming movement, use of mixed alpine pastures and the number of weeks in 2012 a farm had movements registered. The final score for the farm depended on the distribution of the parameters. Different cut offs; 50, 90, 95 and 99%, were explored. The final scores ranged between 0 and 5. Validation of the scores against results from the BVD surveillance programme 2013 gave promising results for setting the cut off for each of the five selected farm level criteria at the 50th percentile. Restricting testing to farms with a score ≥ 2 would have resulted in the same number of detected BVD positive farms as testing all farms, i.e., the outcome of the 2013 surveillance programme could have been reached with a smaller survey. CONCLUSIONS: The seasonality and time dependency of the activity of single farms in the networks requires a careful assessment of the actual time period included to determine farm level criteria. However, selecting farms in the sample for risk-based surveillance can be optimized with the proposed scoring system. The system was validated using data from the BVD eradication program. The proposed method is a promising framework for the selection of farms according to the risk of infection based on animal movements.