Studies on genetic diversity of bovine viral diarrhea viruses in Danish cattle herds.

Scandinavian countries have successfully pursued bovine viral diarrhea virus (BVDV) eradication without the use of vaccines. In Denmark, control and eradication of BVDV were achieved during the last two decades, but occasionally new BVDV infections are detected in some Danish cattle herds. The aim of this study was to determine recent BVDV subtypes isolated from 4 Danish herds (A, B, C, and D) isolated in 2009-2012 and to analyze the genetic variation of these isolates within the same herd and its relation with those of other herds. The results showed that three herds (B, C, D) were BVDV 1-b and only one herd (herd A) was BVDV 1-d, no other subtypes were detected. The deduced E2 amino acids result showed a high identity percent (99-100 %) between isolates originating from the same herd, but with higher variation compared to isolates of the other herds. Some of these new Danish strains have closer relationship to BVDVs from outside Denmark than to older Danish strains indicating that these are new introductions to Denmark. In conclusion, BVDV-1 subtypes recently detected in Denmark were only subtypes 1b and 1d, and BVDV infections established in a herd is genetically stable over a long time period.

Intranasal Inoculation with Classical Swine Fever Virus Provided a More Consistent Experimental Disease Model Compared to Oral Inoculation.

The severity of disease resulting from classical swine fever virus (CSFV) infection is determined by several factors, including virus strain and host factors. The different outcomes of experimental studies in pigs with the same strain of CSFV emphasize the need to elucidate the influence of individual factors within experimental protocols. In this study, we investigated the outcome of disease after oral and intranasal inoculation with a moderately virulent CSFV strain in young pigs. To compare the two routes of inoculation, various infection parameters were examined during a period of two weeks. While all intranasally inoculated pigs (n = 5) were directly infected, this was only the case for two out of five pigs after oral inoculation. In addition, the intranasally inoculated pigs developed a more pronounced clinical disease and pathological lesions, as well as markedly more change in hematological and immunological parameters than the orally inoculated pigs. The wide variation among the orally inoculated pigs implied that statistical evaluation was markedly impaired, leaving this route of application less suitable for comparative studies on classical swine fever. Furthermore, our study provides additional details about the immunomodulatory effects of CSFV on the kinetics of CRP, TNF-α, and leukocyte sub-populations in pigs after infection with the CSFV strain Paderborn.

Experimental Infections of Pigs with African Swine Fever Virus (Genotype II); Studies in Young Animals and Pregnant Sows.

African swine fever is an important viral disease of wild and domestic pigs. To gain further knowledge of the properties of the currently circulating African swine fever virus (ASFV), experimental infections of young pigs (approximately 8 weeks of age) and pregnant sows (infected at about 100 days of gestation) with the genotype II ASFV Georgia/2007 were performed. The inoculated young pigs developed typical clinical signs of the disease and the infection was transmitted (usually within 3-4 days) to all of the "in contact" animals that shared the same pen. Furthermore, typical pathogical lesions for ASFV infection were found at necropsy. Inoculation of pregnant sows with the same virus also produced rapid onset of disease from post-infection day three; two of the three sows died suddenly on post-infection day five, while the third was euthanized on the same day for animal welfare reasons. Following necropsy, the presence of ASFV DNA was detected in tonsils, spleen and lymph nodes of some of the fetuses, but the levels of viral DNA were much lower than in these tissues from the sows. Thus, only limited transplacental transmission occurred during the course of this experiment. These studies contribute towards further understanding about the spread of this important viral disease in domestic pigs.

Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial.

Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance.

Development of Primer-Probe Energy Transfer real-time PCR for the detection and quantification of porcine circovirus type 2.

A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in post-weaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 10(7) copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.

Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon.

This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products could be a valuable instrument for virus rescue, conservation and further characterization.

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus.

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.

Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer.

Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous detection and differentiation of three Office International des Epizooties (OIE) classified vesicular viruses: foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease, causing clinically indistinguishable vesicular diseases in swine. The multiplex assay consists of extraction of total RNA from clinical samples; reverse transcription to cDNA using random primers and one-tube real-time amplification of cDNA using multiplex PriProET with specific fluorescent-labelled primers and probes for detection of the three viruses from the vesicular disease complex. The probes are labelled with unique reporter fluorophores, which during amplification are excited by donor fluorophores incorporated in the 5' end of specific amplicons by primer extension. The sensitivity of the multiplex assay was approximately 100 TCID(50), which is 10-fold lower compared to the individual PriProET assays for the three vesicular viruses.

Persistent BVDV infection in mousedeer infects calves. Do we know the reservoirs for BVDV?

Bovine virus diarrhoea virus (BVDV)-1f was isolated from a Lesser Malayan Mousedeer in Copenhagen Zoo during a routine screening. Analysis of animals related to the Copenhagen mousedeer revealed that its mother and all siblings were virus positive, a pattern also seen for persistently infected (PI) cattle. BVDV could be transmitted from the PI mousedeer to a calf after indirect contact. The host spectrum for BVDV seems to be even wider than expected; the implications for BVDV control are discussed.

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels.

BACKGROUND: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR® BVDV-Ab indirect ELISA. The latter has successfully been used to eradicate BVD in Sweden. Data (2003-2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples. The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools. RESULTS: During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level. Moreover, we found that when testing bulk milk, the SVANOVIR® BVDV-Ab can detect a lower prevalence of seropositive lactating cows, compared to the Danish blocking ELISA (0.78 % vs. 50 %). Values in the SVANOVIR® BVDV-Ab better relate to low concentrations of antibody positive milk (R(2) = 94-98 %), than values in the blocking ELISA (R(2) = 23-75 %). For sera, the two ELISAs performed equally well. CONCLUSIONS: The SVANOVIR ELISA is recommended for analysis of bulk milk samples in the current Danish situation, since infected dairy herds e.g. due to import of infected cattle can be detected shortly after BVDV introduction, when only few lactating cows have seroconverted. In sera, the two ELISAs can be used interchangeably.

Determination of the sequence of the complete open reading frame and the 5'NTR of the Paderborn isolate of classical swine fever virus.

The classical swine fever (CSF) epidemic in the Netherlands in 1997-1998 lasted 14 months, during which 429 infected and 1300 at risk herds were culled, at an estimated economical cost of 2 billion US dollars. Despite the overwhelming scale of the epizootic, the CSF virus (CSFV) strain causing the outbreak has remained largely uncharacterized. The Dutch epizootic is epidemiologically linked to a small CSF outbreak in 1997, in Paderborn in Germany. E2 and partial 5' NTR sequencing has shown that the index Paderborn isolate, and several Dutch isolates taken during the 1997-1998 epizootic, are virtually identical, confirming that the Paderborn isolate triggered the Dutch outbreak, and furthermore showing that this single isolate was stable throughout the whole Dutch outbreak (the above reviewed in [C. Terpstra, A. J. de Smit, Veterinary Microbiol. 77 (2000) 3-15]). We determined the nucleotide sequence of the 5' NTR (by 5' RACE) and the complete open reading frame of the Paderborn isolate (GenBank AY072924). Our sequence was identical to previously published partial 5'NTR and E2 sequences for the index Paderborn 1997 and Dutch 1997 (Venhorst) isolates, confirming the identity of the virus we sequenced. Phylogenetic analysis based on the complete open reading frame showed that Paderborn is genetically very different from common European laboratory reference strains. Neutralization studies showed that Paderborn is also antigenically very different from common laboratory strains such as Alfort 187. Paderborn is the only recent European CSFV field isolate for which a complete sequence is available, and given Paderborns genetic and antigenic uniqueness, the Paderborn sequence may have practical use for diagnostic and vaccine antigen development.

Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA.

We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.

DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif.

Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does not include any CSFV components. In the present study, the DIVA vaccine properties of CP7_E2gif were evaluated in comparison to the conventional live attenuated Riemser C-strain vaccine. Sera and tonsil samples obtained from pigs immunised with these two vaccines were analysed. No viral RNA was found in serum after vaccination with CP7_E2gif, whereas some serum samples from C-strain vaccinated animals were positive. In both vaccinated groups, individual viral RNA-positive tonsil samples were detected in animals euthanised between 7 and 21 days post vaccination. Furthermore, serum samples from these animals, together with archival samples from pigs vaccinated with CP7_E2gif and subsequently CSFV challenged, were analysed for specific antibodies using ELISAs and for homologous neutralising antibodies. In animals vaccinated with CP7_E2gif, neutralising antibodies were detected from day 10. However, the sera remained negative for anti-CSFV E2-specific antibodies whereas pigs vaccinated with C-strain seroconverted against CSFV by 14 days after vaccination, as determined by a CSFV-E2 specific blocking ELISA. One week after subsequent CSFV challenge, a strong anti-CSFV E2 reaction was detected in CP7_E2gif vaccinated pigs and anti-E(rns) antibodies were detected from 10 days after infection. In conclusion, CP7_E2gif has the potential to be used as a DIVA vaccine in combination with detection of anti-CSFV E2-specific antibodies.

Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus.

Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental infection of calves with atypical pestivirus, BVDV-1 and a combination of both viruses have been examined by routine and new diagnostic tests to validate their robustness and sensitivity. As expected, virus neutralization tests using homologous virus were able to differentiate the two groups infected by BVDV-1 or atypical pestivirus, whereas the animals inoculated with a mixture of these two viruses had a reaction pattern very similar to the homologous virus alone. It was found that immunoassays using whole virus and polyclonal antibodies are the most robust, but all tests examined were able to detect antibodies also from cattle infected with atypical pestivirus a few weeks after infection. The detection, however, was at a lower level and slightly delayed. Statistical validation of the threshold suggested by the manufacturer showed that in some cases the reduction of the cut-off values would improve the test sensitivity.

Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs.

Host-virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population.

Efficacy of marker vaccine candidate CP7_E2alf in piglets with maternally derived C-strain antibodies.

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain "Koslov". CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could be isolated from the tonsils of the vaccinated piglets. Two weeks after vaccination at the time of challenge, the vaccinated piglets only, had an increase in the ELISA antibody titer. Interestingly, the maternally derived immunity in the mock-vaccinated control piglets seems to neutralize the challenge virus. Thus, the previously observed 100% mortality in naïve (negative for antibodies to CSFV) piglets infected with CSFV Koslov was reduced in the control piglets of this study to 30% for challenge at the age of 7 weeks and 50% at the age of 10 weeks, respectively. In conclusion, CP7_E2alf proved to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8 weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4 weeks before farrowing with one dose of C-strain vaccine.

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1.

Atypical bovine pestiviruses related to bovine viral diarrhoea virus (BVDV) have recently been detected in cattle from South America, Asia and Europe. The purpose of this study was to compare the clinical and virological aspects of dual infection with BVDV-1 (Horton 916) and an Asian atypical bovine pestivirus (Th/04_KhonKaen) in naïve calves, in comparison to single infections. Milder clinical signs were observed in the animals infected with single Th/04_KhonKaen strain. Leukocytopenia and lymphocytopenia were observed in all infected groups at a similar level which correlated with the onset of viraemia. Co-infection with both viruses led to prolonged fever in comparison to single strain inoculated groups and simultaneous replication of concurrent viruses in blood and in the upper respiratory tract. Following the infections all the calves seroconverted against homologous strains. Atypical pestiviruses pose a serious threat to livestock health and BVDV eradication, since they may have the potential to be widely spread in cattle populations without being detected and differentiated from other BVDV infections.

Diagnostic value of meat juice in early detection of Classical swine fever virus infection.

To evaluate the diagnostic potential of meat juice for early detection of Classical swine fever virus (CSFV), meat juice and serum samples from pigs experimentally infected with different strains of CSFV were compared for virus load. From all samples, viral RNA was extracted by automated procedure before real-time reverse transcription polymerase chain reaction analysis was performed. Viral RNA was detected in meat juice, but at a lower level than in corresponding serum. Sensitivity was calculated to 91% and specificity to 97%. Disagreements between meat juice and serum results were found when samples originated from pigs infected with low virulence CSFV strains and/or when samples were collected within the first days after infection. In conclusion, while not the first choice for sample material for CSFV diagnosis, meat juice may constitute a useful alternative for herd-based studies or when blood and/or target organ material is not available. Strain virulence and time points for sample collection after infection are factors of importance for diagnostic success.

Implementation and validation of a sensitive PCR detection method in the eradication campaign against Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs (n=299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.