Border Disease Virus Strains Research Articles

Distribution and Genetic Characterization of Border Disease Virus Circulating in Sardinian Ovine Flocks.

Border Disease (BD) is a worldwide distributed pathology accountable for significant losses in the sheep and goat farming industry. The etiological agent is a Pestivirus within the family Flaviviridae called border disease virus (BDV). Despite the Sardinian ovine population being by far larger than any other Italian region, the prevalence and distribution of BD on the island are unknown. Here, we aim to determine the distribution of BDV in sheep flocks and to genetically characterize the circulating strains in Sardinia. The geographical distribution, antibody positivity, and viral genome presence have been analysed for 1286 sheep flocks distributed all over the island from bulk tank milk sampled between May 2014 and 2015. Of the flocks tested, 11.28% (95% CI 9.66–13.12) resulted positive for the presence of anti-pestivirus antibodies with an uneven distribution between Sardinian provinces. In addition, using RT-PCR, nine BDV genomes were amplified from milk pellets of the seropositive samples. Phylogenetic analysis revealed that all the viruses amplified clustered in the same group classified as BDV-7. This represents the first study on the distribution of pestivirus infection and genetic characterization of BDV strains circulating in the Sardinian sheep population. Future studies are needed to clarify the origin, the evolution, and the epidemiology of BDV-7 in Sardinia.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

Sanitary Emergencies at the Wild/Domestic Caprines Interface in Europe.

Simple SummaryEven if it is an important achievement from a biodiversity conservation perspective, the documented increase in abundance of the four native European wild Caprinae (Rupicapra rupicapra, R. pyrenaica, Capra ibex, C. pyrenaica) can also be a matter of concern, since tighter and more frequent contact with sympatric livestock implies a greater risk of transmission of emerging and re-emerging pathogens. This article reviews the main transmissible diseases that, in a European scenario, are of greater significance from a conservation perspective. Epidemics causing major demographic downturns in wild Caprinae populations during recent decades were often triggered by pathogens transmitted at the livestock/wildlife interface. Population density and distribution of the four native European wild Caprines (Rupicapra rupicapra, Rupicapra pyrenaica, Capra ibex, Capra pyrenaica) have increased in recent decades. The improved conservation status of this valuable wildlife, while a welcome event in general terms, is at the same time a matter of concern since, intuitively, frequent and tighter contacts with sympatric livestock imply a greater risk of cross-transmission of emerging and re-emerging pathogens, and offer unexpected opportunities for pathogens to spread, persist and evolve. This article recalls the transmissible diseases that are perceived in Europe to be of major significance from a conservation perspective, namely brucellosis (BRC) by Brucella melitensis, infectious kerato-conjunctivitis (IKC) by Mycoplasma conjunctivae, pestivirosis (PV) by the border disease virus strain 4 and mange by Sarcoptes scabiei. Special emphasis has been put on the epidemiological role played by small domestic ruminants, and on key knowledge needed to implement evidence-based prevention and control strategies. Remarkably, scientific evidence demonstrates that major demographic downturns in affected wild Caprinae populations in recent decades have often been triggered by pathogens cross-transmitted at the livestock/wildlife interface.

Characterization of internal ribosome entry sites according to secondary structure analysis to classify border disease virus strains

Applying palindromic nucleotide substitutions (PNS) method, variable loci of the internal ribosome entry site (IRES) secondary structure in the 5’ untranslated region (UTR) of Border disease virus sequences were analysed allowing their allocation into ten IRES classes within the species. Sequence characteristics of Turkish and Chinese strains were highly divergent from other genogroups, indicating geographic segregation and micro-evolutive steps within the species. Observed heterogeneity in the BDV species has to be considered for potential implications on diagnostic tests, control and preventive measures.

First report of border disease virus in Melophagus ovinus (sheep ked) collected in Xinjiang, China.

Melophagus ovinus (sheep ked) is a blood-sucking ectoparasite that is parasitic primarily on sheep. It is widely distributed in different geographical regions worldwide. In China, it has been mainly found in Xinjiang, Gansu, and Tibet in recent years. In addition to causing direct damage to the animal hosts, M. ovinus also carries pathogens and serves as a vector for disease transmission. Border disease virus (BDV) is a positive-sense, single-stranded RNA pestivirus that mainly infects and causes border disease (BD) in sheep and goats worldwide. Since 2012, this disease has been reported in 4 provinces in China. In the present study, we investigated the presence of BDV in M. ovinus from Xinjiang and Gansu. Frozen M. ovinus collected during 2017 and 2018 from Xinjiang and Gansu and preserved in our laboratory were studied. First, total RNA of M. ovinus was extracted, followed by reverse transcription, PCR (RT-PCR) amplification of the 5′-UTR of BDV, and sequencing of the amplified products. Finally, the sequencing results were analyzed using DNAStar, MEGA 5.0 molecular biology software, and the BLAST online platform. The results from RT-PCR and sequencing analyses showed that among the samples included in the study, only the M. ovinus collected from Qinghe County in Alta, Xinjiang in 2018 tested positive for BDV. BLAST analysis showed that the viral strain with the most similar nucleotide identity to the sequence of the China/BDV/2018 fragment was the goat-derived BDV strain AH12-02 collected in Anhui, China, in 2012. A phylogenetic-tree analysis showed the strain to exhibit a BDV-3 genotype. This is the first report globally on BDV detected in M. ovinus and is also the first report of BDV discovered in Xinjiang, China. This study reconfirms the presence of BDV in China.

Experimental infection with high- and low-virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease.

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free-ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high-virulence (Cadí-6) and low-virulence (Freser-5) BDV strains was performed. Pregnant and non-pregnant animals with and without antibodies against BDV were included in each group. Cadí-6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser-5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí-6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí-6 group. Chamois from both groups presented lesions in brain but the ones infected with the low-virulence Freser-5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low-virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross-protection in chamois against high-virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.

New insights on pestivirus infections in transhumant sheep and sympatric Pyrenean chamois (Rupicapra p. pyrenaica)

Border Disease Virus (BDV) causes health and economic impact on livestock and is also of importance in wildlife conservation as it causes high mortality outbreaks in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Pastoral practices are known as a main interspecies pathogen transmission. Hence, the presence of pestivirus in transhumant sheep flocks and sympatric chamois was assessed in areas with different epidemiological scenarios of chamois BDV infections. Moreover, the present study had also the goal to identify if inter-specific infections occurred and when they happened. Five sheep flocks grazing in two alpine areas in the Pyrenees with two different BDV epidemiological scenarios in chamois populations were studied during two transhumant seasons. Sheep were sampled before and after transhumance. Pyrenean chamois sera and spleen samples from both areas where also studied during the same period. Antibodies against BDV were assessed by means of ELISA and VNT. A qRT-PCR was used in order to detect the virus. Seroprevalence in sheep ranged between 0 and 91.1% at the flock level. Chamois were found to have high seroprevalences (52.9–77.7%) in both areas, and four new BDV isolates were sequenced. One sheep farm presented persistent BDV circulation and three showed low BDV circulation. The after-transhumance period was identified as the moment when viral transmission occured in the first farm, associated to BDV strains of domestic origin, according to VNT results. However, the BDV isolate was genetical closely related to previous BDV strains from chamois origin. In another farm, antibodies in two of the three positive sera were associated to infection with a chamois-like BDV strain. Altogether indicates that occasional viral transmission from chamois to sheep may occur.

Complete Genome Sequences of Three Border Disease Virus Strains of the Same Subgenotype, BDSwiss, Isolated from Sheep, Cattle, and Pigs in Switzerland.

ABSTRACTWe report here the complete genome sequences of three border disease virus (BDV) strains of the same subgenotype isolated in Switzerland from a sheep, a cow, and a pig, respectively. This is the first report of full-length sequences of a tentatively new subgenotype isolated from three different species of cloven-hoofed farm animals.

Co-existence of bovine viral diarrhea and border disease viruses in a sheep flock suffering from abortus and diarrhea

Pestivirus infections have a huge economic impact on livestock production. In 2014, an aborted fetus from a sheep flock suffering from abortus and diarrhea was submitted for virological diagnosis. Due to the positive result of the sample for pestivirus and continuing clinical symptoms in the flock, all of the animals were sampled individually. Blood samples for serum and peripheral blood mononuclear cells were collected from 93 animals (5 rams, 26 lambs, and 62 sheep). During sampling 1 ocular and 4 rectal swab samples were obtained from lambs that had a clinical eye problem and diarrhea, respectively. Additionally 5 aborted fetuses were submitted after the initial sampling. Thirteen of the 93 blood samples tested positive for pestivirus by antigen-detection ELISA. Propagation of noncytopathogenic virus was detected in blood samples from 6 lambs and in 1 aborted fetus sample by using the indirect immunoperoxidase monolayer assay. Pestivirus RNA was detected in 10 of 13 samples by RT-PCR employing pan-pestivirus primers. Border disease virus (BDV) RNA was identified with PBD1/PBD2 specific primers in all 10 samples that tested positive for pan-pestivirus primers. Differentiating RT-PCR further identified BVDV-1 sequences in 3 of the 10 samples. The Sequenced BDV strain (KY-57) was located in the cluster of BDV-7 (Aydin-like) while the BVDV strain was close close to BVDV-1c. The results of this study highlight the possibility of dual infection in sheep with BDV and BVDV-1.

A new genotype of border disease virus with implications for molecular diagnostics.

Border disease virus (BDV) is a (+) single-stranded RNA pestivirus affecting mainly sheep and goats worldwide. Genetic typing of BDV has led to the identification of at least seven major genotypes. This study reports the detection of a BDV strain from a goat in northwestern Italy during routine investigations. Sequence analysis revealed mutations in the 5'-UTR of the virus with implications for BDV molecular diagnostics. Moreover, subsequent phylogenetic analysis based on the combined 5'-UTR and Npro/partial C genes, showed divergence from known BDV genotypes, revealing the detection of a novel pestivirus group, for which we propose the name BDV genotype 8.

Chinese border disease virus strain JSLS12-01 infects piglets and down-regulates the antibody responses of classical swine fever virus C strain vaccination

During 2012 and 2013, several border disease virus (BDV) strains were identified from Chinese goat and sheep herds. At the same time, pigs from the same areas were found to be seropositive to BDV by ELISA, without showing clinical signs (unpublished data). To examine the susceptibility of pigs to the Chinese BDV strains, BDV isolate JSLS12-01, isolated from naturally infected sheep, was used to infect pigs. Antibody responses, viremia, clinical signs and pathological changes of the infected animals were examined. It confirmed that the current BDV strain could infect the domestic pigs, the animals showed viremia during 4 to 14 days post infection (dpi) and sero-conversion from 14dpi; no clinical and pathological changes were observed. In addition, CSFV maternal antibody did not influence BDV infection. Subsequently, pigs were infected with the BDV isolate and vaccinated with Hog cholera lapinized virus (HCLV) 21 days later to determine the effect of BDV infection on antibody induction of CSFV vaccination. The specific CSFV antibody and neutralizing antibody titers of the BDV infected group remained negative after the primary vaccination. Even after the boost vaccination, they were still significantly lower than those of the uninfected groups (p<0.05). These results indicated that BDV infection could down-regulate the antibody responses of CSFV C-strain vaccination. It should be paid attention that BDV prevalence in pig herds and in live vaccines might hamper the vaccination of CSF.

Antigenic and molecular characterisation of Border disease virus associated with high mortality in lambs in Spain

Introduction Border disease virus (BDV) causes congenital disorders in sheep and results in severe, but underestimated, economic losses worldwide. However, information about BDV strains affecting several ruminants worldwide is scarce....

Characterization of one sheep border disease virus in China.

BackgroundBorder disease virus (BDV) causes border disease (BD) affecting mainly sheep and goats worldwide. BDV in goat herds suffering diarrhea was recently reported in China, however, infection in sheep was undetermined. Here, BDV infections of sheep herds in Jiangsu, China were screened; a BDV strain was isolated and identified from the sheep flocks in China. The genomic characteristics and pathogenesis of this new isolate were studied.ResultsIn 2012, samples from 160 animals in 5 regions of Jiangsu province of China were screened for the presence of BDV genomic RNA and antibody by RT-PCR and ELISA, respectively. 44.4% of the sera were detected positively, and one slowly grown sheep was analyzed to be pestivirus RNA positive and antibody-negative. The sheep kept virus positive and antibody negative in the next 6 months of whole fattening period, and was defined as persistent infection (PI). The virus was isolated in MDBK cells without cytopathic effect (CPE) and named as JSLS12-01. Near-full-length genome sequenced was 12,227 nucleotides (nt). Phylogenetic analysis based on 5'-UTR and Npro fragments showed that the strain belonged to genotype 3, and shared varied homology with the other 3 BDV strains previously isolated from Chinese goats. The genome sequence of JSLS12-01 also had the highest homology with genotype BDV-3 (the strain Gifhorn). Experimental infections of sheep had mild clinical signs as depression and short-period mild fever (5 days). Viremia was detected in 1–7 days post-infection (dpi), and seroconversion began after 14 dpi.ConclusionsThis study reported the genomic and pathogenesis characterizations of one sheep BDV strain, which confirmed the occurrence of BDV infection in Chinese sheep. This sheep derived BDV strain was classified as BDV-3, together with the goat derived strains in China. These results might be helpful for further understanding of BDV infection in China and useful for prevention and control of BDV infections in the future.

First isolation of border disease virus in Japan is from a pig farm with no ruminants

The first isolation of border disease virus (BDV) in Japan was from a pig farm of the farrow-to-finishing type that kept no small ruminants or cattle. The infection was detected in the course of sero-surveillance for classical swine fever virus (CSFV) in Japan. The infected pigs had no clinical symptoms of CSFV or other disease; nevertheless, a high sero-positive rate of 58.5% was identified. A persistently infected pig with the BDV was found and suspected to be the cause of sero-prevalence in the farm. The isolated BDV was genetically close to BDV strains from New Zealand, but there was no epidemiological evidence concerning the route(s) of the invasion into the farm.

Molecular characterization of border disease virus strain Aveyron

For the pestivirus border disease virus (BDV) at least seven major genotypes have been described (BDV-1–BDV-7). So far, complete genomic sequences have been reported for four BDV genotypes (BDV-1–BDV-4). In this study we report the entire genomic sequence of the noncytopathogenic (ncp) BDV-5 reference strain Aveyron. The viral genome encompasses 12,284 nucleotides (nt) and contains one large open reading frame (11,700nt) flanked by a 370 nt long 5′-untranslated region (UTR) and a 214nt long 3′-UTR. The genome organization as well as the lengths of the viral polyprotein (3899 amino acids) and the 5′-UTR are very similar to the ones of other BDV strains, while the 3′-UTR of BDV Aveyron is considerably shorter when compared to other BDV strains. Comparative analysis of complete coding sequences revealed that BDV Aveyron shares nucleotide sequence identities of 76.9% to 79.0% with the other BDV strains, and less than 72% identity with other pestiviruses. In contrast to other BDV strains, a unique insertion of four amino acids (KAPD) of unknown origin is present in the C-terminal part of the viral autoprotease NS2 encoded by BDV Aveyron. Immunoblot analysis revealed that infection of cells with the ncp BDV strain Aveyron comprising this unique insertion in NS2 resulted in the expression of high amounts of NS3 and thereby showed that BDV Aveyron significantly differs from other ncp BDV strains in terms of NS2-3 processing and production of NS3.

Complete Genome Sequence of Border Disease Virus Genotype 3 Strain Gifhorn

The complete genome sequence of the genotype 3 border disease virus strain Gifhorn has been determined; this strain was originally isolated from pigs. This represents the consensus sequence for the virus used to produce the bacterial artificial chromosome (BAC) cDNA clone pBeloGif3, which yields a virus that is severely attenuated in cell culture.

Genome Sequence of Border Disease Virus Strain JSLS12-01, Isolated from Sheep in China

Border disease virus (BDV) is a recognized virus in the genus Pestivirus and causes border disease (BD) in sheep and goats. Here, a novel BDV strain, JSLS12-01, was identified from sheep in Jiangsu Province, China. The complete coding sequence (CDS) was finished, which provides a better understanding of the molecular evolution of BDV isolates.

Detection of border disease virus (BDV) in goat herds suffering diarrhea in eastern China

BackgroundBorder disease virus (BDV) is an important pathogen in sheep and goat production. Neither epidemiological investigation nor any reports of BDV infection was available in China. During Jan to Apr, 2012, several herd goats in Anhui and Jiangsu provinces in eastern China suffered unremitting diarrhea, with morbidity and mortality of about 28-37% and 10-15%, respectively. In the present study, sera and tissue samples from diseased goats of four farms were taken for BDV detection, isolation and identification.ResultsPanpesti generic primers and border disease virus (BDV)-specific primers targeting the 5’-UTR region produced RT-PCR positive bands for sera (24/28) and tissue samples (7/30). Twenty positive sera and tissue samples were inoculated onto Madin-Darby bovine kidney (MDBK) cells for virus isolation. Finally, three different strains of BDV, named AH12-01, AH12-02 and JS12/04, were successfully isolated as identified by RT-PCR using 5’-UTR and Npro gene primers, sequencing and electron microscopy. Sequences of 5’-UTR and Npro genes of them were used for phylogenetic analysis and comparison to other reference sequences available in GenBank. The results indicated AH12-01, AH12-02 and JS12/04 possess high relationship with the BDV 3 group viruses and differed with each other.ConclusionThis is the first detection of BDV from goats with diarrhea and confirmation of BDV infection in China.

Two different epidemiological scenarios of border disease in the populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the first disease outbreaks.

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.

SURVEILLANCE OF BORDER DISEASE IN WILD UNGULATES AND AN OUTBREAK IN PYRENEAN CHAMOIS (RUPICAPRA PYRENAICA PYRENAICA) IN ANDORRA

The Principality of Andorra is surrounded by areas in which Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations were severely affected by infection with border disease virus (BDV) which caused disease outbreaks between 2001 and 2009. Nevertheless, the Andorran chamois populations were not affected during this period. In light of the severe impact of BDV on several of the neighboring Pyrenean chamois populations, we monitored local Andorran populations in an effort to detect pestivirus antibodies and BDV in wild ungulates. In addition, an episode of mortality between 2009 and 2010 in chamois was investigated. We analyzed samples (spleen or serum) from 175 Pyrenean chamois, 284 European mouflon (Ovis orientalis musimon), 13 roe deer (Capreolus capreolus capreolus), and five wild boars (Sus scrofa castilianus). With the exception of three dead chamois found between 2009 and 2010, all samples came from healthy animals hunted during the hunting season. A commercial blocking enzyme-linked immunosorbent assay (ELISA) was used to test sera for antibodies against pestivirus. Positive sera were tested with a comparative virus neutralization test (VNT) using three BDV strains and a bovine viral diarrhea virus strain. Reverse-transcription-polymerase chain reaction (RT-PCR) was performed on all sera and spleen homogenates. Antibodies against pestivirus were detected by ELISA in four of the 69 chamois (5%; 95% CI= 1.29-13.11). The VNT confirmed three of these chamois were infected with a BDV. Viral RNA was detected by RT-PCR in three chamois-one apparently healthy animal hunted in 2009 and two dead animals. Viral sequences showed that the three chamois were infected with a BDV-4, the same genotype that was involved in previous episodes of mortality in the Pyrenees. Although Pyrenean chamois from Andorra had had little contact with the pestiviruses until 2009, in this year BDV was associated with a severe disease outbreak.