An investigation into the transmission and control of pestivirus in sheep in Australia.

Border disease virus (BDV) is a member of the pestivirus genus that primarily affects sheep, causing reproductive losses through abortion, still births and the birth of weak lambs. The key characteristic of this disease is the birth of persistently infected (PI) lambs which, after surviving transplacental infection, are born antibody negative, yet virus positive, and thus shed the virus for their entire life and are the primary source of spread within a flock. The cornerstones of BDV control are detection and elimination of PI animals, biosecurity measures to prevent re-infection, and surveillance programs. Recommendations for the control of BDV in sheep are centred around the approach to bovine viral diarrhoea virus (BVDV), the prominent cattle pestivirus species, due to a lack of specific research into BDV control and elimination. In this study, two aspects of a BDV control program were investigated: the effectiveness of the BVDV vaccine, Pestigard®, and the rate of seroconversion in a flock deliberately exposed to known PI lambs. The vaccine appeared to be safe, and the optimal dose was the full cattle dose (2 mL). While vaccination induced high virus neutralising titres to BVDV when administered as either a quarter, half or full dose registered for cattle, the BDV titres achieved were low and unlikely to prevent transplacental infection. In a second study, after exposure of between 2 and 15 days exposure to two PI lambs in confined conditions, only 3 of 66 previously naïve sheep demonstrated seroconversion. This demonstrated a very low rate of transmission and suggested that deliberate exposure to PI lambs at low-risk times for less than 15 days was not likely to be an effective means of achieving seroconversion throughout a flock and, therefore, not provide protection against BDV challenge during gestation.

New Potential Role of European Mouflon (Ovis aries musimon) in the Epidemiology of Border Disease in the Pyrenees.

Since 2001, high-mortality outbreaks of border disease (BD) have negatively affected populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Studies in the affected areas determined that sympatric wild ruminants did not seem to have an epidemiologic role in the circulation of border disease virus (BDV). However, the recent increase in European mouflon (Ovis aries musimon) densities might enhance the risk of pathogen transmission among chamois and mouflons. We conducted a serologic and virologic investigation of BDV in European mouflon from the Spanish Pyrenees, with the aim of determining potential changes in the role of this species in BDV epidemiology. From 2018 to 2022, we detected antibodies against BDV in 31/185 (16.7%) animals but did not detect BDV RNA in any spleen sample (0/65). These results indicate that BDV infection is occurring in these mouflon populations to a greater extent than previously described, which could shift the current understanding of BD epidemiology in the Pyrenees and cause an unpredictable effect on both chamois and mouflon populations. Further studies on the molecular identification of BDV in mouflon and chamois are required to better understand the contribution of mouflon in the epidemiology of BD.

Investigation into an outbreak of Border disease virus in pigs in England.

Border disease (BD) was first reported in 1959 in lambs from the border region of England and Wales. The causative virus (BD virus; BDV) has since been identified in several other ruminant species and pigs. The virus is prevalent in sheep flocks of UK, Europe and USA and has potential to inflict substantial economic losses. Natural BDV infection of pigs was first reported in the UK in 1992 from pigs with haemorrhagic lesions and more recently from healthy pigs in Spain and Japan. Here, a persistent problem of poor growth and anaemia in a small proportion of growing pigs on a mixed pig and sheep holding was investigated and tissues were tested in a pan viral microarray. The microarray detected BDV RNA in several tissues which was further confirmed by sequencing, specific BDV PCR and immunohistochemistry. Phylogenetically, the virus clustered with other BDVs in the sub-genotype 1b. This investigation highlights likely interspecies transmission of pestiviruses and their impact on pestivirus detection and eradication programs.

Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

Global Distribution and Genetic Heterogeneity of Border Disease Virus.

Border disease virus (BDV) belongs to the genus Pestivirus of the family Flaviviridae. Interspecies transmission of BDV between sheep, cattle, and pigs occurs regularly, sometimes making diagnosis a challenge. BDV can yield substantial economic losses, including prenatal and postnatal infections in lambs, which are the primary source of infection and maintenance of the virus in the population. Since BDV is antigenically and genetically related to bovine viral diarrhea virus (BVDV), it might pose a significant risk to cattle, influencing BVDV eradication campaigns. Similarly, the presence of BDV in swine herds due to pestivirus spillover between small ruminants and pigs might cause uncertainty in classical swine fever virus (CSFV) diagnostics. Therefore, knowledge of BDV epidemiology in different geographical regions will help prevent its spread and optimize control measures. Previous epidemiological studies have shown that various BDV genotypes are predominant in different countries. This review provides an overview of the spread of BDV world-wide in different host species.

Near-complete nucleotide sequence of a border disease virus genotype BDV-7 isolate from Italy.

To gain further insight into the genomic features of border disease virus (BDV), we determined the nearly complete genome sequence of isolate TO/121/04 from an aborted ovine fetus. Its genome contains a single open reading frame (ORF), which comprises 11,681 nucleotides encoding a polyprotein of 3893 amino acids. Phylogenetic analysis of the near full-length genome sequence showed that the BDV isolate differed significantly from all ovine pestiviruses identified so far, thus re-affirming the presence in Italy of this novel genetic group, termed BDV-7.

Histological and virological findings in severe meningoencephalitis associated with border disease virus in Alpine chamois (Rupicapra rupicapra rupicapra) in Aosta Valley, Italy.

In 2015, a young female Alpine chamois (Rupicapra rupicapra rupicapra), originated from the Aosta Valley Region, Northernwestern Italy, was conferred to the National Reference Centre for Wild Animal Diseases for pathologic examinations. Histological analysis revealed a severe meningoencephalitis characterized by lymphocytic and plasmacellular infiltration, gliosis, perivascular cuffs, and leptomeningitis at the level of brain and brain stem. Laboratory investigations included polymerase chain reaction, sequencing and characterization by phylogenetic analysis, and evaluation of the internal ribosome entry site secondary structure in the 5' untranslated region. These tests identified the pathological agent as border disease virus, a known health risk in domestic small ruminants. Genetic characteristics of the isolated strains, closely related to ovine and caprine strain sequences from neighboring regions of Piedmont, France, and Switzerland, suggested geographic segregation and micro-evolutive steps within the species.

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.

Development and evaluation of real-time RT-PCR using ear hair for specific detection of sheep persistently infected with border disease virus (BDV).

The aim of this study was to develop an improved border disease virus (BDV) specific real time RT-PCR and to evaluate its performance on manually plucked hairs from sheep persistently infected with BDV that may act as a non-invasive alternate sample. The BDV real time RT-PCR assay reported here showed a high analytical sensitivity (100.6 TCID50/ml), specificity (no reactivity with BVDV-1, BVDV-2, HoBi-like pestivirus and CSFV) and reproducibility. When the assay was validated on 210 samples from BDV-infected and uninfected sheep, it showed a 100% diagnostic sensitivity and specificity with virus isolation. Further evaluation of the assay on manually plucked hair follicles from ear (mid-lateral, mid-medial) and tail tip from sheep persistently infected with BDV showed that a minimum of 20 hair follicles need to be tested for correct diagnosis of BDV. The BDV load was comparatively higher in hairs from mid-medial ear than those from other tested locations. Evaluation on other samples from PI sheep demonstrated that the test performance was similar to that of pestivirus generic real-time RT-PCR, but improved than the currently available BDV specific real-time RT-PCR. Although more number of PI animals need to be evaluated, the results of the study showed that manually plucked hairs from mid-medial ear pinna is a suitable alternative sample in real-time RT-PCR for detection of BDV persistently infected sheep. Use of the non-invasive ear hair samples and the improved BDV specific real-time RT-PCR reported here may be useful for BDV surveillance in several sheep rearing countries.

Border disease in cattle.

Within the family Flaviviridae, viruses within the genus Pestivirus, such as Border disease virus (BDV) of sheep, can cause great economic losses in farm animals. Originally, the taxonomic classification of pestiviruses was based on the host species they were isolated from, but today, it is known that many pestiviruses exhibit a broad species tropism. This review provides an overview of BDV infection in cattle. The clinical, hematological and pathological-anatomical findings in bovines that were transiently or persistently infected with BDV largely resemble those in cattle infected with the closely related pestivirus bovine viral diarrhoea virus (BVDV). Accordingly, the diagnosis of BDV infection can be challenging, as it must be differentiated from various pestiviruses in cattle. The latter is very relevant in countries with control programs to eradicate BVDV in Bovidae, as in most circumstances, pestivirus infections in sheep, which act as reservoir for BDV, are not included in the eradication scheme. Interspecies transmission of BDV between sheep and cattle occurs regularly, but BDV in cattle appears to be of minor general importance. Nevertheless, BDV outbreaks at farm or local level can be very costly.

First description of enhanced expression of transforming growth factor-alpha (TGF-α) and glia maturation factor-beta (GMF-ß) correlate with severity of neuropathology in border disease virus-infected small ruminants.

Border disease (BD) is caused by Pestivirus and characterized by severe neuropathology, and histopathologically observed severe hypomyelination. We have previously shown that small ruminants infected with border disease virus (BDV) play an important role for neuropathology and pathogenesis of severe oxidative damage in brain tissue, neuronal mtDNA; in the production of high pathologic levels of nitric oxide; in glial cell activation and stimulation of intrinsic apoptosis pathway. This study aimed to investigate the relationship between glia maturation factor beta (GMF-ß) and transforming growth factor alpha (TGF-α) expressions and the causes of BDV-induced neuropathology and to investigate their role in neuropathogenesis in a way that was not presented before. Expression levels of GMF-ß and TGF-α were investigated. Results of the study revealed that the levels of GMF-ß (P < 0.005) and TGF-α (P < 0.005) expression in the brain tissue markedly increased in the BDV-infected animals compared to the non-infected healthy control group. While TGF-α expressions were predominantly observed in neurons, GMF-ß expressions were found in astrocytes, glial cells and neurons. These results were reasonable to suggest that BDV-mediated increased GMF-ß might play a pivotal role neuropathogenesis and a different type of role in the mechanism of neurodegeneration/neuropathology in the process of BD. The results also indicated that increased levels of GMF up-regulation in glial cells and neurons causes neuronal destruction, suggesting pathological pathway involving GMF-mediated brain cell cytotoxicity. It is clearly indicated that the cause of astrogliosis is due to severe TGF-a expression. This is the first study to demonstrate the expression of GMF-ß and TGF-α in neurons and reactive glial cells and its association with neuropathology in BD.

Seroprevalence of border disease virus and other pestiviruses in sheep in Algeria and associated risk factors.

BACKGROUND: Border disease virus (BDV) is a pestivirus responsible for significant economic losses in sheep industry. The present study was conducted between 2015 and 2016 to determine the flock seroprevalence of the disease in Algeria and to identify associated risk factors. 56 flocks from nine departments were visited and 689 blood samples were collected from adult sheep between 6 and 24 months of age (n = 576) and from lambs younger than 6 months (n = 113). All samples were tested by RT-PCR as well as by Ag-ELISA, to detect Persistently Infected (PI) animals. Serum samples from adults were tested by Ab-ELISA (Enzyme Linked Immuno-Sorbent Assay), to detect specific antibodies against pestivirus and 197 of them were further characterized by VNT (virus neutralization test) for the detection of neutralizing antibodies specific for BDV and for Bovine virus diarrhea virus (BVDV-1 and BVDV-2). RESULTS: No PI animals were found among the 689 sheep tested. 144/197 sera were positive in VNT for BDV, and 2 sera were strongly positive BVDV-2. Fifty-five flocks (98%) had at least one seropositive animal and the apparent within-flock seroprevalence was estimated to be 60.17% (95% C.I.: 52.96-66.96). The true seroprevalence based on estimated sensitivity and specificity of the Ab-ELISA was 68.20% (95% C.I.; 60.2-76.3). Several risk factors were identified as linked to BDV such as climate, landscape, flock management and presence of other ruminant species in the farm. CONCLUSION: These high seroprevalence rates suggest that BDV is widespread and is probably endemic all over the country. Further studies are needed to detect and isolate the virus strains circulating in the country and understand the distribution and impact of pestiviruses in the Algerian livestock.

Insemination with border disease virus-infected semen results in seroconversion in cows but not persistent infection in fetuses.

BACKGROUND: This study examined various health variables in cows after artificial insemination with Border disease virus (BDV)-infected semen and the occurrence of persistent infection in ensuing fetuses. Five cows were inseminated (day 0) with BDV-infected semen as well as with semen from a fertile Eringer bull. One cow, inseminated with virus-free semen only, served as a control. Clinical examination, assessment of eating and rumination activities, measurement of intraruminal temperature and leukocyte count were used to monitor the health of the cows. Blood samples were collected at regular intervals for the detection of viral RNA and antibodies against BDV, and the cows were slaughtered on day 56. The uteri, placentae and fetuses were examined macroscopically, histologically, immunohistochemically and by means of molecular methods for the presence of pestiviruses. RESULTS: The demeanour, eating and rumination activities and intraruminal temperature were not affected by insemination with BDV-infected semen, whereas the total leukocyte and lymphocyte counts dropped transiently and were significantly lower on day 6 than on day 0. Seroconversion occurred by day 28 in the five infected cows but not in the control cow. The uteri, placentae and fetuses had no macroscopic or histological lesions, and immunohistochemical examination and RT-PCR were negative for pestiviruses. CONCLUSIONS: The findings showed that cows inseminated with BDV-infected semen seroconverted and fetuses thus produced were not persistently infected. Transmission of BDV to cattle through infected semen, therefore, seems to be of minor importance.

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.

Border Disease Virus Infection of Bovine Placentas.

Subsequent to a previous study of border disease virus (BDV) horizontal transmission from a persistently BDV-infected calf to 6 seronegative pregnant heifers, the heifers were slaughtered 60 days after exposure to the infected calf, and their fetuses and placentas were examined. Immunohistochemical examination of fetal organs and placenta showed positive labeling of moderate intensity for pestivirus antigen in 3 of 6 heifers. BDV infection in these 3 animals was confirmed by the detection of BDV RNA in different organs using reverse transcription quantitative polymerase chain reaction. In the placenta, the positive cells were visualized mostly on the fetal side. In those 3 heifers that harbored an infected fetus, the placental tissue in the placentome region showed a moderate to severe mononuclear and fibrosing placentitis and, in severe cases, necrotic areas. The inflammatory population was composed predominantly of T and B cells, a substantial number of macrophages, and, to a lesser extent, plasma cells. This is a novel report of placentitis in persistently BDV-infected fetuses from pregnant heifers that became acutely infected by cohousing with a calf persistently infected with BDV, which extends previous reports on bovine viral diarrhea virus-infected and BDV-infected cattle and sheep, respectively.

Detection of border disease virus in Mexican cattle.

The genus Pestivirus within Flaviviridae is comprised of four recognized species, namely, bovine viral diarrhoea virus 1 (BVDV-1), bovine viral diarrhoea virus 2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). BDV, while primarily infecting sheep and goats, has also been reported in cattle and wild animals. Infections of sheep and goats result in economic loss due to abortions and the birth of persistently infected animals that have poor production and reduced life expectancy. In this study, we report the detection of BDV in cattle serum collected as part of pestivirus surveillance programme from six regions of Mexico, where a 67.1% of BVDV seroprevalence was calculated previously. Phylogenetic analyses based on comparison of the 5'UTR region typed the Mexican strains as BDV-1. Border disease (BD) is listed as an exotic disease in Mexico, and the origin of BDV found in these cattle is unclear. This is the first identification of BDV in Mexican cattle.

Evidence of circulation of the novel border disease virus genotype 8 in chamois.

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

Spatial and Temporal Phylogeny of Border Disease Virus in Pyrenean Chamois (Rupicapra p. pyrenaica).

Border disease virus (BDV) affects a wide range of ruminants worldwide, mainly domestic sheep and goat. Since 2001 several outbreaks of disease associated to BDV infection have been described in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain, France and Andorra. In order to reconstruct the most probable places of origin and pathways of dispersion of BDV among Pyrenean chamois, a phylogenetic analysis of 95 BDV 5'untranslated sequences has been performed on chamois and domestic ungulates, including novel sequences and retrieved from public databases, using a Bayesian Markov Chain Monte Carlo method. Discrete and continuous space phylogeography have been applied on chamois sequences dataset, using centroid positions and latitude and longitude coordinates of the animals, respectively. The estimated mean evolutionary rate of BDV sequences was 2.9×10-3 subs/site/year (95% HPD: 1.5-4.6×10-3). All the Pyrenean chamois isolates clustered in a unique highly significant clade, that originated from BDV-4a ovine clade. The introduction from sheep (dated back to the early 90s) generated a founder effect on the chamois population and the most probable place of origin of Pyrenean chamois BDV was estimated at coordinates 42.42 N and 1.9 E. The pathways of virus dispersion showed two main routes: the first started on the early 90s of the past century with a westward direction and the second arise in Central Pyrenees. The virus spread westward for more than 125 km and southward for about 50km and the estimated epidemic diffusion rate was about 13.1 km/year (95% HPD 5.2-21.4 km/year). The strong spatial structure, with strains from a single locality segregating together in homogeneous groups, and the significant pathways of viral dispersion among the areas, allowed to reconstruct both events of infection in a single area and of migrations, occurring between neighboring areas.

[PESTIVIRUSES IN RUMINANTS].

The genus Pestivirus includes four species: bovine viral diarrhea virus 1, bovine viral diarrhea virus 2, classical swine fever disease virus, and ovine border disease virus. Pestiviruses infect many species of domestic and wild animals. Bovine viral diarrhea virus is a prototypical representative of the pestiviruses of ruminant animals. Recently, new candidates appeared for including in this genus: two viruses of the wild ruminant animals that have not been officially classified and one HoBi-like virus discovered for the first time in the bovine fetal serum. The circulation of the ruminant animal pestiviruses within population of domestic and wild animals, the presence of these viruses in bioproducts stimulates studies of the infection reservoirs and their influence on the effect of the bovine viral diarrhea control programs.