DNAJC14-Independent Replication of the Atypical Porcine Pestivirus.

Atypical porcine pestiviruses (APPV; Pestivirus K) are a recently discovered, very divergent species of the genus Pestivirus within the family Flaviviridae. The presence of APPV in piglet-producing farms is associated with the occurrence of so-called "shaking piglets," suffering from mild to severe congenital tremor type A-II. Previous studies showed that the cellular protein DNAJC14 is an essential cofactor of the NS2 autoprotease of all classical pestiviruses. Consequently, genetically engineered DNAJC14 knockout cell lines were resistant to all tested noncytopathogenic (non-cp) pestiviruses. Surprisingly, we found that the non-cp APPV can replicate in these cells in the absence of DNAJC14, suggesting a divergent mechanism of polyprotein processing. A complete laboratory system for the study of APPV was established to learn more about the replication of this unusual virus. The inactivation of the APPV NS2 autoprotease using reverse genetics resulted in nonreplicative genomes. To further investigate whether a regulation of the NS2-3 cleavage is also existing in APPV, we constructed synthetic viral genomes with deletions and duplications leading to the NS2 independent release of mature NS3. As observed with other pestiviruses, the increase of mature NS3 resulted in elevated viral RNA replication levels and increased protein expression. Our data suggest that APPV exhibit a divergent mechanism for the regulation of the NS2 autoprotease activity most likely utilizing a different cellular protein for the adjustment of replication levels. IMPORTANCE DNAJC14 is an essential cofactor of the pestiviral NS2 autoprotease, limiting replication to tolerable levels as a prerequisite for the noncytopathogenic biotype of pestiviruses. Surprisingly, we found that the atypical porcine pestivirus (APPV) is able to replicate in the absence of DNAJC14. We further investigated the NS2-3 processing of APPV using a molecular clone, monoclonal antibodies, and DNAJC14 knockout cells. We identified two potential active site residues of the NS2 autoprotease and could demonstrate that the release of NS3 by the NS2 autoprotease is essential for APPV replication. Defective interfering genomes and viral genomes with duplicated NS3 sequences that produce mature NS3 independent of the NS2 autoprotease activity showed increased replication and antigen expression. It seems likely that an alternative cellular cofactor controls NS2-3 cleavage and thus replication of APPV. The replication-optimized synthetic APPV genomes might be suitable live vaccine candidates, whose establishment and testing warrant further research.

New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria.

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population.

In the Search of Marine Pestiviruses: First Case of Phocoena Pestivirus in a Belt Sea Harbour Porpoise.

Pestiviruses are widespread pathogens causing severe acute and chronic diseases among terrestrial mammals. Recently, Phocoena pestivirus (PhoPeV) was described in harbour porpoises (Phocoena phocoena) of the North Sea, expanding the host range to marine mammals. While the role of the virus is unknown, intrauterine infections with the most closely related pestiviruses- Bungowannah pestivirus (BuPV) and Linda virus (LindaV)-can cause increased rates of abortions and deaths in young piglets. Such diseases could severely impact already vulnerable harbour porpoise populations. Here, we investigated the presence of PhoPeV in 77 harbour porpoises, 277 harbour seals (Phoca vitulina), grey seals (Halichoerus grypus) and ringed seals (Pusa hispida) collected in the Baltic Sea region between 2002 and 2019. The full genome sequence of a pestivirus was obtained from a juvenile female porpoise collected along the coast of Zealand in Denmark in 2011. The comparative Bayesian phylogenetic analyses revealed a close relationship between the new PhoPeV sequence and previously published North Sea sequences with a recent divergence from genotype 1 sequences between 2005 and 2009. Our findings provide further insight into the circulation of PhoPeV and expand the distribution from the North Sea to the Baltic Sea region with possible implications for the vulnerable Belt Sea and endangered Baltic Proper harbour porpoise populations.

Comparative Analysis of Tunisian Sheep-like Virus, Bungowannah Virus and Border Disease Virus Infection in the Porcine Host.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Proposed Update to the Taxonomy of Pestiviruses: Eight Additional Species within the Genus Pestivirus, Family Flaviviridae.

Pestiviruses are plus-stranded RNA viruses belonging to the family Flaviviridae. They comprise several important pathogens like classical swine fever virus and bovine viral diarrhea virus that induce economically important animal diseases. In 2017, the last update of pestivirus taxonomy resulted in demarcation of 11 species designated Pestivirus A through Pestivirus K. Since then, multiple new pestiviruses have been reported including pathogens associated with disease in pigs or small ruminants. In addition, pestivirus sequences have been found during metagenomics analysis of different non-ungulate hosts (bats, rodents, whale, and pangolin), but the consequences of this pestivirus diversity for animal health still need to be established. To provide a systematic classification of the newly discovered viruses, we analyzed the genetic relationship based on complete coding sequences (cds) and deduced polyprotein sequences and calculated pairwise distances that allow species demarcation. In addition, phylogenetic analysis was performed based on a highly conserved region within the non-structural protein NS5B. Taking into account the genetic relationships observed together with available information about antigenic properties, host origin, and characteristics of disease, we propose to expand the number of pestivirus species to 19 by adding eight additional species designated Pestivirus L through Pestivirus S.

Atypical Porcine Pestiviruses: Relationships and Conserved Structural Features.

For two decades, the genus pestivirus has been expanding and the host range now extends to rodents, bats and marine mammals. In this review, we focus on one of the most diverse pestiviruses, atypical porcine pestivirus or pestivirus K, comparing its special traits to what is already known at the structural and functional level from other pestiviruses.

Genomic characterization of pestiviruses isolated from bovine, ovine and caprine foetuses in Turkey: A potentially new genotype of Pestivirus I species.

This study was carried out to investigate the frequency and genetic diversity of pestiviruses in abortion cases in cattle and small ruminants in Turkey. During January 2012 and December 2017, a total of 2029 aborted foetuses (553 bovine foetuses, 1,388 sheep foetuses and 88 goat foetuses) were collected from different regions of Turkey. Real-time RT-PCR (RRT-PCR) assays were used to detect pestiviral RNA in aborted foetuses. To confirm the cause of abortion, pestivirus-positive foetuses were also examined for the presence of Brucella spp., Campylobacter spp., Chlamydophila abortus (C. abortus), akabane virus, bluetongue virus and Schmallenberg virus by molecular detection methods. Pestiviral RNA was detected in 61 (11%) of the 553 bovine foetuses, 124 (8.9%) of the 1,388 sheep foetuses and 3 (3.4%) of the 88 goat foetuses. Furthermore, C. abortus DNA was detected in 3 pestivirus-positive sheep foetuses, whereas other infectious agents were not detected in pestivirus-positive foetuses. Genetic characterization of the pestivirus RRT-PCR positive samples was conducted by sequencing 5' untranslated (5' UTR) and non-structural autoprotease (Npro ) genomic regions. A total of 68 sequences were obtained, and phylogenetic analyses revealed that all sequences belonged to BVDV-1, including 1b (8/68), 1f (2/68), 1l (4/68), 1r (10/68), Aydin-like pestivirus (20/68) and one unknown genotype (24/68). The 5' UTR and Npro sequences of this unknown genotype differed from pestiviruses previously described, providing evidence for the presence of an emerging genotype within the species Pestivirus I, tentatively named as 'Konya-like' pestivirus. 'Konya-like' pestivirus was the dominant genotype in sheep foetuses, whereas Aydin-like pestivirus was found to be the predominant genotype in bovine foetuses. To the best my knowledge, this is the first report of Aydin-like pestivirus infection in cattle. The information provided in this study contributes to the understanding the dissemination and evolution of pestiviruses and could be beneficial for developing more effective vaccines.

HoBi-like pestivirus infection leads to bovine death and severe respiratory disease in China.

HoBi-like pestivirus is an emerging atypical pestivirus in cattle and small ruminants, causing clinical signs similar to those observed in bovine viral diarrhoea virus infections. Natural infection of HoBi-like pestivirus has been reported in cattle herds and small ruminants in multiple countries in South America, Europe and Asia. However, HoBi-like pestiviruses were only identified from contaminated bovine serum and small ruminants in China. So far, no clinical cases induced by HoBi-like pestivirus infection were reported in Chinese cattle herds. Here, for the first time, we reported natural infection of HoBi-like pestivirus in a cattle herd in China. Sick cattle with severe respiratory and diarrhoea and high fatality rate were found in a beef cattle herd in Shandong province in November 2017. RT-PCR, viral isolation, sequencing and phylogenetic analysis showed that the primary causative agent was HoBi-like pestivirus. The isolated HoBi-like pestivirus strain, SDJN-China-2019, shared 94.1%-97.5% homology with the LV168-20_16RN strain from Brazil in nucleotide of 5'UTR, Npro and E2 while it shared only 88.5%-92.1% homology with Asian HoBi-like virus strain Th/04-Khonkaen. Multiple unique mutations of amino acid were observed in Npro and E2 proteins of SDJN-China-2019, which were different from that of other reference strains. In summary, this study provides the first evidence of HoBi-like pestivirus infection in Chinese cattle herds, raising potential threat to the cattle industry in China.

Special Issue: Bovine Viral Diarrhea Virus and Related Pestiviruses.

The genus Pestivirus, encompassing small positive-strand RNA viruses in the family Flaviviridae, comprises four viruses of very significant economic impact to the cattle, swine and sheep industries worldwide: bovine viral diarrhoea virus (BVDV) type 1 and type 2, classical swine fever virus (CSFV) and border disease virus (BDV). Both BVDV- and CSFV-related disease syndromes have been recognised for over 70 years and major progress has been made in elucidating the pathogenesis of these important infections of ruminants and pigs [...].

Pestivirus K (Atypical Porcine Pestivirus): Update on the Virus, Viral Infection, and the Association with Congenital Tremor in Newborn Piglets.

The atypical porcine pestivirus (APPV) belongs to the species Pestivirus K of the genus Pestivirus and the family Flaviviridae, and it has been associated with congenital tremor (CT) type A-II in newborn piglets. Although APPV was discovered in 2015, evidence shows that APPV has circulated in pig herds for many years, at least since 1986. Due to the frequently reported outbreaks of CT on different continents, the importance of this virus for global pig production is notable. Since 2015, several studies have been conducted to clarify the association between APPV and CT. However, some findings regarding APPV infection and the measures taken to control and prevent the spread of this virus need to be contextualized to understand the infection better. This review attempts to highlight advances in the understanding of APPV associated with type A-II CT, such as etiology, epidemiology, diagnosis, and control and prevention measures, and also describes the pathophysiology of the infection and its consequences for pig production. Further research still needs to be conducted to elucidate the host's immune response to APPV infection, the control and prevention of this infection, and the possible development of vaccines.

Prevalence and Genetic Diversity of Atypical Porcine Pestivirus (APPV) Detected in South Korean Wild Boars.

Atypical porcine pestivirus (APPV), currently classified as pestivirus K, causes congenital tremor (CT) type A-II in piglets. Eighteen APPV strains were identified from 2297 South Korean wild boars captured in 2019. Phylogenetic analysis of the structural protein E2 and nonstructural proteins NS3 and Npro classified the APPV viruses, including reference strains, into Clades I, II and III. Clade I was divided into four subclades; however, the strains belonging to the four subclades differed slightly, depending on the tree analysis, the NS3, E2, and Npro genes. The maximum-likelihood method was assigned to South Korean wild boar APPV strains to various subclades within the three trees: subclades I.1 and I.2 in the E2 tree, subclade I.1 in the Npro tree, and subclades I.1 and I.4 in the NS3 ML tree. In conclusion, APPV among South Korean wild boars belonging to Clade I may be circulating at a higher level than among the South Korean domestic pig populations.

Phylogenetic and codon usage analysis of atypical porcine pestivirus (APPV).

Atypical porcine pestivirus (APPV) has been identified as the main causative agent for congenital tremor (CT) type A-II in piglets, which is threatening the health of the global swine herd. However, the evolution of APPV remains largely unknown. In this study, phylogenetic analysis showed that APPV could be divided into three phylogroups (I, II, and III). Phylogroups I and II included viral strains from China, while phylogroup III contained strains from Europe, North America, and Asia. Phylogroups I and II are tentatively thought to be of Chinese origin. Next, compositional property analysis revealed that a high frequency of nucleotide A and A-end codons was used in the APPV genome. Intriguingly, the analysis of preferred codons revealed that the AGA[Arg] and AGG[Arg] were overrepresented. Dinucleotide CC was found to be overrepresented, and dinucleotide CG was underrepresented. Furthermore, it was found that the weak codon usage bias of APPV was mainly dominated by selection pressures versus mutational forces. The codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses showed that the codon usage patterns of phylogroup II and III were more similar to the one of a pig than phylogroup I, suggesting that phylogroup II and III may be more adaptive to pigs. Overall, this study provides insights into APPV evolution through phylogeny and codon usage pattern analysis.

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.

Survey for pestiviruses in backyard pigs in southern Brazil.

The Pestivirus genus comprises species that affect animal health and productivity worldwide. Members of the Suidae family are hosts for classical swine fever virus (CSFV), an important pathogen tracked by the World Organization for Animal Health (OIE). However, swine are also susceptible to other pestivirus species that can result in disease or compromise CSFV detection. We searched for pestivirus infection in swine sera collected from 320 backyard pig herds in southern Brazil. We used reverse-transcription PCR primers for Bungowannah virus; atypical porcine pestivirus (APPV); and a panpestivirus pair that detects bovine viral diarrhea virus (BVDV)-1, -2, and HoBi-like pestivirus (HoBiPeV), border disease virus (BDV), and CSFV. Two samples were positive using the panpestivirus primer pair and were classified as BVDV-1d and -2a, respectively. Serum samples were tested for virus neutralization against BVDV-1a, -1b, and -2 strains, resulting in 28 (4.4%) positive samples. Of those, 16 samples had the highest titers against BVDV-1a (2), BVDV-1b (5), and BVDV-2 (9). Our results indicate that Bungowannah virus, APPV, CSFV, BDV, and HoBiPeV have not been circulating in these specific backyard swine populations. However, ruminant pestiviruses were detected and must be considered in future pestivirus control programs conducted in Brazil.

Molecular Survey and Phylogenetic Analysis of Atypical Porcine Pestivirus (APPV) Identified in Swine and Wild Boar from Northern Italy.

Atypical porcine pestivirus (APPV) is a newly recognized member of the Flaviviridae family. This novel porcine pestivirus was first described in 2015 in the USA, where it has been associated with congenital tremor type A-II in new-born piglets. APPV is widely distributed in domestic pigs in Europe and Asia. In this study, a virological survey was performed in Northern Italy to investigate the presence of APPV using molecular methods. Testing of 360 abortion samples from pig herds revealed two APPV strains from distinct provinces in the Lombardy region and testing of 430 wild boar blood samples revealed three strains, one from Lombardy and two from Emilia Romagna. The nucleotide sequencing of a fragment of the nonstructural protein 3-coding region revealed a high similarity to the previously detected European strains (Spanish, German, and Italian) of APPV.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1.

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility.

Genotyping of Pestivirus A (Bovine Viral Diarrhea Virus 1) detected in faeces and in other specimens of domestic and wild ruminants at the wildlife-livestock interface.

Pestiviruses are widespread in the world among ungulates and infect both domestic and wild animals causing severe economic losses in livestock. Bovine Viral Diarrhea Virus type 1 (BVDV-1), now re-designated as Pestivirus A, causes diseases mainly in cattle, while few data are available about infection in wild ruminants and about the role of these animals in viral maintenance and spread. In order to investigate BVDV-1 infection in domestic and wild ruminants, especially at the wildlife/livestock interface, bulk tank milk from dairy cattle and sheep and spleen from red deer, roe deer and fallow deer were analysed. Furthermore, faecal samples from Apennine chamois and from wild deer were evaluated as a suitable sample for detecting and genotyping pestiviruses. BVDV-1 RNA was found in all animal species tested but not sheep. Genotyping based on partial 5'UTR and Npro sequences detected BVDV-1a in samples from Apennine chamois, red deer, roe deer and pasture-raised cattle, while BVDV-1c was found in a faecal sample from Apennine chamois and in a spleen sample from roe deer. For the first time BVDV-1 RNA was found and genotyped from faecal samples of wild ruminants and of cattle. BVDV-1a detection in Apennine chamois, red deer, roe deer and pasture-raised cattle suggests that the eventuality of viral transmission at the wildlife/livestock interface should be carefully evaluated. BVDV subgenotype 1c was found for the first time in roe deer and Apennine chamois in Central Italy, therefore the epidemiological role of these animals and the viral ecology should be further investigated.

Isolation and Full-Length Sequence Analysis of a Pestivirus from Aborted Lamb Fetuses in Italy.

Pestiviruses are distributed worldwide and are responsible for a variety of economically important diseases. They are not very host-specific, and thus sheep can be infected by well-known pestiviruses like bovine viral diarrhea virus (BVDV) and border disease virus (BDV), as well as by other recently discovered pestivirus species. The aim of this study is to describe the isolation and characterization of four pestivirus strains detected in aborted lamb fetuses from a single farm in the Brescia province (Northern Italy). A total of twelve aborted fetuses were collected and examined. After necropsy, organs were tested for the presence of infectious agents known as potential causes of abortion (Brucella spp., Listeria spp., Coxiella burnetii, Chlamydophila spp., Mycoplasma spp., Neospora caninum, and Toxoplasma gondii), and submitted to viral identification by isolation on Madin Darby bovine kidney (MDBK) cell culture and by PCR assay for Schmallenberg virus and pan-pestivirus RT-PCR real time assay. Three viral strains (Ovine/IT/1756/2017, Ovine/IT/338710-2/2017, and Ovine/IT/338710-3/2017) were isolated in the absence of cytopathic effects (CPEs) in cell cultures and identified with RT-PCR. Another pestivirus strain (Ovine/IT/16235-2/2018) was detected by PCR, but was not successfully isolated. Complete sequence genomic data of the three isolated viruses showed that they were highly similar, differed genetically from known pestivirus species, and were closely related to classical swine fever virus (CSFV). Beyond the identification of new ovine pestiviruses, this study indicates that a systematic diagnostic approach is important to identify the presence and map the distribution of both known and emerging pestiviruses.

Long-Term Circulation of Atypical Porcine Pestivirus (APPV) within Switzerland.

In 2015, a new pestivirus was described in pig sera in the United States. This new "atypical porcine pestivirus" (APPV) was later associated with congenital tremor (CT) in newborn piglets. The virus appears to be distributed worldwide, but the limited knowledge of virus diversity and the use of various diagnostic tests prevent direct comparisons. Therefore, we developed an APPV-specific real-time RT-PCR assay in the 5'UTR of the viral genome to investigate both retro- and prospectively the strains present in Switzerland and their prevalence in domestic pigs. Overall, 1080 sera obtained between 1986 and 2018 were analyzed, revealing a virus prevalence of approximately 13% in pigs for slaughter, whereas it was less than 1% in breeding pigs. In the prospective study, APPV was also detected in piglets displaying CT. None of the samples could detect the Linda virus, which is another new pestivirus recently reported in Austria. Sequencing and phylogenetic analysis revealed a broad diversity of APP viruses in Switzerland that are considerably distinct from sequences reported from other isolates in Europe and overseas. This study indicates that APPV has already been widely circulating in Switzerland for many years, mainly in young animals, with 1986 being the earliest report of APPV worldwide.

Pathogen spread and globalization: The case of Pestivirus heterogeneity in southern Italy.

Pestiviruses are responsible for widespread diseases affecting cattle, pigs and other ruminants, presenting a wide range of clinical manifestations, with significant impact on animal production. Given the recent various reports of a relatively high number of new strains and atypical genomic variants, in the present study, ninety-seven genomic sequences from southern Italy have been evaluated applying the palindromic nucleotide substitutions method, based on 5'-UTR secondary structure alignment and computing genetic distance among strains in the internal ribosome entry site. Sequence analysis revealed a highly heterogeneous virus population, indicating the introduction of virus variants of Bovine viral diarrhea virus and Border disease virus species from foreign countries. The application of different analytical procedures was useful to avoid interpretation difficulties. Circulation of heterogeneous virus populations showed the need for more accurate epidemiological investigations and stringent veterinary controls to protect animal health and welfare.