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 2.9x10(-3) subs/site/year (95% HPD: 1.5-4.6x10(-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

Proposed Revision to the Taxonomy of the Genus Pestivirus; Family Flaviviridae

We propose the creation of seven new species in the genus Pestivirus (family Flaviviridae) in addition to the four existing species, and naming species in a host-independent manner using the format Pestivirus X. Only the virus species names would change; virus isolates would still be referred to by their original names. The original species would be re-designated as Pestivirus A (original designation Bovine viral diarrhea virus 1), Pestivirus B (Bovine viral diarrhea virus 2), Pestivirus C (Classical swine fever virus) and Pestivirus D (Border disease virus). The seven new species (and example isolates) would be Pestivirus E (pronghorn pestivirus), Pestivirus F (Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H (Hobi-like pestivirus), Pestivirus I (Aydin-like pestivirus), Pestivirus J (rat pestivirus) and Pestivirus K (atypical porcine pestivirus). A bat-derived virus and pestiviruses identified from sheep and goat (Tunisian sheep pestiviruses), which lack complete coding region sequences, may represent two additional species

PLASMD BEARING A CDNA COPY OF THE GENOME OF BOVINE VIRAL DIARRHEA VIRUS, CHIMERIC DERIVATIVES THEREOF, AND METHOD OF PRODUCING AN INFECTIOUS BOVINE WRAL DARRHEAVIRUS USING SAD PLASMID

A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a vector in many advantageous applications

In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD-420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.Fil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Faverin, Claudia. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Boynak, Natalia. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vena, M. M.. Biogénesis-Bagó S. A.; ArgentinaFil: Zabal, O.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentin

Acute BVDV infection inhibits expression of interferon-stimulated genes during pregnancy recognition in bovine endometrium

Bovine viral diarrhea virus (BVDV) can evade host detection by downregulation of interferon signaling pathways. Infection of cows with noncytopathic (ncp) BVDV can cause early embryonic mortality. Upregulation of type I interferon stimulated genes (ISGs) by blastocyst-secreted interferon tau (IFNT) is a crucial component of the maternal recognition of pregnancy (MRP) in ruminants. This study investigated the potential of acute BVDV infection to disrupt MRP by modulating endometrial ISG expression. Endometrial cells from 10 BVDV-free cows were cultured and treated with 0 or 100 ng/ml IFNT for 24 h in the absence or presence of ncpBVDV infection to yield four treatment groups: CONT, ncpBVDV, IFNT, or ncpBVDV+IFNT. ncpBVDV infection alone only upregulated TRIM56, but reduced mRNA expression of ISG15, MX2, BST2, and the proinflammatory cytokine IL1B. As anticipated, IFNT treatment alone significantly increased expression of all 17 ISGs tested. In contrast to the limited effect of ncpBVDV alone, the virus markedly inhibited IFNT-stimulated expression of 15 ISGs tested (ISG15, HERC5, USP18, DDX58, IFIH1, IFIT1, IFIT3, BST2, MX1, MX2, RSAD2, OAS1Y, SAMD9, GBP4, and PLAC8), together with ISG15 secreted protein. Only TRIM56 and IFI27 expression was unaltered. IL1B expression was reduced by the combined treatment. These results indicate that acute ncpBVDV infection may decrease uterine immunity and lead to MRP failure through inhibition of IFNT-stimulated endometrial ISG production. This in turn could reduce fertility and predispose cows to uterine disease, while evasion of the normal uterine immune response by ncpBVDV may contribute to maintenance and spreading of this economically important disease

The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.)

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway

Homologous recombination in pestiviruses: Identification of three putative novel events between different subtypes/genogroups

AbstractViruses from the genus Pestivirus of the family Flaviviridae have a non-segmented, single-stranded RNA genome and can cause diseases in animals from the order Artiodactyla. Homologous recombination is rarely reported in this virus family. To detect possible recombination events, all complete pestivirus genomes that are available in GenBank were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Three putative recombinant viruses derived from recombination from different pestivirus subtypes/genogroups were detected: Bovine viral diarrhea virus 1 (BVDV-1) strain 3156, BVDV-2 strain JZ05-1 and Classical swine fever virus (CSFV) strain IND/UK/LAL-290. The present study demonstrated that the pestivirus classification cannot be based only on the analysis of one fragment of the genome because genetic conversions can lead to errors. The designation of the recombinant forms (RF) provides a more informative structure for the nomenclature of the genetic variant. The present work reinforces that homologous recombination occurs in pestivirus populations under natural replication and describes the first evidence of recombination in BVDV-2