Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.Pestiviruses are animal pathogens that are recognized as a separate genus of the family Flaviviridae, which also includes the genera Flavivirus and Hepacivirus (hepatitis C viruses [HCV]), as well as the unassigned GB viruses (32). Pestiviruses are widely used as a surrogate model for studying HCV, which grows poorly in available cell culture systems. Persistent HCV infections are a major cause of liver cirrhosis and hepatocellular carcinoma in humans worldwide.The pestiviral genome is a positive-stranded RNA of 12.3 kb. It is translated into a large polyprotein, which is cotranslationally and posttranslationally processed by viral and cellular proteases. The order of proteins in the polyprotein is NH 2 -N pro -C-E rns -E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B-COOH. The autoprotease N pro generates its C terminus and the N terminus of the downstream core protein C. The proteolytic releases of the structural glycoproteins E rns (RNase secreted), E1, E2, and p7 are mediated by cellular signal peptidases. The nonstructural protein 4A (NS4A)-dependent chymotrypsin-like serine protease in NS3 mediates processing in the NS region downstream of NS3 (32). The mechanism of NS2-3 cleavage was hitherto unknown and is the subject of this study (see below).The pestivirus bovine viral diarrhea virus (BVDV) can establish lifelong persistent infections in animals, which become the primary sources for the horizontal sp...

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.The Flaviviridae family comprises the genera Flavivirus, Pestivirus, and Hepacivirus; the latter includes the human pathogen Hepatitis C virus (HCV) (13). With an estimated 200 million cases of chronic infections worldwide, HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. Due to their close relationship, pestiviruses, especially bovine viral diarrhea virus (BVDV), represent a widely used surrogate system for HCV.The single-stranded RNA genome of BVDV is of positive polarity and has a length of 12.3 kb. Gene expression occurs via translation of one polyprotein which is processed by cellular and viral proteases giving rise to 12 mature proteins (18). Processing of nonstructural protein 2-3 (NS2-3) into NS2 and NS3 is exerted by a recently characterized vital cysteine autoprotease located in NS2 (17). Interestingly, this enzyme is distantly related to the HCV NS2-3 protease which mediates the analogous cleavage in the HCV polyprotein (11,14,18). The NS4A-dependent chymotrypsin-like serine protease in NS3 catalyzes four processing events in the viral polyprotein (29,32,33). Moreover, NS3 has helicase and NTPase activity (28, 31). The enzymatic functions of NS3 are essential for viral RNA replication which is accomplished by a replication complex (replicase) containing NS3 and four other NS proteins including NS5B, the viral RNA-dependent RNA polymerase (34), as essential constituents (3, 12). The NS2 protease-mediated cleavage of NS2-3 is essential for replication of BVDV, since its cleavage product, NS3, cannot be functionally replaced by NS2-3 in the viral replicase (17). Uncleaved NS2-3 plays a c...

Efficient proteolytic release of nonstructural protein 3 (NS3) from the viral polyprotein is considered to be crucial for the cytopathogenicity of pestiviruses. Here we describe a novel cytopathogenic (cp) bovine viral diarrhea virus strain (BVDV CP8) with a complex insertion composed of viral and cell-derived sequences, including two fragments of the cellular J-domain protein Jiv (J-domain protein interacting with viral protein) located in the N-terminal region of the polyprotein. BVDV CP8 expresses a Jiv fusion protein of 513 amino acids in addition to a complete set of viral proteins. This protein has the capacity to induce NS2-3 cleavage in trans. Accordingly, CP8 is a representative of a novel type of cp pestivirus with a cp-specific mutation located outside of the NS2-3 gene.The pestiviral genome is a 12.3-kb single-stranded RNA of positive polarity (5; A. Renard, D. Dino, and J. Martial, 1987, European patent application 86870095.6:publication 02.08672) and encodes the polyprotein NH 2 -N pro -C-E rns -E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B-COOH, which is processed by cellular and virus-encoded proteases (14). The autoprotease N pro generates its own C terminus and thereby the N terminus of the capsid protein (C) (25,29,33). The three envelope glycoproteins E rns , E1, and E2, as well as p7, are released from the polyprotein by cellular proteases (10,12,27). A serine protease located in nonstructural protein 3 (NS3) catalyzes all processing events downstream of NS3 (11,32,34).In the course of lethal mucosal disease, cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains evolve from noncytopathogenic (noncp) ancestors (14, 18). One striking difference between cp and noncp BVDV concerns the expression of free NS3; free NS3 is expressed only by cp BVDV, while only unprocessed NS2-3 can be detected in noncp BVDV-infected cells (9, 21). Mutations responsible for the expression of NS3 include insertions of cellular protein-coding sequences (14,18). A cell-derived insertion termed "Jiv" (formerly cIns; see below) was identified in the NS2 genes of several cp pestiviruses, and its presence was linked to NS2-3 cleavage and cytopathogenicity (2,15,16,18,20,23). The corresponding cellular chaperone protein Jiv (J-domain protein interacting with viral protein) has the capacity to induce NS2-3 cleavage in trans (24). Here we describe a cp BVDV isolate with a Jiv-containing insertion located outside of NS2-3.Genome structure of BVDV strain CP8. Strains CP8 and NCP8 were both isolated from the same animal suffering from mucosal disease and thus represent a so-called virus pair (7). Our first step in the analysis of these viruses was a comparative Northern blot analysis. Five micrograms of total RNA isolated from CP8-and NCP8-infected MDBK cells was separated by denaturing agarose gel electrophoresis and after being blotted onto a membrane was hybridized against different [␣-32 P] dCTP-labeled cDNA probes. In the RNA of NCP8-infected cells, a BVDV-specific probe detected a band of 12.3 kb, the size expected for a sta...

Blood plasma and hepatic parameters were identified that describe the differences between metabolically robust or vulnerable dairy cows grouped according to their past health status. Data from a field study on dairy cows were used from which metabolically challenged dairy cows were selected that had a milk fat percentage of >4.5 mg/g and a fat to protein ratio of >1.5 in their previous early lactation. The selected cows were either classified as metabolically robust or vulnerable based on the occurrence of various metabolic and (re)production disorders in their previous lactations. Blood and liver tissue samples were collected in week 3 ante partum (a.p.) (-3 wk), in week 4 (+4 wk) and in week 13 (+13 wk) post-partum (p.p.). Plasma concentrations of metabolites and hormones and mRNA expression of genes involved in metabolic pathways in the liver were used as variables for a two-group discriminant analysis (DA). Average discriminant scores (centroids) were different (p < 0.05) in -3 wk, +4 wk and in +13 wk. In -3 wk, significant variables that best explained the differences between metabolically robust and vulnerable cows were parity, plasma triglycerides, glucose and mRNA abundance of carnitine palmitoyltransferase 2 (CPT2). In addition, based on the classification matrix, 69% of the dairy cows were correctly classified. In +4 wk, identified significant parameters were parity, plasma glucose and urea, and 67% of the cows were correctly classified. In +13 wk, significant variables that explained the differences between the groups were parity, mRNA abundance of acyl-CoA synthetase long-chain 1 and CPT1, and 66% of the cows were correctly classified. In conclusion, the identified variables may distinguish from metabolically challenged cows, those cows that had a poorer health performance in their previous lactations.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) represent major healthcare concerns. The role of wildlife in the epidemiology of these bacteria is unclear. The purpose of this study was to determine their prevalence in wild boars in Germany and to characterize individual isolates. A total of 375 fecal samples and 439 nasal swabs were screened for the presence of ESBL-/AmpC-E. coli and MRSA, respectively. The associations of seven demographic and anthropogenic variables with the occurrence of ESBL-/AmpC-E. coli were statistically evaluated. Collected isolates were subjected to antimicrobial susceptibility testing, molecular typing methods, and gene detection by PCR and genome sequencing. ESBL-/AmpC-E. coli were detected in 22 fecal samples (5.9%) whereas no MRSA were detected. The occurrence of ESBL-/AmpC-E. coli in wild boars was significantly and positively associated with human population density. Of the 22 E. coli, 19 were confirmed as ESBL-producers and carried genes belonging to blaCTX-M group 1 or blaSHV-12. The remaining three isolates carried the AmpC-β-lactamase gene blaCMY-2. Several isolates showed additional antimicrobial resistances. All four major phylogenetic groups were represented with group B1 being the most common. This study demonstrates that wild boars can serve as a reservoir for ESBL-/AmpC-producing and multidrug-resistant E. coli.