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Bovine viral diarrhea virus (BVDV) is the causative agent of the bovine viral diarrhea-mucosal disease, which is a leading cause of economic losses in the cattle industry worldwide. To date, many underlying mechanisms involved in BVDV-host interactions remain unclear, especially the functions of long noncoding RNAs (lncRNAs). In our previous study, the lncRNA expression profiles of BVDV-infected Madin-Darby bovine kidney (MDBK) cells were obtained by RNA-seq, and a significantly downregulated lncRNA IALNCR targeting MAPK8/JNK1 (a key regulatory factor of apoptosis) was identified through the lncRNA-mRNA coexpression network analysis. In this study, the function of IALNCR in regulating apoptosis to affect BVDV replication was further explored. Our results showed that BVDV infection-induced downregulation of the lncRNA IALNCR in the host cells could suppress the expression of MAPK8/JNK1 at both the mRNA and protein levels, thereby indirectly promoting the activation of caspase-3, leading to cell-autonomous apoptosis to antagonize BVDV replication. This was further confirmed by the small interfering RNA (siRNA)-mediated knockdown of the lncRNA IALNCR . However, the overexpression of the lncRNA IALNCR inhibited apoptosis and promoted BVDV replication. In conclusion, our findings demonstrated that the lncRNA IALNCR plays an important role in regulating host antiviral innate immunity against BVDV infection. IMPORTANCE Bovine viral diarrhea-mucosal disease caused by BVDV is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BVDV-host interactions are complex. To date, most studies focused only on how BVDV escapes host innate immunity. By contrast, how the host cell regulates anti-BVDV innate immune responses is rarely reported. In this study, a significantly downregulated lncRNA , with a potential function of inhibiting apoptosis (inhibiting apoptosis long noncoding RNA, IALNCR ), was obtained from the lncRNA expression profiles of BVDV-infected cells and was experimentally evaluated for its function in regulating apoptosis and affecting BVDV replication. We demonstrated that downregulation of BVDV infection-induced lncRNA IALNCR displayed antiviral function by positively regulating the MAPK8/JNK1 pathway to promote cell apoptosis. Our data provided evidence that host lncRNAs regulate the innate immune response to BVDV infection.

Background: Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the pestivius virus group. BVDV is responsible for significant economic loss in cattle industry worldwide because of reducing reproductive performance, increasing incidence of other diseases and mortality among young stock. The core (C) protein of the Flaviviridae family member is involved in host antiviral immune response through activation of related signaling pathways that affect the viral replication. However, the influence of C protein-interaction partners in BVDV infections is poorly defined., Methods: To explore C-protein-interacting partners, yeast two-hybrid was used to screen the interaction protein of C protein using bovine peripheral blood mononuclear cell (PBMC) cDNA library. The co-immunoprecipitation and confocal assays were manipulated to determine the interaction between potential partners and C protein. Knockdown and overexpression of the partner were used to examine whether the C-protein-interacting partner plays a role in BVDV proliferation and virulence. Meanwhile, qRT-PCR and western blot assays were used to investigate the effect of C protein and C-protein-interacting partner on the immune response of host cells., Results: We identified protein inhibitor of activated STAT 4 (PIAS4) as a novel interacting partner of the BVDV C protein. Co-immunoprecipitation and confocal assays demonstrated a strong interaction between C protein and PIAS4. Silencing of PIAS4 with small interfering RNA suppressed C protein expression and BVDV growth, while overexpression of PISA4 increased C protein expression and BVDV growth. The overexpression of PIAS4 increased the cell apoptosis. Meanwhile, the expressions of STAT4, SOCS3, IFITM, IFN-α were negatively regulated by the expression of PIAS4. The expression of C protein suppressed the antiviral proteins expression, and the inhibition effect was enhanced by interaction of PIAS4 and C protein. These results highlighted the beneficial properties of cellular PIAS4 for BVDV protein expression and growth., Conclusions: This study provides reliable clues for understanding the roles of PIAS4 in the regulation of BVDV growth.

Bovine viral diarrhoea (BVD) is an important disease of cattle, with significant impacts on animal health and welfare. The wide host range of the causative pestiviruses may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVD eradication campaigns. It is likely that the quasispecies nature of these RNA viruses contributes to their interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in persistently infected (PI) animals is, therefore, essential for studies of virus transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from the serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250 bp paired-read technology produced full-length virus consensus sequences from both sources and demonstrated the quasispecies diversity of this pestivirus A genotype 1a field strain within serum and after culture. We report the distribution and diversity of over 800 SNPs and provide evidence for a loss of diversity after only three passages in cell culture, implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies. Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples.

Microgels that can generate antipathogenic levels of hydrogen peroxide (H 2 O 2 ) through simple rehydration in solutions with physiological pH are described herein. H 2 O 2 is a widely used disinfectant but the oxidant is hazardous to store and transport. Catechol, an adhesive moiety found in mussel adhesive proteins, was incorporated into microgels, which generated 1-5 mM of H 2 O 2 for up to four days as catechol autoxidized. The sustained release of low concentrations of H 2 O 2 was antimicrobial against both gram-positive (Staphylococcus epidermidis) and gram-negative (Escherichia coli) bacteria and antiviral against both non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV). The amount of released H 2 O 2 is several orders of magnitude lower than H 2 O 2 concentration previously reported for antipathogenic activity. Most notably, these microgels reduced the infectivity of the more biocide resistant non-envelope virus by 3 log reduction value (99.9% reduction in infectivity). By controlling the oxidation state of catechol, microgels can be repeatedly activated and deactivated for H 2 O 2 generation. These microgels do not contain a reservoir for storing the reactive H 2 O 2 and can potentially function as a lightweight and portable dried powder source for the disinfectant for a wide range of applications. STATEMENT OF SIGNIFICANCE: Researchers have designed bioadhesives and coatings using the adhesive moiety catechol to mimic the strong adhesion capability of mussel adhesive proteins. During catechol autoxidation, hydrogen peroxide (H 2 O 2 ) is generated as a byproduct. Here, catechol was incorporated into microgels, which can generate millimolar levels of H 2 O 2 by simply hydrating the microgels in a solution with physiological pH. The sustained release of H 2 O 2 was both antimicrobial and antiviral, inactivating even the more biocide resistant non-enveloped virus. These microgels can be repeatedly activated and deactivated for H 2 O 2 generation by incubating them in solutions with different pH. This simplicity and recyclability will enable this biomaterial to function as a lightweight and portable source for the disinfectant for a wide range of applications., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

The causes of bovine respiratory disease complex (BRDC) are multifactorial and include infection with both viral and bacterial pathogens. Host factors are also involved as different breeds of cattle appear to have different susceptibilities to BRDC. Infection with bovine pestiviruses, including bovine viral diarrhea virus 1 (BVDV1), BVDV2 and 'HoBi'-like viruses, is linked to the development of BRDC. The aim of the present study was to compare the growth of different bovine pestiviruses in primary testicle cell cultures obtained from taurine, indicine and mixed taurine and indicine cattle breeds. Primary cells strains, derived from testicular tissue, were generated from three animals from each breed. Bovine pestivirus strains used were from BVDV-1a, BVDV-1b, BVDV-2a and 'HoBi'-like virus. Growth was compared by determining virus titers after one passage in primary cells. All tests were run in triplicate. Virus titers were determined by endpoint dilution and RT-qPCR. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey's Multiple Comparison Test (P˂0.05). Significant differences in virus growth did not correlate with cattle breed. However, significant differences were observed between cells derived from different individuals regardless of breed. Variation in the replication of virus in primary cell strains may reflect a genetic predisposition that favors virus replication.

The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1β, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic-polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bovine viral diarrhea viruses (BVDV) are arguably the most important viral pathogen of ruminants worldwide and can cause severe economic loss. Clinical symptoms of the disease caused by BVDV range from subclinical to severe acute hemorrhagic syndrome, with the severity of disease being strain dependent. These viruses are classified as members of the Pestivirus genus of the Flaviviridae. BVDV are considered primarily a pathogen of cattle but can infect most ruminant species. The virus particle consists of a lipid bilayer membrane surrounding the encapsidated genomic RNA. Inserted in the outer membrane are two virus-encoded glycoproteins that contain the major antigenic determinants of the virus as well as receptor binding and cell fusion functions. A third glycoprotein is weakly associated with the virion, but also possesses unique features that play important roles in suppression of innate immunity. The viral proteins are encoded in a single, large open reading frame. The viral proteins are proteolytically cleaved from the polyprotein by different proteases. The structural proteins are processed by cellular signal peptidases while the processing of the nonstructural proteins is by the viral serine protease. The virus is assembled and matures in the endoplasmic reticulum and golgi bodies of the cell. The virus is released via exocytosis, where viral proteins are not exposed on the surface of the cell., (Published by Elsevier Ltd.)

In this study, the antiviral effects of bovine interferon-tau (boIFN-tau) on bovine viral diarrhea virus (BVDV) were examined in vitro and in vivo. In the in vitro experiments, the replication of cytopathic and non-cytopathic BVDV was inhibited in the bovine cells treated with boIFN-tau. The replication of BVDV was completely suppressed by boIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the effect of boIFN-tau on virus propagation in cattle persistently infected (PI) with non-cytopathic BVDV, boIFN-tau was subcutaneously administered to PI cattle at 10(5) U/kg or 10(6) U/kg body weight 5 times per week for 2 weeks. No physical abnormality such as depression was observed in the cattle during the experiment. The mean BVDV titers in the serum of the PI cattle decreased slightly during the boIFN-tau administration period with the dose of 10(6) U/kg. However, the BVDV titers in the serum returned to the pre-administration level after the final boIFN-tau administration. These results suggest that boIFN-tau demonstrates an anti-BVDV effect, reducing the BVDV level in serum transiently when injected into PI cattle.

The incidence of contamination of cell strains used in biological and virological studies and of fetal calf sera (FCS) manufactured by Russian and foreign companies used for cell culturing with noncytocidal bovine viral diarrhea virus (BVDV; Pestivirus, Flaviviridae) was analyzed. The virus was detected by reverse transcription PCR and indirect immunofluorescence with monoclonal antibodies to BVDV virion envelope glycoprotein in 25% of 117 cell strains and 45% of 35 tested FCS lots. The virus multiplied and persisted in a wide spectrum of human cell strains and in monkey, swine, sheep, rabbit, dog, cat, and other animal cells. The levels of BVDV genome RNA in contaminated cell cultures reached 10(2)-10(3) g-eq/cell and in serum samples 10(3)-10(7) g-eq/ml. These facts necessitate testing of cells and FCS for BVDV reproduced in cells without signs of infection detectable by light microscopy. The molecular mechanisms of long-term virus persistence in cells without manifestation of cell destruction are unknown.

The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC(50)) of the initial lead, the thiazolidinone BMS-824, was ~10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC(50), the drug concentration required to reduce cell growth by 50% (CC(50)), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. High-performance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus.Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2)oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus.Virus was not isolated from any samples in treatment group 1.As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.

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.

In this paper we evaluated the distributional effects on actors in the milk market of a hypothetical programme to eradicate bovine viral diarrhoea (BVD) from the Scottish dairy herd. With this in mind, we applied an economic welfare methodology which utilizes data on price, on output quantity, on elasticities of supply and demand and on simulated cost and yield effects of an eradication programme. Our analysis is based on Markov-chain Monte Carlo simulation of BVD spread in the dairy herd. We found that consequent upon the eradication of the disease milk yield per cow increased for all herd sizes in Scotland whereas milk price received by farmers fell. Consequently, milk consumers gained around pound11 million in discounted economic surplus and producers with infected herds gained around pound39 million whereas producers with un-infected herds lost around pound2 million in discounted surplus. On balance, however, the eradication programme generated around pound 47 million in discounted economic gain for Scotland. We found that the results are sensitive to changes in yield gains made by owners of the infected herd.

Objective: To evaluate economic effects and health and performance of the general cattle population after exposure to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) in a feedlot., Animals: 21,743 high-risk calves from the southeastern United States., Procedures: PI status was determined by use of an antigen-capture ELISA (ACE) and confirmed by use of a second ACE, reverse transcriptase-PCR assay of sera, immunohistochemical analysis, and virus isolation from sera. Groups with various amounts of exposure to BVDV PI cattle were used. After being placed in the feedlot, identified PI cattle were removed from 1 section, but PI cattle remained in another section of the feedlot. Exposure groups for cattle lots arriving without PI animals were determined by spatial association to cattle lots, with PI animals remaining or removed from the lot., Results: 15,348 cattle maintained their exposure group. Performance outcomes improved slightly among the 5 exposure groups as the risk for exposure to BVDV PI cattle decreased. Health outcomes had an association with exposure risk that depended on the exposure group. Comparing cattle lots with direct exposure with those without direct exposure revealed significant improvements in all performance outcomes and in first relapse percentage and mortality percentage in the health outcomes. Economic analysis revealed that fatalities accounted for losses of $5.26/animal and performance losses were $88.26/animal., Conclusions and Clinical Relevance: This study provided evidence that exposure of the general population of feedlot cattle to BVDV PI animals resulted in substantial costs attributable to negative effects on performance and increased fatalities.

The complete annotation of the cattle genome allows reliable protein identification by tandem mass spectrometry (MS(2)) and greatly facilitates proteomics. Previously, we reported that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins, involved in immune function of professional antigen-presenting cells (APC) that have been significantly altered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II-related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that cp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation to immunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activated macrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

A 14-month-old heifer with a 17-day history of unresponsive bloody diarrhea was necropsied. There were focal, pink-red erosions of the nares and hard palate; ulcers and fissures of the tongue; and multiple ulcerative lesions of the alimentary canal. Interdigital skin of both rear limbs was ulcerated and bleeding; and the margins of the vulva contained punctiform red ulcers. The gross lesions were consistent with mucosal disease. Histopathology and laboratory testing ruled out rinderpest, foot-and-mouth disease, and vesicular stomatitis, and identified bovine virus diarrhea virus to be the cause of this disease. Lesions of the vulva similar to those seen in some stages of infectious pustular vulvovaginitis were negative for bovine herpesvirus-1 and tested positive for bovine viral diarrhea virus antigen by immunohistochemistry.

In the bovine synepitheliochorial placenta key sites of fetal-maternal interaction are placentomes consisting of maternal caruncles interdigitating with fetal cotyledons. The aim of this study was to establish an epithelial cell line from caruncles of pregnant cows and to develop a model to study restricted trophoblast invasion, pathogenesis of pregnancy associated diseases and pathways of infection and transport. Primary epithelial cells were isolated, successfully subcultured for 32 passages and cryopreserved at various stages. The cultures were termed bovine caruncular epithelial cell line-1 (BCEC-1). Cytokeratin, zonula occludens-1 protein and vimentin but neither alpha-smooth muscle actin nor desmin were detected by immunofluorescence performed every 5 (+/-1) passages. These results were confirmed by Western blotting. BCEC-1 were then cultured either without matrix or on fibronectin or collagen coated Transwell polyester membrane inserts, respectively, enabling separate access to the basal or apical epithelial compartments. Transmission and scanning electron microscopy of BCEC-1 revealed ultrastructural features also observed in vivo, such as apical microvilli and junctional complexes. Transepithelial electrical resistance (TEER) was measured regularly and revealed an increase with advancing confluence in all cultures. Cultures on coated inserts reached confluence and corresponding TEER-levels at an earlier stage. In addition, the cells were tested negative for bovine virus diarrhoea (BVD) virus, but were permissive for the virus. In conclusion, the BCEC-1 cell line retained characteristics of maternal caruncular epithelial cells as observed in vivo and in primary cell cultures and thus will be a highly useful tool for future studies of pathways of invasion, fetal-maternal communication, transport and infection.

Contamination of cell cultures with adventitious viruses may pose serious risks for virology diagnosis, research and vaccine production. This article reports the selection and characterization of three cell lines resistant to bovine viral diarrhea virus (BVDV), a major contaminant of cell cultures. The resistant cells were obtained from canine (MDCK), swine (PK-15) and rabbit (RK-13) parental cell lines by expanding and cloning cells that survived lytic infection with BVDV. All three selected cell lines were resistant to three standard BVDV strains and to 10 field isolates as demonstrated by immunofluorescence for viral antigens and by co-cultivation with susceptible cells. Inoculation of selected cells with BVDV (10 TCID50/cell) resulted in frequencies of infection of <10(-5) (MDCK-R, PK-15R) and 3.3 x 10(-4) (RK-13R). Comparing to the parental cell lines, these cells were >10,000-, >20,000- and 600-fold less susceptible to BVDV, respectively. Inoculation of resistant cells with BVDV in the presence of polyethylene-glycol increased the frequency of infection in the order of >437-, >346- and 87-fold, respectively, indicating that resistance is probably due to a block in viral entry. Nevertheless, each resistant cell line retained the susceptibility to three other viruses that replicate in the parental cells. Thus, these cells may be useful for many virology purposes, with a very low risk of BVDV contamination.

A series of amino acid esters bearing coumarin (3-15) were synthesized and evaluated, in vitro, against HIV-1, and bovine viral diarrhea virus (BVDV). The in vitro cytotoxicity of 3-10 and 12 was assayed against a panel of tumor cell lines consisting of CD4 human T-cells. Compound 14 showed inhibition of HIV-1 with EC50 > 1.6 microg mL(-1), meanwhile compound 9 exhibited activity against leukaemia (MT4) with CC50 = 24 micromol L(-1).

For the identification of herds with cattle persistently infected (PI) with bovine viral diarrhea virus, 1,272 animals from 20 herds were subjected to serum neutralizing (SN) test using the Nose strain and virus isolation. Eighteen PI cattle were detected from 5 herds. On the phylogenetic tree based on the nucleotide sequences of the 5' untranslated region, the isolates from the PI cattle were classified into genotypes-1a or -1b. Of 3 unvaccinated calves aged 6 to 12 months selected from each herd, the probabilities of obtaining 2 or more non-PI cattle with SN antibody titers of 64 or more (P(SN)), one or more PI cattle (P(VI)), and either of the conditions (P(Total)) were calculated using the hypergeometric probability model. P(Total) for the 5 herds with PI cattle was 1.000. P(SN) for 3 herds with many PI cattle within the selected age group was as low as 0.500 or less, and P(VI) was as high as 0.886 or more. P(SN) in the 2 other herds with few PI cattle was 1.000, and P(VI) was as low as 0.375 or less. P(Total) in 13 of 15 herds without PI cattle was 0.000, and was 0.714 or 0.774 for the 2 other herds. These results suggest that herds with PI cattle can be predicted with high accuracy when both SN test and virus isolation are performed on only 3 unvaccinated calves aged 6 to 12 months selected from a herd.

The effect of environmental factors and management routines on the risk of diarrhoea, respiratory disease and other infectious diseases was investigated in 3081 heifer calves 0-90 days old in 122 Swedish dairy herds. The farmers kept records on cases of diseases in their heifer calves and in addition, project veterinarians clinically examined all calves every 2-3 months. At each visit, the veterinarians also measured the ammonia concentration and relative air humidity in the housing facilities for the calves. The cleanliness of the animals and their environment was recorded as a measure of the hygienic status of the farm. The presence or absence of draught (i.e. wind velocity>0.5 m/s) was recorded twice during the study period. The effect of these factors, as well as the placing of the calf pens, the nature of the pen walls, air volume per animal, management factors (such as the status of the caretaker and feeding routines) and presence or absence of a bovine viral diarrhoea virus (BVDV) infection in the herd, was evaluated by means of a two-level variance component logistic model. The placing of calf pens along an outer wall was significantly associated with the risk of diarrhoea (odds ratio (OR): 1.92, P<0.01). The risk for respiratory disease was significantly associated with an ammonia concentration below 6 ppm (OR: 0.42, P<0.05) while the odds ratio for moderately to severely increased respiratory sounds was significantly associated with a BVDV infection in the herd (OR: 2.39, P<0.05) and draught (OR: 3.7, P<0.02). Absence of draught was significantly associated with the risk for infectious diseases other than diarrhoea and respiratory disease (OR: 0.42, P<0.01).

The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.

Objectives: Investigation of natural ethnopharmacologic extracts exhibiting antiviral potential may lead to the discovery of new therapeutics for the treatment of chronic viral hepatitis infections. Traditional Korean medicinal herbs have been identified that exhibit potency against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Research on the antiviral potential of naturally derived extracts is facilitated through the use of appropriate animal and liver cell culture models for these hepatotrophic pathogens. Objectives of this study were to demonstrate antiviral activity of an aqueous extract of herbal formulation KYH-1 in surrogate in vitro assays for HBV and HCV and identify mechanisms of action., Methods: Antiviral potency of KYH-1 was measured in tissue culture systems that support replication of the woodchuck hepatitis virus (WHV), and the bovine viral diarrhea virus (BVDV). These assays serve as surrogate models for HBV and HCV, respectively. A recombinant HBV polymerase gene expression assay was used to define a molecular target., Results: KYH-1 exhibited potent antiviral activity against WHV and to a lesser extent against BVDV. KYH-1 and its constituent components inhibited HBV polymerase priming in vitro. Additionally, KYH-1 suppressed HBV replication in a human hepatoblastoma cell line., Conclusion: Evaluation of naturally derived products for antiviral activity against HBV and HCV in standardized surrogate assays provides a scientific basis for potential use as complementary or alternative medicines. This study provides significant results justifying preclinical evaluation of KYH-1 as an antiviral therapy for HBV infections.

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.

Bovine viral diarrhea virus (BVDV) with deletions in the 5'-nontranslated region (5'-NTR) were tested for their suitability as live BVD vaccines. Firstly, the genetic stability of the mutants was established by culturing over 15 passages in bovine cells. Secondly, two deletion mutants and the parent strain CP7-5A were characterised with respect to in vivo replication competence, attenuation and induction of protective immunity against BVDV. Naïve calves (n = 5 per group) were inoculated with mutants d2-31 and d5-57 or CP7-5A and 5 weeks later, a challenge with the BVDV type 1 strain New York was performed. The mutants were found to be genetically and phenotypically stable. Moreover, the results indicate that the mutants were attenuated with regard to effects including pyrexia and drop in leucocyte counts. Infection with the mutants induced moderate to high titers of BVDV neutralizing antibodies and completely prevented viremia after challenge infection with a heterologous BVDV strain. Taken together, the 5'-NTR deletion mutants combine a good safety profile with good efficacy and are therefore well suited as candidate live vaccines.

In vitro experiments studied the antiviral activity of 11 different drugs against viruses of bovine infective rhinotracheitis (BIRT) and bovine viral diarrhea (BVD). The 50% inhibiting concentrations of the test agents were determined in the monolayers of MDBK and KCT cell cultures. Only did phosprenyl show a virucidal activity against BIRT virus. All the tested drugs significantly inhibited the reproduction of BIRT virus in the sensitive MDBK cell cultures. Thus, bromuridin, acyclovir, ribavirin and methisazonum inhibited the virus by > or = 100,000 times; liposomal ribavirin, gossypolum, anandinum, polyprenolum, phosprenyl, by 1000-10,000 times; eracond and argovit, by 100 times. In experiments on BVD virus, the cultured KCT cells displayed the antiviral activity of bromuridin, phosprenil, polyprenolum, methisazonum, acyclovir, gossypolum, argovit, and ribavirin (in two variants), which caused a statistically significant (100-10,000-fold) decrease in the productive activity of this virus. Eracond and anandid proved to be ineffective.

The objectives of the present work were to estimate the level of bovine viral diarrhoea virus (BVDV) infection in cattle herds at the different Lithuanian districts and to determine factors influencing the course of BVDV infection. The studies were explored in 147 intensive dairy cattle breeding herds from 27 different Lithuanian regions in 1997-2001. BVDV infection was diagnosed in all investigated regions. The existing variations in the structure of cattle population determined different distribution patterns of BVDV infection. The number of seropositive animals ranged from 11.9 to 100%. It must be pointed out that 29.9% of the herds were not infected with BVDV and in 32.7% of the herds from 70 to 100% of cattle were seropositive to BVDV. A positive correlation between the number of seropositive cattle, and the size of herds and age of animals was determined. Sex of animal had no influence on the prevalence of BVDV. It was estimated that the annual incidence risk of infection with BVDV decreases with the animal age.

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.

RT-PCR is used widely as a diagnostic method to detect and differentiate pestiviruses. The construction of two chimeric classical swine fever virus (CSFV) recombinants based on a marker virus constructed previously [J. Virol. 72 (1998) 5318-5322] is described. These viruses, termed vA187CAT&#95;5UTRBVD and vA187CAT&#95;IRESBVD, contain the entire 5' untranslated region (5'UTR) or the internal ribosome entry site (IRES) of bovine viral diarrhea virus (BVDV), respectively. Both chimeric viruses proved to be infectious in cell culture. Hence, the 5'UTR as well as the IRES element only of BVDV can substitute for the corresponding genome region of CSFV. Next, two sets of primers and corresponding dual-labeled TaqMan probes were designed; one detecting specifically a conserved but CSFV-specific area within the 5'UTR of wild-type CSFV, the other one targeting the CAT gene inserted in vA187CAT&#95;5UTRBVD. The two primer/probe sets were combined in a closed-tube multiplex one-step RT-PCR. To monitor the entire extraction and detection process limited amounts of vA187CAT&#95;5UTRBVD were added directly to clinical samples before RNA extraction. The multiplex RT-PCR proved to be as sensitive as the single primer/probe set method, but allowed the validation of each sample tested individually, based on the detection of the CAT marker gene. vA187CAT&#95;5UTRBVD was also used successfully for foot-and-mouth disease virus (FMDV) TaqMan RT-PCR. Therefore, it is considered a universal internal positive control for RT-PCR assays to exclude loss of RNA during extraction, or failure of amplification due to inhibitory substances present in the sample.

A major issue of current virology concerns the characterization of cellular proteins that operate as functional components of the viral multiplication process. Here we describe a group of host factors designated as 'NFAR proteins' that are recruited by the replication machinery of bovine viral diarrhea virus, a close relative of the human pathogen hepatitis C virus. The NFAR proteins associate specifically with both the termini of the viral RNA genome involving regulatory elements in the 5' and 3' non-translated regions. Modification of the protein interaction sites in the 3' non-translated region yielded viral RNAs that were replication deficient. Viral replication was also inhibited by RNAi approaches that reduced the concentration of RNA helicase A, a member of the NFAR group, in the host cell's cytoplasm. Further experimental data suggest that NFAR proteins mediate a circular conformation of the viral genome that may be important for the coordination of translation and replication. Because NFAR proteins are presumed components of the antiviral response, we suspect that viral recruitment may also serve to weaken cellular defense mechanisms.

Monotherapy of hepatitis C virus infection with either alpha interferon or ribavirin alone is rather ineffective, while the use of the two antivirals together is much more efficacious. In vitro drug-drug combination analysis utilizing related members of the family Flaviviridae, bovine viral diarrhea virus and yellow fever virus, revealed significant direct synergistic interactions between these drugs' antiviral activities that might explain this clinical observation.

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is approximately 2.2 microM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 micro M compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 microM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.

Needle puncture and other accidents that occur during surgery and other procedures may lead to viral infections of medical personnel, notably by hepatitis C (HCV) and human immunodeficiency virus (HIV), now that hepatitis B can be prevented by vaccination. A new surgical glove called G-VIR, which contains a disinfecting agent for enveloped viruses, has been developed. Herpes simplex type 1 (HSV) was used as a standard enveloped virus in both in vitro and in vivo tests of the virucidal capacity of the glove. Bovine viral diarrhea virus (BVDV) and feline immunodeficiency virus (FIV) were used as models for HCV and HIV, respectively. For in vitro study, a contaminated needle was passed through a glove and residual virus was titrated; for in vivo studies, animals were stuck with a contaminated needle through a glove. Despite variation in virus enumeration inherent in the puncture technique, statistical evaluation showed that infection was reproducibly and substantially reduced by passage through the virucidal layer. For BVDV, the amount of virus passing through the virucidal glove was reduced in 82% of pairwise comparisons with control gloves that lacked the virucidal agent; when plaque counts were adjusted to a common dilution, the median count for the virucidal glove was on the average reduced >10-fold. In experiments in which the proportion of wells infected with FIV was measured, the ratio of TCID(50) values (control glove to G-VIR) was >15, and probably much higher. For HSV, the amount of virus passing through the virucidal glove was reduced in 81% of comparisons with control gloves; the median of adjusted plaque counts was reduced on the average approximately eightfold or ninefold. In vivo tests with FIV and HSV in cats and mice, respectively, found smaller percentage reductions in infection than the in vitro tests but confirmed the virucidal effect of the gloves., (Copyright 2003 Wiley-Liss, Inc.)

Current standard therapy for the treatment of chronic infections with hepatitis C virus consists of combination therapy with (pegylated) interferon-alpha and ribavirin. 1,5-Anhydrohexitol nucleoside analogues are constrained congeners known to mimic the ribonucleoside conformation. Within this series some analogues are endowed with strong antiviral properties, particularly against herpesviruses. The six-membered anhydrohexitol ring was, therefore, combined with the triazolyl carboxamide moiety of ribavirin, thus providing a new series of ribavirin analogues. None of the newly synthesized compounds elicited any substantial antiviral activity, neither against herpes simplex virus type 1 and 2, nor against bovine viral diarrhoea virus (a surrogate for hepatitis C virus) or the yellow fever virus.

Objective: To correlate tissue distribution with development of lesions after experimental infection with a virulent strain of noncytopathic bovine viral diarrhea virus (BVDV) type 2 in calves., Animals: Ten 14-day-old and two 2-month-old colostrum-deprived calves., Procedure: Calves were intranasally inoculated with BVDV type-2 strain 1373 from an outbreak of clinically severe bovine viral diarrhea (BVD). Two 14-day-old calves served as noninfected controls. Two calves each were euthanatized on postinoculation days 3, 6, and 12, and 1 each on days 8, 9, 13, and 14. Tissues were collected for immunohistologic and histologic examination., Results: Inoculated calves developed nonspecific clinical signs characterized by high fever and decreased numbers of leukocytes and thrombocytes. Viral antigen was detected focally in lymphoid tissues on day 3. On days 6, 8, 9, 12, and 14, viral antigen became increasingly widespread throughout organs and tissues. Viral antigen in lymphoid tissues was associated with severe depletion of all compartments. Lesions in other tissues were not well correlated with distribution of viral antigen. Depletion of lymphoid tissues was observed in a calf on day 13, but viral antigen had been cleared from most tissues and was detected in vascular walls only., Conclusions and Clinical Relevance: Infection with a virulent BVDV strain resulted in wide dissemination of viral antigen in host tissues. Severe lymphoid depletion developed in lymphoid tissues, whereas viral antigen was generally not associated with lesions in other tissues. Findings suggest that development of lesions in acute BVD is not solely a function of viral replication and is also attributable to host reaction to infection.

Retrospective analyses of cases from which bovine viral diarrhea virus (BVDV) was isolated from 1980 to 2000 were conducted. These cases originated from the northwestern US and included both beef and dairy cattle. The results indicated that there was a shift in diseases associated with BVDV infection and in the animal age at onset of disease. Comparative results from the 1980 data indicated a low fetal infection rate (<5%), followed by steady increases of clinical cases and peaking at 6 months (30%). By 2000, the shift of BVDV cases was noticeable and indicated a biphasic occurrence of disease. The first phase was fetal infections, which increased to >25%, followed by a second phase at 6 months (>35%). Phylogenetic analysis was conducted on selected isolates from the time period 1998-2000 (n = 54). There were representative viral isolates from the two genotypes (BVDV1 and BVDV2), as well as subgenotypes, BVDV1a and BVDV1b. The types were further correlated with the clinical manifestation, which were reported as mucosal disease, persistently infected (PI)-poor doer, and abortion-open cows. The results indicated that BVDV were distributed throughout the clinical spectrum of disease, with BVDV2 representing the greatest frequency of isolation, and the greatest association with abortion-open cows. When the BVDV genotypes and subgenotypes were categorized into early (<100 days gestation) versus late (>100 days gestation) fetal infections, there was an inverse relationship noted. It was observed that BVDV1a was associated least with early infection (14%) and most with late infections (86%). BVDV1b was intermediate, followed by BVDV2, which was associated more with early infections (45%) and less with late infections (55%) when compared with BVDV1a and BVDV1b.

Aim: Due to the absence of a cell culture model for HCV, this study evaluated the hypothesis of lethal mutagenic activity of the guanosin analogue ribavirin in HCV, using the BVDV model., Methods: First the capacity of ribavirin to inhibit BVDV replication in cell culture was studied. We then amplified by RT-PCR the 5'NTR and NS5B regions of BVDV in the supernatants of BVDV-infected cell cultures treated with increasing concentrations of ribavirin. The PCR products were then sequenced to detect potential mutations., Results: At a phenotypic level, the inhibition of BVDV replication by ribavirin was most potent when ribavirin was administered soon after BVDV inoculation. These results show a direct antiviral effect of ribavirin on BVDV at an early stage of the viral cycle. At a genotypic level, ribavirin decreased viral RNA levels., Conclusion: This study shows that ribavirin directly inhibits BVDV replication. These results could explain the synergistic effect of ribavirin and IFN in combined therapy in chronic carriers of HCV. This effect must be confirmed with subgenomic replicons of HCV.

Although transplacental virus infections account for considerable morbidity and mortality in both animals and humans, very little is so far known about the pathways whereby virus reaches the conceptus, the subsequent virus-host interactions in the early phases of the infections, and the establishment of persistent non-lethal infection. Using a natural animal model we recently demonstrated that bovine pestivirus can spread from the site of infection to the ovine fetus within 72 h, despite the expression of interferon in the reproductive tract [1]. In the present study we demonstrate that pestivirus first establishes infection and spread within the allantoic and amniotic membranes and then the fetus, followed several days later by infection of the uterine glands. However, virus replication and spread within the fetus is, at least in part, controlled by fetal developmental factors. In fetuses less than 25 days of gestational age, the virus remains restricted to the bulbis cordis, the first brachial pouch and occasionally the aorta. Over the next few days the virus spreads to multiple tissues, in addition to becoming more widespread and pronounced within the initially infected tissues. A potential role for the binucleated cells of the allantochorion in the spread of the virus from the fetal to the maternal tissues was also found. These cells expressed high levels of viral antigen just prior to and during the time period in which virus antigen became detectable in the epithelial cells of the uterine glands, in endothelial cells of uterine vessels and in scattered macrophage-like cells in the uterine stroma. Most likely this relatively late virus transfer is inconsequential for the mother, since it occurs at a time when a maternal virus-specific antibody response is becoming measurable. This is in contrast to the fetus, where the infection will have established itself widely prior to the development of lymphoid tissues and a functional immune response, thus setting the scenario for development of specific tolerance to the persisting virus., (Copyright 2002 Academic Press.)

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 x 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. At the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different strains of virus. Further, the quantity of BVDV associated with washed embryos from both initial and secondary cultures varied among strains, but the variation was unrelated to difference in genotype (SD-1 and PA-131 greater than NY-1 and CD-87). Although all strains of BVDV replicated in UTC in the initial in vitro cultures and remained associated with washed blastocysts, susceptible UTC in the secondary in vitro cultures were seldom infected by any strain of virus.

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5' NTR mutants correlated with reduced amounts of accumulated viral RNAs.

The primary aim of a bovine virus diarrhea virus (BVDV) vaccine is to prevent transplacental transmission of virus. We studied the efficacy of two experimental conventionally inactivated vaccines, based on BVDV strain Singer and containing a different antigen amount, against three antigenically different BVDV strains in a vaccination-challenge experiment in sheep. We also studied the efficacy of an experimental multivalent E2 subunit vaccine against four antigenically different BVDV strains. The vaccine contained the glycoproteins E2 of BVDV strains that belong to antigenic groups IA, IB and II. All three vaccines induced neutralizing antibodies against all challenge strains. Only the conventional vaccine that contained the highest antigen amount induced complete protection against homologous challenge. Neither of the conventional vaccines provided complete protection against heterologous challenge. The multivalent subunit vaccine induced partial protection against the homologous challenge strains. However, the immune response did inhibit virus replication in ewes, as shown by the results of the virus titrations.

Transmission of viruses by animal sera represents a considerable risk for humans and animals particularly when the serum is used for the production of pharmaceutical products such as vaccines. Procedures applicable for inactivating large numbers of different viruses, both enveloped and non-enveloped, are therefore mandatory. For this purpose we have developed and validated UVC irradiation as the virus-inactivation procedure of choice for serum to be used in an industrial setting. Spiking experiments in foetal calf serum (FCS) were performed by independent contract laboratories and revealed constantly high clearance rates for various viruses such as bovine parvovirus, parainfluenza type III virus, bovine diarrhoea virus, foot-and-mouth disease virus and different forms of mycoplasmas. UVC-treated sera maintained their growth-promoting activities for various cell types (MRC-5, Vero, CHO). Conventional growth curves generated in the presence of 10% and 1% UVC-treated FCS differed only slightly from controls, indicating the lack of significant damage during UVC exposure. Experiments using a sensitive photometric-based acid phosphatase assay (APA), which correlates well with the more tedious cell counting procedure, confirmed these findings even in the presence of minimal serum requirements. UVC treatment of animal sera appears advantageous compared to currently recommended inactivation procedures, such as Gamma irradiation, for at least three reasons: (i) it possesses a high inactivation capacity for parvoviruses, a pathogen that cannot be destroyed easily by conventional methods; (ii) it causes no noticeable impairment in cell growth and (iii) it can be performed in a controlled manner at the production site.

Systematic eradication of BVDV without vaccination started in Scandinavia in 1993. In principle, the schemes include; (1) identification of non-infected and infected herds using different combinations of serological herd tests such as bulk milk tests and spot tests (sample of animals in a certain age), (2) monitoring/certification of non-infected herds by repeated sampling, applying one of the above-mentioned methods and (3) virus clearance in infected herds aimed at removing persistently infected (PI) animals in a cost- and time-efficient manner. In the virus clearance protocol described, an initial test is performed on all animals with subsequent follow-up of calves born as well as of dams seronegative in the initial test. It is generally recommended to perform an initial antibody test on all samples. This should be done not only to screen for seronegative animals on which virus isolation should be attempted (i.e. possible PI animals), but more in order to identify non-immune animals in reproductive age, that is, the key animals in herd-level persistence of infection. In Sweden, a common finding has been self-clearance, where the infection ceases without any other intervention than controlled introduction of new animals. Other epidemiological observations concern the course of events following virus introduction. Important risk factors for spreading BVDV are discussed, where livestock trade is perceived as the most central to control. Live vaccines, imported semen and embryos constitute special hazards, since they may act as vehicles for the introduction of new BVDV strains. The importance of making farmers aware of herd biosecurity and their own responsibility for it is stressed, and in order to maintain a favourable situation after a scheme has been concluded, effort must be put into establishing such a persisting attitude in the farming community.

A series of experiments was performed to assess the ability of the heat treatment step used in the manufacture of diaspirin crosslinked hemoglobin (DCLHb) to inactivate viruses. In-process solutions (reaction mixtures after the crosslinking process) from six different manufacturing lots were used as test media in a 1:680 scaled down system in which the key process parameters used in the large scale production were duplicated. The inactivation of five different viruses (Bovine Viral Diarrhea Virus, Pseudorabies Virus, Human Immunodeficiency Virus 1, Porcine Parvovirus and Hepatitis A Virus) was evaluated. Each validation experiment consisted of spiking the solution at 37 degrees C with virus, heating to 74 +/- 1 degrees C over a period of 30 minutes, holding at 74 +/- 1 degrees C for 90 minutes and cooling from 74 +/- 1 degrees C to less than 10 degrees C over a period of 30 minutes. Duplicate experiments were performed with each of the viruses with the exception of Human Immunodeficiency Virus 1, for which three experiments were performed. In each experiment samples were removed before, during, and after heating for the purpose of determining virus titer and evaluating key process parameters. The results obtained from these experiments confirmed that the key process parameters in these experiments using the scaled down test system reproduced those of the large scale manufacturing process. The results of the virus assays showed at least a 7 log reduction was accomplished by the heat treatment for each of the viruses tested.