ABSTRACT
Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO2-TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO2-TSC. Moreover, resistance selection and characterization showed that N4-TSCR mutants were highly resistant to N4-TSC but remained susceptible to 6NO2-TSC. In contrast, 6NO2-TSCR mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSCR mutants, whereas I261 M was found in 6NO2-TSCR mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a "hot spot" for BVDV non-nucleoside inhibitors.
Subject(s)
Antiviral Agents , Drug Resistance, Viral , Genotype , Indans , Thiosemicarbazones , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Animals , Cattle , Thiosemicarbazones/pharmacology , Thiosemicarbazones/chemistry , Indans/pharmacology , Indans/chemistry , Drug Resistance, Viral/genetics , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/genetics , Diarrhea Viruses, Bovine Viral/drug effects , Diarrhea Viruses, Bovine Viral/genetics , Cell Line , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Diarrhea Virus 2, Bovine Viral/genetics , Diarrhea Virus 2, Bovine Viral/drug effects , Virus Replication/drug effects , Mutation , RNA, Viral/geneticsABSTRACT
Bovine viral diarrhea virus (BVDV) and bovine alphaherpesvirus type 1 (BoHV-1) are responsible for major economic losses of livestock worldwide, making their eradication an important objective of veterinary research. Vaccines against these infectious agents are commercially available but have some limitations due to the specific features of these viral agents. The development of new antiviral drugs is therefore essential. Native banana lectin (BanLec) is a lectin isolated from banana fruit (Musa acuminata) and has a high affinity for mannose glycans found in several viral envelopes. The inhibitory properties of this lectin against several viruses has already been demonstrated. The aim of this work was therefore to test the antiviral and virucidal activities of BanLec against BVDV-1 and BoHV-1. Its antiviral activity was assessed by measuring the viral titer and viability of susceptible Madin-Darby Bovine Kidney cells (MDBK) treated with BanLec before and after viral infection. The virucidal properties of BanLec were determined by preincubation of the lectin with the viruses, followed by measurement of the viral load in exposed cells. Treatment with 25 µg/mL BanLec resulted in high levels of inhibition against BVDV-1 (99.98%) and BoHV-1 (99.68%) without affecting cell viability, demonstrating promising potential as an antiviral agent.
Subject(s)
Antiviral Agents/pharmacology , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Viruses, Bovine Viral/drug effects , Lectins/pharmacology , Musa/chemistry , Antiviral Agents/chemistry , Cell Line , Dose-Response Relationship, Drug , Hemagglutination Tests , Lectins/chemistryABSTRACT
Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. RESULTS: showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p<0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.
Subject(s)
Autophagy , Diarrhea Virus 1, Bovine Viral/pathogenicity , Animals , Autophagy/drug effects , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Cattle Diseases/virology , Cytopathogenic Effect, Viral , Diarrhea Virus 1, Bovine Viral/classification , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/isolation & purification , Dogs , Madin Darby Canine Kidney Cells , Sirolimus/pharmacology , Species Specificity , Viral Proteins/metabolism , Virus ReplicationABSTRACT
The aim of this study was to examine the effect of treating of chromium(III) and iron(III) and their combinations on Herpes Simplex Virus type 1 (HSV-1) and Bovine Viral Diarrhoea virus (BVDV) replication. The antiviral efficacies of chromium(III) and iron(III) on HSV-1 and BVDV were evaluated using Real Time PCR method. Moreover, the cytotoxicity of these microelements was examined using the MTT reduction assay. The IC50 (50% inhibiotory concentration) for the chromium chloride was 1100 µM for Hep-2 cells and 1400 µM for BT cells. The IC50 for the iron chloride was 1200 µM for Hep-2 cells and more than1400 µM for BT cells. The concentration-dependent antiviral activity of chromium chloride and iron chloride against HSV-1 and BVDV viruses was observed. In cultures simultaneously treated with (1) 200 µM of CrCl3 and 1000 µM of FeCl3, (2) 1000 µM of CrCl3 and 200 µM of FeCl3, (3) 400 µM of CrCl3 and 800 µM of FeCl3, (4) 800 µM of CrCl3 and 400 µM of FeCl3 a decrease in number of DNA or RNA copies was observed compared with control cells and cells incubated with chromium(III) and iron(III) used separately. The synergistic antiviral effects were observed for chromium(III) and iron(III) against HSV-1 and BVDV.
Subject(s)
Antiviral Agents/pharmacology , Chlorides/pharmacology , Chromium Compounds/pharmacology , Diarrhea Virus 1, Bovine Viral/drug effects , Ferric Compounds/pharmacology , Herpesvirus 1, Human/drug effects , Virus Replication/drug effects , Animals , Cattle , Cell Line , Cell Line, Tumor , DNA, Viral/antagonists & inhibitors , DNA, Viral/biosynthesis , Diarrhea Virus 1, Bovine Viral/genetics , Diarrhea Virus 1, Bovine Viral/growth & development , Dose-Response Relationship, Drug , Drug Synergism , Epithelial Cells/drug effects , Epithelial Cells/virology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/growth & development , Humans , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesisABSTRACT
The objective of this study was to compare reproductive protection in cattle against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) provided by annual revaccination with multivalent modified-live viral (MLV) vaccine or multivalent combination viral (CV) vaccine containing temperature-sensitive modified-live BoHV-1 and killed BVDV when MLV vaccines were given pre-breeding to nulliparous heifers. Seventy-five beef heifers were allocated into treatment groups A (n=30; two MLV doses pre-breeding, annual revaccination with MLV vaccine), B (n=30; two MLV doses pre-breeding, annual revaccination with CV vaccine) and C (n=15; saline in lieu of vaccine). Heifers were administered treatments on days 0 (weaning), 183 (pre-breeding), 366 (first gestation), and 738 (second gestation). After first calving, primiparous cows were bred, with pregnancy assessment on day 715. At that time, 24 group A heifers (23 pregnancies), 23 group B heifers (22 pregnancies), and 15 group C heifers (15 pregnancies) were commingled with six persistently infected (PI) cattle for 16days. Ninety-nine days after PI removal, cows were intravenously inoculated with BoHV-1. All fetuses and live offspring were assessed for BVDV and BoHV-1. Abortions occurred in 3/23 group A cows, 1/22 group B cows, and 11/15 group C cows. Fetal infection with BVDV or BoHV-1 occurred in 4/23 group A offspring, 0/22 group B offspring, and 15/15 group C offspring. This research demonstrates efficacy of administering two pre-breeding doses of MLV vaccine with annual revaccination using CV vaccine to prevent fetal loss due to exposure to BVDV and BoHV-1.
Subject(s)
Abortion, Spontaneous/prevention & control , Abortion, Veterinary/prevention & control , Bovine Virus Diarrhea-Mucosal Disease/prevention & control , Infectious Bovine Rhinotracheitis/prevention & control , Infectious Disease Transmission, Vertical/prevention & control , Pregnancy Complications, Infectious/prevention & control , Viral Vaccines/administration & dosage , Abortion, Spontaneous/immunology , Abortion, Spontaneous/virology , Abortion, Veterinary/immunology , Abortion, Veterinary/virology , Animals , Antibodies, Viral/biosynthesis , Bovine Virus Diarrhea-Mucosal Disease/immunology , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/immunology , Diarrhea Virus 1, Bovine Viral/pathogenicity , Female , Fetus , Herpesvirus 1, Bovine/drug effects , Herpesvirus 1, Bovine/immunology , Herpesvirus 1, Bovine/pathogenicity , Immunization, Secondary , Infectious Bovine Rhinotracheitis/immunology , Infectious Bovine Rhinotracheitis/virology , Pregnancy , Pregnancy Complications, Infectious/immunology , Pregnancy Complications, Infectious/virology , Vaccines, Attenuated , Vaccines, Combined , Vaccines, InactivatedABSTRACT
Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.
Subject(s)
Coordination Complexes/chemical synthesis , Indoles/chemical synthesis , Photosensitizing Agents/chemical synthesis , Radiation Tolerance/physiology , Virus Inactivation , Zinc/chemistry , Adenoviruses, Human/drug effects , Adenoviruses, Human/growth & development , Adenoviruses, Human/radiation effects , Anions , Cations , Coordination Complexes/pharmacology , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/growth & development , Diarrhea Virus 1, Bovine Viral/radiation effects , Enterovirus B, Human/drug effects , Enterovirus B, Human/growth & development , Enterovirus B, Human/radiation effects , Herpesvirus 1, Human/drug effects , Herpesvirus 1, Human/growth & development , Herpesvirus 1, Human/radiation effects , Indoles/pharmacology , Isoindoles , Lasers, Semiconductor , Light , Newcastle disease virus/drug effects , Newcastle disease virus/growth & development , Newcastle disease virus/radiation effects , Photosensitizing Agents/pharmacology , Species Specificity , Static Electricity , Vaccinia virus/drug effects , Vaccinia virus/growth & development , Vaccinia virus/radiation effectsABSTRACT
Nitazoxanide (NTZ) inhibits influenza, Japanese encephalitis, hepatitis B and hepatitis C virus replication but effects on the replication of other members of the Flaviviridae family has yet to be defined. The pestivirus bovine viral diarrhoea virus (BVDV) is a surrogate model for HCV infection and NTZ induced PKR and eIF2α phosphorylation in both uninfected and BVDV-infected cells. This led to the observation that NTZ depletes ATP-sensitive intracellular Ca(2+) stores. In addition to PKR and eIF2α phosphorylation, consequences of NTZ-mediated Ca(2+) mobilisation included induction of chronic sub-lethal ER stress as well as perturbation of viral protein N-linked glycosylation and trafficking. To adapt to NTZ-mediated ER stress, NTZ treated cells upregulated translation of Ca(2+)-binding proteins, including the ER chaperone Bip and the cytosolic pro-survival and anti-viral protein TCTP. Depletion of intracellular Ca(2+) stores is the primary consequence of NTZ treatment and is likely to underpin all antiviral mechanisms attributed to the thiazolide.
Subject(s)
Adenosine Triphosphate/metabolism , Antiviral Agents/metabolism , Calcium/metabolism , Cytoplasm/chemistry , Cytoplasm/drug effects , Thiazoles/metabolism , Animals , Cell Line , Diarrhea Virus 1, Bovine Viral/drug effects , Endoplasmic Reticulum/drug effects , Glycosylation/drug effects , Humans , Nitro Compounds , Protein Transport/drug effects , Tumor Protein, Translationally-Controlled 1 , Virus Replication/drug effectsABSTRACT
2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1µM, 0.05±0.01µM and 0.3±0.04µM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25µM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1µM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication.
Subject(s)
Antiviral Agents/pharmacology , Benzimidazoles/pharmacology , Classical Swine Fever Virus/drug effects , Diarrhea Virus 1, Bovine Viral/drug effects , Enzyme Inhibitors/pharmacology , Pyridines/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Antiviral Agents/toxicity , Benzimidazoles/chemistry , Benzimidazoles/isolation & purification , Benzimidazoles/toxicity , Cattle , Cell Line , Classical Swine Fever Virus/enzymology , Diarrhea Virus 1, Bovine Viral/enzymology , Drug Resistance, Viral , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/toxicity , Hepacivirus/drug effects , Hepacivirus/enzymology , Models, Molecular , Mutation , Protein Conformation , Pyridines/chemistry , Pyridines/isolation & purification , Pyridines/toxicityABSTRACT
The aim of this study was to compare the effectiveness of a needle-free injection device (NF) with a needle and syringe (NS) when used to vaccinate calves against bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV). The study was conducted in two independent phases. Ninety-six crossbred beef calves were vaccinated in the spring and 98 beef calves in the autumn. The calves were vaccinated using a NF or NS at 2 months of age (day 0) and again on day 119, with a modified-live virus vaccine containing IBRV, BVDV (types 1 and 2), parainfluenza-3 virus, and bovine respiratory syncytial virus. In each herd 10 calves were left unvaccinated to determine whether exposure to either BVDV or IBRV occurred. Visible vaccine residue at the surface of the skin/hair was apparent immediately following vaccination with NF in 30% of the spring-born calves following both the primary and booster vaccination. In the autumn, visible vaccine residues occurred in 19% and 8% of NF-vaccinated calves following the primary and booster vaccination. Post-vaccination skin reactions recorded on days 21, 42, 119 and 140 occurred with greater frequency in NF-vaccinated calves than NS-vaccinated ones. Blood samples were collected on days 0, 21, 42, 119, and 140 and tested for antibodies to BVDV and IBRV. Vaccination technique had no significant effect on BVDV or IBRV antibody concentrations at any time point. NF was as effective as NS vaccination in eliciting BVDV and IBRV antibody responses.
Subject(s)
Bovine Virus Diarrhea-Mucosal Disease/prevention & control , Hemorrhagic Syndrome, Bovine/prevention & control , Herpesviridae Infections/veterinary , Infectious Bovine Rhinotracheitis/prevention & control , Vaccination/methods , Animals , Antibodies, Viral/blood , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 2, Bovine Viral/drug effects , Female , Hemorrhagic Syndrome, Bovine/virology , Herpesviridae Infections/prevention & control , Herpesviridae Infections/virology , Herpesvirus 1, Bovine/drug effects , Infectious Bovine Rhinotracheitis/virology , Needles/veterinary , Seasons , Syringes/veterinary , Vaccination/instrumentation , Vaccination/veterinaryABSTRACT
The Hepatitis C virus causes chronic infections in humans, which can develop to liver cirrhosis and hepatocellular carcinoma. The Bovine viral diarrhea virus is used as a surrogate model for antiviral assays for the HCV. From marine invertebrates and microorganisms isolated from them, extracts were prepared for assessment of their possible antiviral activity. Of the 128 tested, 2 were considered active and 1 was considered promising. The best result was obtained from the extracts produced from the Bacillus sp. isolated from the sponge Petromica citrina. The extracts 555 (500 µg/mL, SI>18) and 584 (150 µg/mL, SI 27) showed a percentage of protection of 98% against BVDV, and the extract 616, 90% of protection. All of them showed activity during the viral adsorption. Thus, various substances are active on these studied organisms and may lead to the development of drugs which ensure an alternative therapy for the treatment of hepatitis C.
Subject(s)
Antiviral Agents/pharmacology , Bacillus/chemistry , Diarrhea Virus 1, Bovine Viral/drug effects , Porifera/microbiology , Virus Attachment/drug effects , Animals , Antiviral Agents/isolation & purification , Bacillus/classification , Bacillus/isolation & purification , Cattle , Cell Line , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Diarrhea Virus 1, Bovine Viral/physiology , Microbial Sensitivity Tests , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
Bovine viral diarrhea virus (BVDV) is a widespread bovine pathogen for which there is no specific therapeutic agent. A previous study using 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) to treat calves persistently infected with BVDV resulted in a decrease in the viral load of infected calves but treatment resulted in the rapid selection of drug-resistant mutant isolates. In this article we describe three mutations found in the mutant isolates associated with in vivo and in vitro resistance to DB772. All three mutations are found in the NS5B which functions as the RNA-dependent-RNA-polymerase during viral replication. Growth curves for the mutant isolates were not largely different from those of wild-type isolates when cultured in the absence of DB772. Thus, DB772 appears to act by binding to the specified domain but binding is disrupted or inhibited by the described mutation.
Subject(s)
Antiviral Agents/therapeutic use , Benzimidazoles/therapeutic use , Cattle Diseases/virology , Diarrhea Virus 1, Bovine Viral/genetics , Drug Resistance, Viral , Furans/therapeutic use , Pestivirus Infections/virology , Viral Nonstructural Proteins/genetics , Animals , Antiviral Agents/pharmacology , Benzimidazoles/pharmacology , Cattle , Cattle Diseases/drug therapy , DNA Mutational Analysis , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/isolation & purification , Furans/pharmacology , Molecular Sequence Data , Mutation, Missense , Pestivirus Infections/drug therapy , RNA, Viral/genetics , Selection, Genetic , Sequence Analysis, DNAABSTRACT
Bovine viral diarrhea virus (BVDV) causes lethal mucosal disease of cattle and leads to severe economic loss of cattle production and reproduction worldwide. Over the past decades, vaccination was not very successful in providing prevention of BVDV infection. This reality demands that anti-BVDV drugs should be used as an alternative treatment strategy. In this study, a BAC cDNA of noncytopathic BVDV strain SD-1 is constructed to contain an enhanced green fluorescence protein (eGFP) gene between viral NS3 and NS4A coding sequences. The recombinant reporter virus is generated subsequently by transfection of MDBK cells with the transcripts produced in vitro. The rescued reporter virus is stable in MDBK cells and the eGFP protein is expressed and processed properly. Of most importance, the reporter virus shows a growth property similar to the SD-1 parent and the fluorescent signal intensity increases in parallel to the reporter virus RNA and protein replication. In addition, two known anti-BVDV drug G418 (viral assembly/release inhibitor) and ribavirin (viral RNA replication inhibitor) are identified as hits in a high-throughput format, suggesting that this system is capable of identifying BVDV inhibitors that target different steps in viral life cycle. The cell-based system developed provides a useful and versatile tool which should facilitate the identification of BVDV inhibitors on a large scale.
Subject(s)
Antiviral Agents/pharmacology , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/genetics , Genes, Reporter , High-Throughput Screening Assays/methods , Amino Acid Sequence , Animals , Base Sequence , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Cell Line , Chromosomes, Artificial, Bacterial/genetics , DNA, Complementary/genetics , Diarrhea Virus 1, Bovine Viral/growth & development , Diarrhea Virus 1, Bovine Viral/metabolism , Kidney/cytology , Kidney/virology , Microbial Sensitivity Tests , Molecular Sequence Data , Recombinant Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Bovine viral diarrhea virus (BVDV) is one of the most important pathogens to the cattle industry, causing a significant economic loss throughout the world. Despite the wide use of various control measures for BVDV, the disease remains prevalent. In this study, we achieved an efficient inhibition of NADL strain replication by plasmid-mediated shRNA targeting conserved regions of the viral genome. To further enhance the inhibiting efficiency, a dual shRNA expression plasmid, which could simultaneously express two different shRNA, was established and showed stronger inhibitory effects on virus replication. Moreover, the antiviral activity induced by the dual shRNA expression system was also evident on other BVDV-1 subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). Therefore, the dual shRNA system provides a more powerful strategy for inhibiting BVDV replication in a cross-resistance manner.
Subject(s)
Diarrhea Virus 1, Bovine Viral/drug effects , RNA Interference , RNA, Small Interfering/therapeutic use , Virus Replication/drug effects , Animals , Bovine Virus Diarrhea-Mucosal Disease/drug therapy , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Cell Line , Conserved Sequence/drug effects , Conserved Sequence/genetics , Diarrhea Virus 1, Bovine Viral/genetics , Diarrhea Virus 1, Bovine Viral/physiology , Genotype , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction/veterinary , Virus Replication/geneticsABSTRACT
A phenanthridinone skeleton was derived from our previous researches on thalidomide and retinoids as a multi-template for generation of anti-viral lead compounds. Structural development studies focusing on anti-hepatitis C virus activity afforded 5-butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenanthridin-6(5H)-one (10) and 5-butylbenzo[b]phenanthridin-6(5H)-one (39), which showed EC(50) values of approximately 3.7 and 3.2microM, respectively.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Phenanthridines/chemical synthesis , Phenanthridines/pharmacology , Cell Line , Diarrhea Virus 1, Bovine Viral/drug effects , Drug Design , Gene Expression Regulation, Viral/drug effects , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Hepacivirus/genetics , Humans , Indicators and Reagents , Magnetic Resonance Spectroscopy , Mass Spectrometry , RNA, Viral/biosynthesis , RNA, Viral/drug effects , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, beta-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.
Subject(s)
Clathrin/physiology , Diarrhea Viruses, Bovine Viral/physiology , Endocytosis/physiology , Virus Internalization , Ammonium Chloride/pharmacology , Animals , Cell Line , Chloroquine/pharmacology , Chlorpromazine/pharmacology , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 1, Bovine Viral/pathogenicity , Diarrhea Virus 1, Bovine Viral/physiology , Diarrhea Virus 2, Bovine Viral/drug effects , Diarrhea Virus 2, Bovine Viral/pathogenicity , Diarrhea Virus 2, Bovine Viral/physiology , Diarrhea Viruses, Bovine Viral/drug effects , Diarrhea Viruses, Bovine Viral/pathogenicity , Dose-Response Relationship, Drug , Endocytosis/drug effects , Hydrogen-Ion Concentration , Sheep , Sucrose/pharmacology , Thymus Gland/cytology , Virus Internalization/drug effects , beta-Cyclodextrins/pharmacologyABSTRACT
Unsymmetrical methylene derivatives 5 were prepared following a known method, by reaction of the Mannich bases of 2-naphthols 4 with indoles. All synthesized compounds were tested against a wide panel of viruses, since previous work showed that Mannich bases on 7-hydroxycoumarin 1 and unsymmetrical methylene derivatives 2 were endowed with some antiviral activities. The symmetrical Mannich bases 4 were completely inactive, whereas the unsymmetrical methylene derivatives 5, although possessing a certain degree of toxicity, showed a significant activity against RSV. Some of compounds 5 showed a moderate antiviral activity against HIV-1, BVDV, YFV and CVB-2. The lack of activity of Mannich bases 4 demonstrates the crucial importance for antiviral activity of coumarin moiety present in Mannich bases 1.
Subject(s)
Indoles/pharmacology , Animals , Antiviral Agents , Cell Line , Diarrhea Virus 1, Bovine Viral/drug effects , Enterovirus/drug effects , HIV-1/drug effects , Inhibitory Concentration 50 , Molecular Structure , Respiratory Syncytial Viruses/drug effects , Yellow fever virus/drug effectsABSTRACT
[7-[3-(1,3-Benzodioxol-5-yl)propyl]-2-(2-furyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] (LZ37) was identified as a selective inhibitor of in vitro bovine viral diarrhea virus (BVDV) replication. The EC(50) values for inhibition of BVDV-induced cytopathic effect (CPE) formation, viral RNA synthesis and production of infectious virus were 4.3+/-0.7microM, 12.9+/-1microM and 5.8+/-0.6microM, respectively. LZ37 proved inactive against the hepatitis C virus and the flavivirus yellow fever. LZ37 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carried the F224Y mutation in the viral RNA-dependent RNA polymerase (RdRp). LZ37 showed cross-resistance with the imidazopyrrolopyridine AG110 [which selects for the E291G drug resistance mutation] as well as with the imidazopyridine BPIP [which selects for the F224S drug-resistant mutation]. LZ37 did not inhibit the in vitro activity of purified recombinant BVDV RdRp. Molecular modelling revealed that F224 is located near the tip of the finger domain of the RdRp. Docking of LZ37 in the crystal structure of the BVDV RdRp revealed several potential contacts including: (i) hydrophobic contacts of LZ37 with A221, A222, G223, F224 and A392; (ii) a stacking interaction between F224 side chain and the ring system of LZ37 and (iii) a hydrogen bond between the amino function of LZ37 and the O backbone atom of A392. It is concluded that LZ37 interacts with the same binding site as BPIP or VP32947 at the top of the finger domain of the polymerase that is a "hot spot" for inhibition of pestivirus replication.
Subject(s)
Antiviral Agents/pharmacology , Benzodioxoles/pharmacology , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 2, Bovine Viral/drug effects , Triazoles/pharmacology , Virus Replication/drug effects , Amino Acid Substitution/genetics , Animals , Antiviral Agents/chemistry , Benzodioxoles/chemistry , Cattle , Cell Line , Diarrhea Virus 1, Bovine Viral/physiology , Diarrhea Virus 2, Bovine Viral/physiology , Drug Resistance, Viral , Hepacivirus/drug effects , Inhibitory Concentration 50 , Models, Molecular , Mutation, Missense , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Triazoles/chemistry , Viral Proteins/genetics , Yellow fever virus/drug effectsABSTRACT
BACKGROUND: Aminoglycoside G418 is commonly used to generate stable replicons for RNA viruses, such as hepatitis C virus, West Nile virus, and bovine viral diarrhoea virus (BVDV). This precludes testing 6418's own antiviral activities against those viruses. Here, we report antiviral activity of 6418 against BVDV. METHODS: Cell viability and virus yield reduction assays were used to investigate antiviral effects of G418 against BVDV. The expression of viral proteins and RNA were determined by western blot and real-time quantitive PCR, respectively. RESULTS: We demonstrated that G418 (50% cytotoxicity concentration of 400 microg/ml) improved cell viability of Madin-Darby bovine kidney cells infected with a cytopathic strain of BVDV (NADL) in a dose-dependent manner with 50% effective concentration of 4 microg/ml. Interestingly, close structural analogues with known properties as translation inhibitors similar to G418 - kanamycin and gentamicin - had no antiviral activity against BVDV. In addition, 6418 inhibits virus yield of two different strains of BVDV (NADL and NY-1) without affecting viral RNA replication and translation or viral NS3 protein processing. CONCLUSION: Our data indicate that antiviral activity of G418 could result from interference with either the assembly or release of active virus, rather than the regulation of viral translation and replication. Thus, we propose the use of chemical analogues of G418 as antiviral therapeutics for treatment of viral diseases associated with the Flaviviridae family, such as hepatitis C virus, dengue virus, yellow fever virus, West Nile virus and others.
Subject(s)
Antiviral Agents/pharmacology , Bovine Virus Diarrhea-Mucosal Disease/drug therapy , Diarrhea Virus 1, Bovine Viral/drug effects , Gentamicins/pharmacology , Animals , Cattle , Cell Survival/drug effects , Cells, Cultured , Diarrhea Virus 1, Bovine Viral/physiology , Interferons/pharmacology , Kanamycin/analogs & derivatives , Kanamycin/pharmacology , Kidney/virology , Neutralization Tests , Peptide Hydrolases/analysis , RNA Helicases/analysis , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity Relationship , Viral Nonstructural Proteins/analysis , Virus ReplicationABSTRACT
The antiviral activity of iminocyclitol compounds with a deoxynojirimycin (DNJ) head group and either a straight chain alkyl or alkylcycloalkyl group attached to the nitrogen atom have been tested in vitro against multiple-enveloped viruses. Several of these analogues were superior to previously reported DNJ compounds. Iminocyclitols that inhibit the glycan-processing enzyme endoplasmic-reticular glucosidase have been shown to inhibit the morphogenesis of viruses that bud from the endoplasmic reticulum (ER) at non-cytotoxic concentrations. Bovine viral diarrhoea virus (BVDV) has been used as a surrogate system for study of the hepatitis C virus, which belong to the virus family (Flaviviridae) as West Nile virus (WNV) and dengue virus (DV). N-Nonyl-DNJ (NNDNJ) was previously reported to have micromolar antiviral activity against BVDV, but a limiting toxicity profile. N-Butylcyclohexyl-DNJ (SP169) was shown to be as potent as NNDNJ in assays against BVDV and less toxic. However, it was inactive against hepatitis B virus (HBV). The present study reports efforts to improve the performance profiles of these compounds. Introduction of an oxygen atom into the N-alkyl side chain of DNJ, either as an ether or a hydroxyl functionality, reduced toxicity but sacrificed potency. Introduction of a hydroxyl group at the tertiary carbon junction of the cycloalkyl and linear alkyl group, as in N-pentyl-(1-hydroxycyclohexyl)-DNJ (OSL-9511), led to a structure that was as well tolerated as DNJ (CC50>500 microM), but retained micromolar antiviral activity against all ER morphogenesis budding viruses tested: BVDV, WNV, DV and HBV. The implication of this modification to the development of broad-spectrum antiviral agents is discussed.
Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/drug effects , Diarrhea Virus 1, Bovine Viral/drug effects , Diarrhea Virus 2, Bovine Viral/drug effects , Hepatitis B virus/drug effects , West Nile virus/drug effects , Animals , Cattle , Cell LineABSTRACT
We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.