Design of anti-BVDV drug based on common chemical features, their interaction, and scaffolds of TLR8 agonists.

In the absence of an experimental bTLR8 structure, recent studies have called attention to the fact that bTLR8 can also be activated by hTLR7/hTLR8 agonist, such as antiviral imidazoquinoline derivatives of resiquimod (R848) and imiquimod (R837) as well as some guanine nucleotide analogs with a scaffold structure related to the nucleic acids of ssRNA virus. In particular, the known small agonists (namely CL075, CL097 and R848) have been targeted to determine distinguishable deciding factors in complex with dimeric bTLR8-ECDs in comparison to ligand-induced activated hTLR8-ECDs. According to basic knowledge, the deciding eligibility criteria can be subsequently applied in our bTLR8 model to characterize the 3D-arrangement of chemical features (pharmacophore) and to investigate the distinct restrictions affecting species-specificity on dual TLR7/TLR8 small agonists suggested in previous works. Despite the lack of extensive structural biology studies regarding the interaction of bTLR8-ECDs with the agonists, our complex models of bTLR8-ECDs and the known agonists were applied to identify the deciding factors required for the interactions from agonist-based and (bTLR8-agonist complexes) structure-based pharmacophores. These pharmacophore constraints impose their essential chemical features to active bTLR8 receptors. The characterized pharmacophores all were employed in the virtual screening of candidates with a further acting factor of calf immune enhancer. Two hits were suggested as satisfying all decision factors to identify a potent bTLR8-specific agent with novel scaffolds dissimilar to imidazoquinoline analogues lacking overall homogeneity.

Host factors that interact with the pestivirus N-terminal protease, Npro, are components of the ribonucleoprotein complex.

UNLABELLED: The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. IMPORTANCE: Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that N(pro) is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to N(pro). A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon.

Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Biophysical and microbiological study of high hydrostatic pressure inactivation of Bovine Viral Diarrheavirus type 1 on serum.

The effect of high hydrostatic pressure application on fetal bovine serum components and the model microorganism (Bovine Viral Diarrheavirus type 1 NADL strain) was studied at 132 and 220 MPa pressure for 5 min at 25°C. Protein secondary structures were found to be unaffected by an artificial neural network application on the amide I region for both untreated and HHP treated samples. FTIR spectroscopy study of both the HHP-treated and control samples revealed changes in the intensity of some bands in the finger-print region (1500-900 cm(-1)) originating mainly from lipids which are thought to result from changes in the lipoprotein structure. The virus strain lost its infectivity completely after 220 MPa HHP treatments. These results indicate that HHP can be successfully used for inactivation of pestiviruses while leaving structural and functional properties of serum and serum products unaffected.

Method for detection of extraneous active bovine viral diarrhoea virus and classical swine fever virus in animal viral vaccines by RT-PCR, which amplify negative-strand viral RNA in infected cells.

An oligonucleotide sense primer, Pst324alpha, was designed and used for synthesizing cDNA from negative-strand viral RNA in infected cells and used for rapid detection of active extraneous bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) in animal viral vaccines by culturing a sample in cells followed by reverse transcriptase-polymerase chain reaction (RT-PCR). Active and inactivated viruses of BVDV No. 12-43 strain and CSFV GPE(-)strain were inoculated to bovine testicle and swine testicle cells for incubation. After the complete extraction of RNA from these cells, cDNA was synthesized using Pst324alpha, and PCR was carried out using primers 324 and 326 (novel RT-PCR). Amplification of novel RT-PCR products was observed in cells infected with active viruses but not in cells inoculated with inactivated viruses, inoculums and cultured media after incubation. This novel RT-PCR was able to amplify viral sequences from cells infected with only a small number of infectious particles (less than 10 TCID50) at three days postinoculation and was as sensitive as the general RT-PCR using a random primer and the interference and immunofluorescent antibody (FA) methods. The results of experiments on detection of BVDV RNA from vaccines contaminated with active and inactivated BVDV showed that the sensitivity of the novel RT-PCR was almost the same as the sensitivities of the interference and FA methods. These results suggest that the novel RT-PCR is easier and more rapid than the interference method for detection of active BVDV and that the novel RT-PCR is a reliable means for detection of active extraneous BVDV for quality control of animal vaccines.