Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs

MDBK cells infected with wt BVDV and BVDV R viruses.

<p>MDBK cells were infected with wt BVDV p0, BVDV R₁, R_2, or R₃ (MOI: 0.1) and IF-stained using a monoclonal antibody against the NS3 protein at 24, 48 and 72 h p.i. The nucleus was stained with DAPI. Images were taken with a 20× objective (numerical aperture 0.40) and processed using ImageJ software (the nuclei were pseudocolored with red for better illustration).</p

Infection with BVDV R₂, R₃ and R₅ leads to a decrease in dead cells.

<p>MDBK cells were infected with wt BVDV p0 or BVDV R_1–5 (MOI 0.01) and at 48 h p.i. apoptotic cells were measured as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100528#pone-0100528-g002" target="_blank">Figure 2</a>. Considering the differences in CPE between each viral infection, the total cells that would die by viral CPE was estimated (see Materials and Methods Section). The results are summarized in a bar graph as mean ± SD of % dead cells in relation to the mock-infected culture. *<b><i>p</i></b><0.050; Student's t test vs wt BVDV p0.</p

Antiviral activity of TSC against BVDV R_1–5.

<p>BVDV-TSC^r T_1–5 were propagated in MDBK cells in the absence of TSC during 20 passages. The antiviral activity of 80 µM of TSC against the viral populations obtained (BVDV R₁–R₅) and wt BVDV p0 was evaluated by plaque reduction assays. White and black bars indicate the number of viral plaque forming units (PFUs) formed in the presence or in the absence of TSC, respectively. (TSC vs untreated: R₁<b><i>p</i></b> =  0.560; R₂<b><i>p</i></b> = 0.054; R₃<b><i>p</i></b> =  0.449; R₄<b><i>p</i></b> =  0.382; R₅<b><i>p</i></b> =  0.002; p0 <b><i>p</i></b> =  0.003).</p

Oligonucleotides used for retrotranscription (RT) reactions and PCR amplifications.

<p>Sequences and positions in the genome of BVDV NADL of the oligonucleotides designed and used for RT reactions and PCR amplifications of Npro, glycoprotein Erns and non-structural proteins coding region. Positions in the BVDV genome are according to BVDV NADL genome sequence GenBank accession no AJ133738.1.</p

Molecular characterization of BVDV R_1–5.

<p>The complete nucleotide sequence of the Erns, Npro and NS region from wt BVDV p0, BVDV-TSC^r T_1–5 and BVDV R_1–5 genomes was obtained by direct sequencing. The nucleotide sequences are arranged in three or four codons and the position in the genome of the first nucleotide of each group of sequences is indicated above according to the complete genome of BVDV (GenBank accession no. NC_001461.1). Only the genome regions which showed mutations are displayed. Each BVDV-TSC^r T-R pair is shown in gray or white rows and, for non-synonymous mutations, amino acid changes are indicated below the corresponding nucleotide sequence, indicating the number of amino acids for each protein. Viruses that showed altered CPE (BVDV R₂, R₃ and R₅) are indicated in boldface.</p

Apoptosis measurements.

<p>MDBK cells were infected with wt BVDV p0 or BVDV R_1–5 (MOI 0.01) and at 48 h p.i. apoptotic cells were measured by incorporation of PE Annexin V and 7AAD dyes and analyzed by flow cytometry. Mock-infected cultures were added as control. Representative density plots (20,000 events) obtained with WinMDI software are displayed. Quadrants delimitating positive (pos) and negative (neg) events are shown and the percentage of events in each quadrant is indicated. The populations of PE Annexin V−/7AAD−, PE Annexin V+/7AAD− and PE Annexin V+/7AAD+ correspond to viable cells, early apoptotic cells, and late apoptotic cells.</p

Viral RNA production.

<p>MDBK cells were infected with wt BVDV p0, or BVDV R₁, R₂ or R₃ (MOI 1) and the intracellular viral RNA (ivRNA) was quantified at 24 h p.i. by real-time PCR. For each infection with BVDV R viruses, the results are expressed as ivRNA relative to wt BVDV p0. Mock-infected cells were added as control in each experiment. The results are shown as mean ± SD from two independent experiments in triplicate. *<b><i>p</i></b><0.050; Student's t test vs wt BVDV p0.</p

Choice and Outcomes of Rate Control versus Rhythm Control in Elderly Patients with Atrial Fibrillation: A Report from the REPOSI Study

Background: Among rate-control or rhythm-control strategies, there is conflicting evidence as to which is the best management approach for non-valvular atrial fibrillation (AF) in elderly patients. Design: We performed an ancillary analysis from the \u2018Registro Politerapie SIMI\u2019 study, enrolling elderly inpatients from internal medicine and geriatric wards. Methods: We considered patients enrolled from 2008 to 2014 with an AF diagnosis at admission, treated with a rate-control-only or rhythm-control-only strategy. Results: Among 1114 patients, 241 (21.6%) were managed with observation only and 122 (11%) were managed with both the rate- and rhythm-control approaches. Of the remaining 751 patients, 626 (83.4%) were managed with a rate-control-only strategy and 125 (16.6%) were managed with a rhythm-control-only strategy. Rate-control-managed patients were older (p&nbsp;=&nbsp;0.002), had a higher Short Blessed Test (SBT; p&nbsp;=&nbsp;0.022) and a lower Barthel Index (p&nbsp;=&nbsp;0.047). Polypharmacy (p&nbsp;=&nbsp;0.001), heart failure (p&nbsp;=&nbsp;0.005) and diabetes (p&nbsp;=&nbsp;0.016) were more prevalent among these patients. Median CHA2DS2-VASc score was higher among rate-control-managed patients (p&nbsp;=&nbsp;0.001). SBT [odds ratio (OR) 0.97, 95% confidence interval (CI) 0.94\u20131.00, p&nbsp;=&nbsp;0.037], diabetes (OR 0.48, 95% CI 0.26\u20130.87, p&nbsp;=&nbsp;0.016) and polypharmacy (OR 0.58, 95% CI 0.34\u20130.99, p&nbsp;=&nbsp;0.045) were negatively associated with a rhythm-control strategy. At follow-up, no difference was found between rate- and rhythm-control strategies for cardiovascular (CV) and all-cause deaths (6.1 vs. 5.6%, p&nbsp;=&nbsp;0.89; and 15.9 vs. 14.1%, p&nbsp;=&nbsp;0.70, respectively). Conclusion: A rate-control strategy is the most widely used among elderly AF patients with multiple comorbidities and polypharmacy. No differences were evident in CV death and all-cause death at follow-up