Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication:

Practical cell-based assays can accelerate anti-Zika (ZIKV) and anti-dengue (DENV) virus drug discovery. We developed an immunodetection assay (IA), using a pan-flaviviral monoclonal antibody recognizing a conserved envelope domain. The final protocol includes a direct virus yield reduction assay (YRA) carried out in the human Huh7 cell line, followed by transfer of the supernatant to a secondary Huh7 culture to characterize late antiviral effects. Sofosbuvir and ribavirin were used to validate the assay, while celgosivir was used to evaluate the ability to discriminate between early and late antiviral activity. In the direct YRA, at 100, 50, and 25 TCID50, sofosbuvir IC50 values were 5.0 ± 1.5, 2.7 ± 0.5, 2.5 ± 1.1 µM against ZIKV and 16.6 ± 2.8, 4.6 ± 1.4, 2.6 ± 2.2 µM against DENV; ribavirin IC50 values were 6.8 ± 4.0, 3.8 ± 0.6, 4.5 ± 1.4 µM against ZIKV and 17.3 ± 4.6, 7.6 ± 1.2, 4.1 ± 2.3 µM against DENV. Sofosbuvir and ribavirin IC50 values determined in the secondary YRA were reproducible and comparable with those obtained by direct YRA and plaque reduction assay (PRA). In agreement with the proposed mechanism of late action, celgosivir was active against DENV only in the secondary YRA (IC50 11.0 ± 1.0 µM) and in PRA (IC50 10.1 ± 1.1 µM). The assay format overcomes relevant limitations of the gold standard PRA, allowing concurrent analysis of candidate antiviral compounds against different viruses and providing preliminary information about early versus late antiviral activity

Agreement between an in-house replication competent and a reference replication defective recombinant virus assay for measuring phenotypic resistance to HIV-1 protease, reverse transcriptase, and integrase inhibitors

BACKGROUND: Although clinical management of drug resistance is routinely based on genotypic methods, phenotypic assays remain necessary for the characterization of novel HIV-1 inhibitors, particularly against common drug-resistant variants. We describe the development and assessment of the performance of a recombinant virus assay for measuring HIV-1 susceptibility to protease (PR), reverse transcriptase (RT), and integrase (IN) inhibitors. METHODS: The system is based on the creation of replication-competent chimeric viruses through homologous recombination between patient or laboratory virus-derived PCR fragments and the corresponding NL4-3 vector where the whole Gag-PR, RT-RNaseH or IN coding regions has been deleted through inverse PCR. The susceptibility to nucleoside (NRTIs) and non-nucleoside (NNRTIs) RT inhibitors and to IN inhibitors (INIs) is calculated through a single-round infection assay in TZM-bl cells, while protease inhibitor (PI) activity is determined through a first round of infection in MT-2 cells followed by infection of TZM-bl cells with MT-2 supernatants. RESULTS: The assay showed excellent reproducibility and accuracy when testing PI, NRTI, NNRTI, and INI susceptibility of drug-resistant clones previously characterized through the reference pseudoparticle-based Phenosense assay. The coefficient of interassay variation in fold change (FC) resistance was 12.0%-24.3% when assaying seven drug/clones pairs in three runs. FC values calculated by the Phenosense and in-house for 20 drug/clones pairs were in good agreement, with mean±SD ratio of 1.14±0.33 and no cases differing by more than twofold. CONCLUSIONS: The described phenotypic assay can be adopted to evaluate the antiviral activity of licensed and investigational HIV-1 drugs targeting any of the three HIV-1 enzymes

The HIV-1 integrase E157Q polymorphism per se does not alter susceptibility to raltegravir and dolutegravir in vitro

The HIV-1 integrase E157Q natural polymorphism has been reported to cause one case of raltegravir (RAL) and dolutegravir (DTG) failure. Six recombinant viruses were constructed containing integrase from clinical HIV-1 isolates found to harbour E157Q as the only integrase strand inhibitor (INSTI) resistance-related mutation. Phenotypic analysis showed that E157Q results in minimal changes in RAL and DTG susceptibility. Together with data retrieved from the Stanford HIV database, our results indicate that E157Q is not a relevant INSTI resistance mutation per se. The previously reported case of E157Q-based resistance must have resulted from combined as yet unidentified integrase polymorphisms

Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro

A strong correlation between Zika virus (ZIKV) infection and severe neurological disease in newborns and occasionally adults has emerged in the Brazilian outbreak. Efficient human cell-based assays are required to test candidate inhibitors of ZIKV replication. The aim of this work was to investigate ZIKV propagation and quantification in different cell lines. The human (U87, A549, Huh7), mosquito (C6/36) and monkey (VERO E6) cell lines tested were all permissive to ZIKV infection. When assessed by plaque forming units (PFU) in three different target cell lines, the maximal production of ZIKV was achieved in Huh7 at day 3 post-infection (6.38 ± 0.44 log10PFU/ml). The C6/36 cell line showed a low and slow production of virus when compared with other cell lines. A549 readout cells generated a larger number of plaques compared to Huh7 but not to VERO E6 cells. ZIKV PFU and RNA titers showed the highest correlation when Huh7 and A549 were used as the producer and readout cells, respectively. Also, U87 cells produced ZIKV RNA titers which were highly correlated with PFU independently from the readout cell line. Using the best virus-cell system, sofosbuvir and ribavirin EC50were 1.2 μM and 1.1 μM when measured through plaque assay, and 4.2 μM and 5.2 μM when measured by quantitative real time PCR (qRT-PCR), respectively. In summary, ZIKV can efficiently infect different human cell lines and rapidly reach peak viral titers. Overall, A549 cells appear to be as efficient as the VERO E6 gold standard for plaque assay allowing the use of human, rather than simian, cells for evaluating candidate anti-ZIKV compounds by the reference assay. The possibility to replace the labor-intensive plaque assay with the more rapid and easy-to-perform qRT-PCR is appealing and warrants further investigation

Time Course of Neutralizing Antibody in Health Care Workers With Mild or Asymptomatic COVID-19 Infection

We describe the time course of neutralizing antibody (NtAb) titer in a cohort of health care workers with mild or asymptomatic severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. NtAb levels decreased over time; however, serum neutralizing activity remained detectable after a median of 7 months from SARS-CoV-2 diagnosis in the majority of cases

Comparable in vitro activity of second-generation HIV-1 integrase strand transfer inhibitors (INSTIs) on HIV-1 clinical isolates with INSTI resistance mutations

Second-generation HIV-1 integrase strand transfer inhibitors (INSTIs) dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) showed a high genetic barrier to resistance and limited cross-resistance with first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG). In this study, DTG, BIC, and CAB demonstrated a comparable activity on a panel of INSTI-resistant strains isolated from patients exposed to RAL, EVG, and/or DTG, with a significantly reduced susceptibility only with the pathway Q148H/K/R plus one to two additional INSTI mutations

In Vitro Anti-SARS-CoV-2 Activity of Selected Metal Compounds and Potential Molecular Basis for Their Actions Based on Computational Study

Metal-based drugs represent a rich source of chemical substances of potential interest for the treatment of COVID-19. To this end, we have developed a small but representative panel of nine metal compounds, including both synthesized and commercially available complexes, suitable for medical application and tested them in vitro against the SARS-CoV-2 virus. The screening revealed that three compounds from the panel, i.e., the organogold(III) compound Aubipyc, the ruthenium(III) complex KP1019, and antimony trichloride (SbCl3), are endowed with notable antiviral properties and an acceptable cytotoxicity profile. These initial findings prompted us to perform a computational study to unveil the likely molecular basis of their antiviral actions. Calculations evidenced that the metalation of nucleophile sites in SARS-CoV-2 proteins or nucleobase strands, induced by Aubipyc, SbCl3, and KP1019, is likely to occur. Remarkably, we found that only the deprotonated forms of Cys and Sec residues can react favorably with these metallodrugs. The mechanistic implications of these findings are discussed

Single-dose BNT162b2 mRNA COVID-19 vaccine significantly boosts neutralizing antibody response in health care workers recovering from asymptomatic or mild natural SARS-CoV-2 infection

Objectives: To measure SARS-CoV-2 neutralizing antibody (NtAb) titres in previously infected or uninfected health care workers who received one or two doses of BNT162b2 mRNA COVID-19 vaccine. Methods: NtAbs were titrated as dose-inhibiting 50% virus replication (ID50) by live virus microneutralization. We evaluated 41 health care workers recovering from mild or asymptomatic infection at first vaccination dose (T1_inf) and 21 days later (T2_inf). Sixteen uninfected health care workers were evaluated 20 days after first dose (T2_uninf) and 20 days after second vaccine dose (T3_uninf). Results: At T2_inf, but not at T1_inf, there was a significant correlation between days from diagnosis (median 313, interquartile range 285–322) and NtAb levels (P = 0.011). NtAb titres increased at T2_inf with respect to T1_inf (1544 (732–2232) vs 26 (10–88), P < 0.001). Similarly, there was a significant increase in NtAb titres at T3_uninf compared with T2_uninf (183 (111–301) vs 5 (5–15), P < 0001). However, NtAb levels at T2_inf were significantly higher than those at T2_uninf and T3_uninf (P < 0.0001 for both analyses). Conclusions: A single vaccination in people with mild or asymptomatic previous infection further boosts SARS-CoV-2 humoral immunity to levels higher than those obtained by complete two-vaccination in uninfected subjects