456 research outputs found
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
No full textBACKGROUND:
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
Supplemental_Matherial_Cell_based_Immunodetection_Assay_DENV_ZIKV_Antivirals_by_Vicenti_et_al_rev_2 – Supplemental material for Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication
No full textSupplemental material, Supplemental_Matherial_Cell_based_Immunodetection_Assay_DENV_ZIKV_Antivirals_by_Vicenti_et_al_rev_2 for Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication by Ilaria Vicenti, Filippo Dragoni, Alessia Giannini, Federica Giammarino, Michele Spinicci, Francesco Saladini, Adele Boccuto and Maurizio Zazzi in SLAS Discovery</p
Novel antiviral strategies against emerging viral diseases
No full textNovel antiviral strategies against emerging viral diseases
Doctoral Research School of Biochemistry and Molecular Biology-Cycle XXXII; Tutor: Luisa Bracci; Supervisor: Maurizio Zazzi; Candidate: Adele Boccuto
Background
Zika virus (ZIKV) Dengue virus (DENV) and West Nile virus (WNV) belong to the Flaviviridae family. The World Health Organization has ranked DENV as the most critical mosquito-borne viral disease and declared ZIKV an international public health emergency. Indeed, WNV is widely established in USA and some European areas and is associated to neuroinvasive disease. Despite intensive work, no specific antiviral therapy is available for ZIKV, DENV or WNV. In addition, increasing co-infections with different flaviviruses or serotypes cocirculating within the same area complicate the clinical outcome and treatment options. The high degree of conservation of some viral enzymes or host factor essential for Flaviviridae replication makes it reasonable to search for “broad-spectrum” antivirals.
Materials and Methods
The half-maximal cytotoxic concentration (CC50) of candidate inhibitors was calculated by Cell Titer-Glo cell viability assay. We determined the drug concentration inhibiting 50% of viral replication (IC50) of ZIKV, DENV and/or WNV by infection of Huh7 (hepatoma cell line) at a multiplicity of infection (MOI) of 0.005 (DENVZIKV) or 0.0025 (WNV) for 1 h and at the end of incubation treating Huh7 cells with serial dilutions of candidate compound at 37°C and 5% CO2. After 72 h (DENV-ZIKV) or 48h (WNV), the IC50 of compounds interfering with the virus life cycle up to protein production, but not at later steps, was directly measured by immune-detection assay (direct yield reduction assay, DYRA).
Otherwise, to determine the impairment of viral replicative capacity exerted at late steps of viral replication, e.g. viral assembly and budding, we performed an additional replicative cycle or by infection of pre-seeded Huh7 (Secondary yield reduction assay, SYRA) with supernatants obtained by first round of infection or by Plaque reduction assay (PRA) as previously described (Vicenti et al., 2018). Selectivity indexes (SI) were calculated as ratio between CC50 and IC50. For in vitro ZIKV resistance selection to sofosbuvir, ZIKV was propagated in the Huh-7 cell line with increasing
concentrations of sofosbuvir. Viral clones replicating at each drug increment step were collected and sequenced to detect emergent mutations in the polymerase (NS5) region and sofosbuvir IC50 was measured by DYRA.
Results and discussion
Evaluation of anti-DENV compounds targeting cellular DEAD-box 3 RNA helicase. Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3 (DDX3) belongs to a family of ATP-dependent RNA helicases and is involved in many aspects of RNA metabolism as well as potentially participating to the antiviral innate immune signaling pathways. On the other hand, many studies highlight DDX3 as an essential host factor for the replication of clinically relevant viruses. As part of the activities planned in the UNAVIR and PANVIR project and in collaboration with First Health Pharmaceuticals, we evaluated the effect of DDX3 helicase inhibitors focusing on antiviral activity against DENV. Overall we tested 51 compounds, 31 showed anti-DENV activity and among these 11 molecules showed promising antiviral activity with median SI 66 (IQR 59-74). Overall, the preliminary data obtained so far support targeting DDX3 protein as a potentially effective strategy for inhibition of DENV replication and feasible broad-spectrum option to minimize the possibilities of generating resistance. The evaluation of the mechanism of action of a DDX3 inhibitor compound are currently under evaluation.
Evaluation of candidate antivirals agents targeting viral entry/fusion.
Virus entry by enveloped viruses is dependent not only on cellular receptors but also on cellular lipids playing multifaceted roles in viral infections as structural components and as necessary cofactors for viral replication. In this scenario, the possibility to identify broad-spectrum antiviral molecules acting on viral envelopes is of particular interest. We tested derivatives entry inhibitors of the previously identified MAS family (Cagno et al., 2018; Tintori et al., 2018) expected to affect virus-cell membrane fusion during DENV and ZIKV infection. We evaluated the
antiviral activity of MAS compounds adapting DYRA and incubating Huh7 cells with serial dilution of MAS molecules for 30 minutes before infection with ZIKV or DENV. Of the ten compounds investigated, five showed antiviral activity both against ZIKV (median SI 16 μM; IQR 12-26) and DENV (median SI 7; IQR 6-14), 3 showed antiviral activity against ZIKV in DYRA (median SI 8; IQR
6.5-17) and anti-DENV activity in SYRA (median SI 11; IQR 9-14), while 2 compounds were completely inactive . Interestingly, the three compound active only against ZIKV were found active also against DENV in SYRA (median SI 11; IQR 9-14). We further evaluated the antiviral activity of MAS9 showing IC50 values of 2.1±1.6 (SI 14) and 2.6±2.2 (SI 12) for DENV and ZIKV, respectively. The evaluation of the mechanism of action confirmed the antiviral activity of MAS9 in viral entry/fusion step of viral replication and through a higher selectivity for viral membranes. These preliminary results support the strategy to target selectively the viral membrane, possibly based on the efficiency of the repair activity of host cell, but not virus, membranes. This class of compounds warrants further investigations to prove and characterize the mechanism of action and the ability to inhibit different emerging enveloped viruses.
Evaluation of candidate anti-flaviviral agents targeting the NS3-NS5 interaction.
A family of 2,6-diaminopurine derivatives targeting a conserved allosteric pocket on DENV/ZIKV NS5 polymerase, which is required to bind NS3 and generate the functional replication complex, has been recently discovered (Vincettiet al., 2019, 2015). Herein, we analysed a new series of derivatives molecules structurally modified to increase the affinity for the allosteric pocket on NS5 and reduce their cellular toxicity. Of the 21 derivatives compounds investigated against ZIKV and DENV, 5 were cytotoxic, 16 showed anti-ZIKV activity (median SI 9 [IQR 7-26.5]) and 13 showed anti-DENV activity (median SI 11 [IQR 6-13]) in SYRA. The best SI was obtained for compound MR333 with SI of 182 and 77 for ZIKV and DENV, respectively. Moreover, MR333 was found to be active also against WNV with comparable antiviral activity in PRA and SI of 55. This class of compounds appears to be attractive and warrants further evaluation of the mechanism of action and the genetic barrier to the emergence of resistance mutations. Biochemical assays investigating MR333 are underway to confirm and characterize the expected inhibition of multiple viruses based on binding to the highly conserved pocket on the thumb of Flavivirus polymerases.
Characterization of in vitro ZIKV resistance to sofosbuvir.
Given the high degree of NS5 homology observed
among members of the Flaviviridae family (Boldescu et al., 2017; Lim et al., 2013), sofosbuvir has been recently evaluated as an anti-Flavivirus lead candidate. Indeed, the anti-ZIKV activity of sofosbuvir has been showed in vitro with cell-based assay and in animal models. However, sofosbuvir drug genetic barrier has been not yet evaluated with ZIKV. The virus breakthrough time grew with increasing sofosbuvir concentration of 5, 10, 20 and 40 μM (5, 15, 22 and 40±9 days, respectively) with exception of 80 μM (20±2 days). No sequence variations were found up to
20 μM sofosbuvir, while the mutations V360L and V607I were selected in presence of 40 μM sofosbuvir and 80 μM. The mutations C269Y and H289T were individually selected in two experiments with 80 μM sofosbuvir. Globally, majority of mutations observed in Sanger sequencing were confirmed by NGS with addition of N407T, T833I, K834C and W835R mutations which were not associated to sofosbuvir drugpressure. By DYRA we demonstrated the reduced susceptibility to sofosbuvir of viruses carrying the C269Y, V360L and V607I mutations. Moreover, the analysis of NS5 gene showed that the V607I is located near the well-known mutation S282T in SGxxxT consensus sequence of HCV NS5B, within the motif B, and is implicated in drug resistance of HCV toward nucleotide inhibitors (Gane et al., 2016). Interestingly, the V360L mutation is located in a well-known stretch of amino acids termed β-nuclear localization sequence (positions 320-368), whereas, the C269Y is located in the poorly conserved 10-residue linker domain (aminoacidic positions 263 to 272) which is essential for the adequate NS5 conformation. Therefore, the mutations detected are probably related to emergence of ZIKV resistance to sofosbuvir in vitro and, certainly, require further biochemical investigations.
References
Boldescu, V., Behnam, M.A.M., Vasilakis, N., Klein, C.D., 2017. Broad-spectrum agents for flaviviral infections: Dengue, Zika and beyond. Nat. Rev. Drug Discov. https://doi.org/10.1038/nrd.2017.33Cagno, V., Tintori, C., Civra, A., Cavalli, R., Tiberi, M., Botta, L., Brai, A., Poli, G., Tapparel, C., Lembo, D.,
Botta, M., 2018. Novel broad spectrum virucidal molecules against enveloped viruses. PLoS One 13, 1– 18. https://doi.org/10.1371/journal.pone.0208333
Gane, E.J., Shiffman, M.L., Etzkorn, K., Morelli, G., Stedman, C., Davis, M.N., Hinestrosa, F., Dvory-Sobol, H., Huang, K.C., Osinusi, A., McNally, J., Brainard, D., McHutchison, J., Thompson, A., Sulkowski, M., 2016. Sofosbuvir/Velpatasvir in Combination with Ribavirin for 24 Weeks is Effective Retreatment for Patients who failed Prior NS5A Containing DAA Regimens: Results of the GS-US-342-1553 Study. J.Hepatol. 64, S147–S148. https://doi.org/10.1016/s0168-8278(16)00037-4
Lim, S.P., Koh, J.H.K., Seh, C.C., Liew, C.W., Davidson, A.D., Chua, L.S., Chandrasekaran, R., Cornvik, T.C., Shi,P.Y., Lescar, J., 2013. A crystal structure of the dengue virus non-structural protein 5 (NS5) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities. J. Biol. Chem. 288, 31105–31114. https://doi.org/10.1074/jbc.M113.508606
Tintori, C., Iovenitti, G., Ceresola, E.R., Ferrarese, R., Zamperini, C., Brai, A., Poli, G., Dreassi, E., Cagno, V., Lembo, D., Canducci, F., Botta, M., 2018. Rhodanine derivatives as potent anti-HIV and anti-HSV microbicides. PLoS One 13, 1–19. https://doi.org/10.1371/journal.pone.0198478
Vicenti, I., Boccuto, A., Giannini, A., Dragoni, F., Saladini, F., Zazzi, M., 2018. Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro. Virus Res. 244, 64–70. https://doi.org/10.1016/j.virusres.2017.11.003
Vincetti, P., Caporuscio, F., Kaptein, S., Gioiello, A., Mancino, V., Suzuki, Y., Yamamoto, N., Crespan, E., Lossani, A., Maga, G., Rastelli, G., Castagnolo, D., Neyts, J., Leyssen, P., Costantino, G., Radi, M., 2015. Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases. J. Med. Chem. 58, 4964–4975. https://doi.org/10.1021/acs.jmedchem.5b00108
Vincetti, P., Kaptein, S.J.F., Costantino, G., Neyts, J., Radi, M., 2019. Scaffold Morphing Approach to Expand the Toolbox of Broad-Spectrum Antivirals Blocking Dengue/Zika Replication. ACS Med. Chem. Lett. 10, 558–563. https://doi.org/10.1021/acsmedchemlett.8b0058
Development of an internally controlled quantitative PCR to measure total cell-associated HIV-1 DNA in blood
No full textComparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro
No full textA 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 Î1⁄4M and 1.1 Î1⁄4M when measured through plaque assay, and 4.2 Î1⁄4M and 5.2 Î1⁄4M 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
The HIV-1 integrase E157Q polymorphism per se does not alter susceptibility to raltegravir and dolutegravir in vitro
No full textThe 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
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Adele Shtern: Catalog of Works and Images
No full textAdele Shtern was born in Montreal and received her BA in Design from Concordia University. She was awarded a Design Canada grant to study Graphic Design in the MFA program at Yale University. She has been a Professor of Art at the Tyler School of Art, the University of Bridgeport, FIT, and Baruch College/CUNY. She is currently an adjunct Professor in the Music and Art Department at BMCC/CUNY. She lives in Long Island City, NY where she continues to be an active artist. She is the co-author of an oral history book entitled Nicky D from LIC.
Shtern is considered a pioneer in computer art. Her involvement with digital art began in 1983 at the New York Institute of Technology on a University of Bridgeport grant. Committed to the process of art as a form of healing, Shtern conceived Lotus Cusp, a multimedia presentation re 9.11 with her BMCC/CUNY students. Several of her images are now on the 9.11 online Memorial Artists’ Registry. Her work has been exhibited extensively in solo and group shows in the US, Canada, Europe and Japan. It has been published internationally and has received many awards.
Adele Shtern embraces her calling as a multi-disciplinary artist using traditional and digital media. She takes pleasure in discovering visually interesting sights in diverse loci. Her creative process involves opening herself to the revelation of seeing the familiar in new ways. Painterly and abstract aspects of the environment catch her eye.
Shtern’s digital photos are part of an ongoing series of photographic explorations. Some of the sites have since been transformed or no longer exist. This record preserves the memory of what once existed, as a visual and cultural archive.
Types of work include: photographs, photomontages, journals of drawings, digital montage, gouache paintings, handmade paper, poetry and music
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No full textSecond-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 susceptibility of HIV-1 CRF02_AG to temsavir, the active compound of the attachment inhibitor fostemsavir
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