Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-8 (of 8 Records) |
Query Trace: Rhoden E[original query] |
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An automated high-throughput enterovirus D68 microneutralization assay platform
Rhoden EE , Mainou BA , Konopka-Anstadt JL , Oberste MS . J Virol Methods 2022 308 114590 Virus neutralization assays, widely used to detect and quantify antibodies induced by virus infection, are considered the gold standard for enterovirus serology testing. Conventional microneutralization assays have been used to assess enterovirus D68 (EV-D68) seroprevalence. While manual or automated 96-well assays are valuable, higher-density assays that increase throughput provide the opportunity to more efficiently screen large, population-based serology collections, as well as to test sample sets against multiple virus strains on the same plate or within the same run. Here, automation was implemented for bulk reagent dispensing, serial dilutions, and luminescence measurement to develop a 384-well enterovirus microneutralization assay that increases overall testing throughput, maintains the reproducibility of the standard 96-well assay, and reduces sample volume usage. EV-D68 strains Fermon, 14-18953, and 18-23087 were used to evaluate the automated 384-well microneutralization assay and compare to the conventional 96-well assay. Sensitivity and specificity were evaluated using pooled human sera and positive and negative control antisera. The Lower Limit of quantitation (LLOQ) was the same as for the 96-well assay and coefficients of variations (CV) of 7.35%, 5.97%, and 2.85% for the three EV-D68 strains respectively, were well below the typical goal of 20% CV for accuracy. Z-factor analysis yielded results of 0.694, 0.638, and 0.852, for the three EV-D68 strains respectively, indicating a high level of precision, reliability, and robustness. Intra-assay (7.25%) and inter-assay (7.12%) variability were well below 20% CV. Moreover, the 96-well and 384-well versions of the assay were highly concordant, with a 0.955 correlation coefficient in titers obtained for 50 sera tested. Validation of this automated 384-well microneutralization will support its use in large serology screens assessing the presence of EV-D68 neutralizing antibodies in human populations. |
Antifungal triazole posaconazole targets an early stage of the parechovirus A3 life cycle.
Rhoden E , Ng TFF , Campagnoli R , Nix WA , Konopka-Anstadt J , Selvarangan R , Briesach L , Oberste MS , Weldon WC . Antimicrob Agents Chemother 2019 64 (3) Viruses in species Parechovirus A (Picornaviridae) are associated with a wide variety of clinical manifestations. Parechovirus A3 (PeV-A3) is known to cause sepsis-like illness, meningitis, and encephalitis in infants and young children. To date, no specific therapies are available to treat PeV-A3-infected children. We had previously identified two FDA-cleared antifungal drugs, itraconazole (ITC) and posaconazole (POS) with potent and specific antiviral activity against PeV-A3. Time-of-addition and synchronized infection assays revealed that POS targets an early stage of the PeV-A3 life cycle. POS exerts an antiviral effect, evidenced by a reduction in viral titer following the addition of POS to Vero-P cells before infection, coaddition of POS and PeV-A3 to Vero-P cells, incubation of POS and PeV-A3 prior to Vero-P infection, and at attachment. POS exerts less of an effect on virus entry. A PeV-A3 ELISA inhibition experiment, using an anti-PeV-A3 monoclonal antibody (mAb), suggested that POS binds directly to the PeV-A3 capsid. POS-resistant PeV-A3 strains developed by serial passage in the presence of POS, acquired substitutions in multiple regions of the genome, including the capsid. Reverse genetics confirmed substitutions in capsid proteins VP0, VP3, VP1 and nonstructural proteins 2A and 3A. Single mutants VP0_K66R, VP0_A124T, VP3_N88S, VP1_Y224C, 2A_S788L and 3A_T1I were respectively 4-, 9-, 12-, 34-, 51-, and 119-fold more resistant to POS than its susceptible prototype strain. Our studies demonstrate that POS may be a valuable tool in developing an antiviral therapy for PeV-A3. |
In vitro antiviral activity of new oxazoline derivatives as potent poliovirus inhibitors
Madia VN , Messore A , Pescatori L , Saccoliti F , Tudino V , De Leo A , Scipione L , Fiore L , Rhoden E , Manetti F , Oberste MS , Di Santo R , Costi R . J Med Chem 2018 62 (2) 798-810 The final stages of polio eradication are proving more difficult than the early phases, and the development of effective drugs and treatments is considered a priority; thus, the research is ongoing. A screening of our in-house chemical library against poliovirus Sabin strains led to the identification of compounds 5 and 6 as hits active at submicromolar concentrations. Derivatives of these compounds were synthesized as a preliminary structure-activity-relationship study. Among them, 7 and 11 were highly active against poliovirus Sabin 1-3. Compound 11 was also very potent against a large panel of wild and vaccine-derived polioviruses. Time-of-addition experiments suggest that 5 and 7 could be active at an early stage of viral replication, whereas 11 was active at same concentration at all stages of viral replication. A ligand-based approach was applied to find the common structural features shared by the new compounds and already-known poliovirus inhibitors. |
Antifungal azoles itraconazole and posaconazole exhibit potent in vitro antiviral activity against clinical isolates of parechovirus A3 (Picornaviridae)
Rhoden E , Nix WA , Weldon WC , Selvarangan R . Antiviral Res 2017 149 75-77 Parechovirus A3 (Par-A3, formerly human parechovirus 3) is an emerging viral infection of the central nervous system in children. We used an automated, homogeneous, cell based assay to identify itraconazole and posaconazole as inhibitors of Par-A3, with antiviral activity below concentrations clinically attainable in pediatric patients. Currently, there is no approved antiviral treatment for Par-A3 infection, despite numerous reports of serious Par-A3 disease in neonates and infants. |
Antiviral activity of pocapavir in a randomized, blinded, placebo-controlled human oral poliovirus vaccine challenge model
Collett MS , Hincks JR , Benschop K , Duizer E , van der Avoort H , Rhoden E , Liu H , Oberste MS , McKinlay MA , Hartford M . J Infect Dis 2017 215 (3) 335-343 Background: Immunodeficient individuals who excrete vaccine-derived polioviruses threaten polio eradication. Antivirals address this threat. Methods: In a randomized, blinded, placebo-controlled study, adults were challenged with monovalent oral poliovirus type 1 vaccine (mOPV1) and subsequently treated with capsid inhibitor pocapavir or placebo. The time to virus negativity in stool was determined. Results: A total of 144 participants were enrolled; 98% became infected upon OPV challenge. Pocapavir-treated subjects (n = 93) cleared virus a median duration of 10 days after challenge, compared with 13 days for placebo recipients (n = 48; P = .0019). Fifty-two of 93 pocapavir-treated subjects (56%) cleared virus in 2-18 days with no evidence of drug resistance, while 41 of 93 (44%) treated subjects experienced infection with resistant virus while in the isolation facility, 3 (3%) of whom were infected at baseline, before treatment initiation. Resistant virus was also observed in 5 placebo recipients (10%). Excluding those with resistant virus, the median time to virus negativity was 5.5 days in pocapavir recipients, compared with 13 days in placebo recipients (P < .0001). There were no serious adverse events and no withdrawals from the study. Conclusions: Treatment with pocapavir was safe and significantly accelerated virus clearance. Emergence of resistant virus and transmission of virus were seen in the context of a clinical isolation facility. Clinical Trials Registration: EudraCT 2011-004804-38. |
In vitro efficacy of antiviral compounds against enterovirus D68
Rhoden E , Zhang M , Nix WA , Oberste MS . Antimicrob Agents Chemother 2015 59 (12) 7779-81 In 2014, the United States experienced a large outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68). We used a homogeneous, cell-based assay to assess antiviral activity of compounds developed for enterovirus/rhinovirus infection or other indications. Four of 16 compounds were highly active against all four strains tested (prototype and three 2014 strains), with EC50 of 0.0012 - 0.0051 muM. Additional studies are needed to assess their in vivo efficacy against EV-D68. |
H1PVAT is a novel and potent early-stage inhibitor of poliovirus replication that targets VP1
Tijsma A , Thibaut HJ , Spieser SA , De Palma A , Koukni M , Rhoden E , Oberste S , Purstinger G , Volny-Luraghi A , Martin J , Marchand A , Chaltin P , Neyts J , Leyssen P . Antiviral Res 2014 110c 1-9 A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core beta-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game. |
Anti-poliovirus activity of protease inhibitor AG-7404, and assessment of in vitro activity in combination with antiviral capsid inhibitor compounds
Rhoden E , Liu HM , Wang-Chern SW , Oberste MS . Antiviral Res 2013 98 (2) 186-91 The National Research Council has recommended that at least one, preferably two, polio antiviral drugs be developed as a supplement to the tools currently available for control of polio outbreaks post-eradication. The primary application of such drugs is expected to be the resolution of chronic poliovirus excretion in persons with primary immunodeficiency disorders. We have assessed the in vitro activity of AG-7404 (also known as "compound 1"), an inhibitor of picornaviral 3C protease, against a large panel of programmatically important poliovirus strains and its activity in combination with two poliovirus capsid inhibitors, V-073 and BTA798. AG-7404 was active against all viruses in this panel, with EC50 values ranging from 0.080 to 0.674muM. Similarly, BTA798 was active against all viruses in this panel, with EC50 values ranging from 0.003 to 0.591muM. By comparison, values for V-073 were 0.003-0.126muM. BTA798 was active against V-073-resistant variants with an alanine to valine change in VP3 at position 24. However, BTA798 was inactive against the V-073-resistant strains with amino acid substitutions at VP1 amino acids 194 (equivalent to 192 in type 3) and 236. As expected from its different mechanism of action, AG-7404 was fully active against all V-073-resistant variants, with EC50 values ranging from 0.218 to 0.819muM, compared to values of 0.202-0.407muM for the V-073-susceptible parental strains. In vitro drug combination experiments demonstrated synergy between AG-7404 and either V-073 or BTA798, whereas the combination of the two capsid inhibitors acted additively. |
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