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Antiviral Research (v.86, #2)
Efficacy of favipiravir (T-705) and T-1106 pyrazine derivatives in phlebovirus disease models by Brian B. Gowen; Min-Hui Wong; Kie-Hoon Jung; Donald F. Smee; John D. Morrey; Yousuke Furuta (pp. 121-127).
Several studies have reported favipiravir (T-705) to be effective in treating a number of viral diseases modeled in rodent systems. Notably, the related pyrazine derivative, T-1106, was found to be more effective than T-705 in treating yellow fever virus infection in hamsters. Based on these findings, we hypothesized that T-1106 may be more effective in treating hepatotropic Punta Toro virus (PTV, Phlebovirus) infection in rodents. In cell culture, the inhibitory concentrations of the compounds against various phleboviruses ranged from 3 to 55μM for T-705 and from 76 to 743μM for T-1106. In PTV-challenged hamsters, a model that generally presents with high liver viral loads, T-1106 was more effective at reducing mortality. However, in mice infected with PTV, a model wherein systemic infection is more prominent, the greater efficacy exhibited by T-1106 in the hamster system was not apparent. In contrast, T-705 was superior in preventing mortality in hamsters challenged with Pichinde virus (PICV, Arenavirus), an infection characterized as diffuse and pantropic. Remarkably, T-1106 has proven more active in vivo than would have been expected from our cell culture results, and our in vivo findings suggest that it is more effective in infections characterized predominantly by high levels of hepatic viral burden.
Keywords: Punta Toro virus; Rift Valley fever; Arenavirus; Phlebovirus; T-705; T-1106
Antiviral drug profile of seasonal influenza viruses circulating in Portugal from 2004/2005 to 2008/2009 winter seasons by Vanessa Correia; Helena Rebelo de Andrade; Luís A. Santos; Angie Lackenby; Maria Zambon (pp. 128-136).
A research project on antiviral drug resistance of influenza viruses circulating in Portugal has been carried out since 2007. Here, the first results obtained regarding the evaluation of susceptibility to amantadine and oseltamivir are presented. Information about antiviral prescription and exposure was available through the National Influenza Surveillance Programme. Amantadine susceptibility was evaluated by pyrosequencing for known resistance markers on 178 influenza A strains from 2004/2005 to 2006/2007. Susceptibility to oseltamivir was evaluated by 50% inhibitory concentration determination on 340 virus strains from 2004/2005 to 2008/2009, 134 of which were further analyzed by sequencing of the neuraminidase gene. This study revealed that influenza antiviral drugs were rarely prescribed at national level. Resistance to amantadine was observed on only A(H3N2) strain isolated during 2005/2006 and on 38 (74.5%) of the 51 A(H3N2) strains from 2006/2007, all carrying the mutation S31N in their M2 sequence. Oseltamivir resistance was observed in 6 (20.7%) of the 29 A(H1N1) strains from 2007/2008 and in all strains from 2008/2009, which exhibited extremely high IC50 values and carrying the mutation H275Y in their neuraminidase sequence. The national data generated and analyzed in this study may contribute to increase the knowledge on influenza antiviral drug resistance which is a problem of global concern.
Keywords: Influenza; Antiviral drugs; Amantadine; Oseltamivir; Resistance; Portugal
Effect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiation by Giuseppe Caso; Izolda Mileva; Margaret A. Mcnurlan; Dennis C. Mynarcik; Frank Darras; Marie C. Gelato (pp. 137-143).
Protease inhibitors (PIs) have been implicated in the development of HIV-associated lipodystrophy through a reduction in the differentiation of preadipocytes. While atazanavir (ATV) is associated with fewer clinical metabolic abnormalities in the short-term, the effects of long-term exposure are not known. ATV effects on preadipocyte replication or differentiation would indicate the potential for long-term problems. This study compared ritonavir (RTV) and ATV effects on preadipocyte replication and differentiation in human primary cultures.Preadipocytes from subcutaneous fat were studied in the presence of therapeutic concentrations of RTV and ATV for replication, differentiation, and adipokine secretion. The effects of the drugs on the expression of PPARγ and related genes during differentiation were also assessed by real-time quantitative PCR. RTV induced a significant inhibition of preadipocyte proliferation, differentiation and adiponectin secretion. ATV at concentrations within the range of therapeutic levels did not affect differentiation or adiponectin secretion, but did have inhibitory effects on preadipocyte proliferation. Inhibition of differentiation by PIs was associated with decreased expression of PPARγ, C/EBPα, and aP2 genes.In summary, although ATV at therapeutic levels has a smaller impact on adipogenesis, alterations in preadipocyte proliferation suggest the potential for adverse effects with long-term use.
Keywords: Adipogenesis; Adiponectin; HIV protease inhibitors; Preadipocyte; PPARγ
A novel Hepatitis C virus p7 ion channel inhibitor, BIT225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-α-2b and nucleoside analogues by Carolyn A. Luscombe; Zhuhui Huang; Michael G. Murray; Michelle Miller; John Wilkinson; Gary D. Ewart (pp. 144-153).
The novel small molecule, BIT225 ( N-[5-(1-methyl-1 H-pyrazol-4-yl)-napthalene-2-carbonyl]-guanidine: CAS No. 917909-71-8), was initially identified using a screening strategy designed to detect inhibitors of Hepatitis C virus (HCV) p7 ion channel activity. Here we report that BIT225 has potent stand-alone antiviral activity against the HCV model pestivirus bovine viral diarrhea virus (BVDV) with an IC50 of 314nM. Combinations of BIT225 with recombinant interferon alpha-2b (rIFNα-2b) show synergistic antiviral action against BVDV and the synergy is further enhanced by addition of ribavirin. Synergy was also observed between BIT225 and two nucleoside analogues known to inhibit the HCV RNA-dependent RNA polymerase.BIT225 has successfully completed a phase Ia dose escalating, single dose safety trial in healthy volunteers and a phase Ib/IIa trial to evaluate the safety and pharmacokinetics of repeated dosing for selected doses of BIT225 in HCV-infected persons. A modest, but statistically significant drop in patient viral load was detected over the 7 days of dosing (ref.www.biotron.com.au). Given the critical role of the p7 protein in the HCV life cycle and pathogenicity, our data indicate that molecules like BIT225, representing a new class of antiviral compounds, may be developable for therapeutic use against HCV infection, either as monotherapy, or in combination with other HCV drugs.
Keywords: Novel antiviral compound; Flavivirus inhibitor; BVDV; HCV; p7
In vitro antiviral activity of some uridine derivatives of 2-deoxy sugars against classical swine fever virus by Ewelina Krol; Ilona Wandzik; Wieslaw Szeja; Grzegorz Grynkiewicz; Boguslaw Szewczyk (pp. 154-162).
Classical swine fever virus glycoproteins: E2, Erns (E0) and E1 are detected on the external part of viral particles and play a major role in the initial stages of viral infection. They form heterodimeric and homodimeric complexes needed to effectively infect host cells. Some glycosylation inhibitors, such as tunicamycin, which act at the early stages of glycan chain processing, can influence, not only glycosylation, but also the stability of E2 and Erns glycoproteins, effectively inhibiting the formation of glycoprotein complexes and virus yield. In our study we tested two of newly designed uridine derivatives of 2-deoxy sugars, IW3 and IW7 mimicking part of tunicamycin. We showed that inhibitors effectively arrest viral growth with IC50 of 9 and 7μg/ml respectively, without significant toxicity for mammalian cells. Moreover, IW3 and IW7 reduced the formation of viral glycoproteins E2 and Erns in a dose-dependent manner. These compounds were further studied in order to elucidate the molecular mechanism of the antiviral effect using mammalian SK6 and insect Sf9 cell lines. We found that they inhibit N-glycosylation process of viral proteins at the late stage of glycan modification characteristic for mammalian cells. Due to the observed antiviral effect accompanied by low cytotoxicity, these inhibitors are potential candidates for anti-pestivirus therapy.
Keywords: Abbreviations; Asn; asparagine; BDV; border disease virus; BVDV; bovine viral diarrhea virus; CC; 50; concentration of the compound required to reduce cell viability by 50%; CSF; classical swine fever; CSFV; classical swine fever virus; DMSO; dimethyl sulfoxide; E; rns; (E0), E2, E1; CSFV envelope proteins; Endo H; endoglycosidase H; ER; endoplasmic reticulum; EU; European Union; FBS; fetal bovine serum; GTs; glycosyltransferases; IC; 50; concentration of the compound required to reduce virus plaque formation by 50%; IPMA; immunoperoxidase monolayer assay; MOI; multiplicity of infection; NR; neutral red; PBS; phosphate-buffered saline; PNGase F; peptide:N-glycosidase F; PRV; pseudorabies virus; PVDF; Polyvinylidene fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ser; serine; Sf9; Spodoptera frugiperda; insect cell line; S.I.; selectivity index; SK6; swine kidney cells; Thr; threonine; Xaa; any amino acidClassical swine fever virus; Glycoproteins; Glycosylation inhibition; Tunicamycin derivatives
A high-throughput assay using dengue-1 virus-like particles for drug discovery by Min Qing; Wei Liu; Zhiming Yuan; Feng Gu; Pei Yong Shi (pp. 163-171).
Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50–100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using viral structural proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the structural proteins. In both approaches, the key to produce high titer VLP (3×106foci-forming unit/ml) is to use low temperature (30°C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against viral envelope protein and by an inhibitor of viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
Keywords: Abbreviations; VLP; virus-like particle; DENV; dengue virus; HTS; high-throughput screen; C; capsid protein; prM; premembrane; E; envelope; NS; non-structural protein; FFU; focus-forming unit; VEEV; Venezuelan equine encephalitis virus; SFV; Semliki forest virus; Rluc; Renilla luciferase; FMDV2A; foot-and-mouth disease virus 2A autocleavage siteVLP (virus-like particle); Dengue; Luciferase reporter; HTS (high-throughput screen)
A peptide derived from hepatitis C virus E2 envelope protein inhibits a post-binding step in HCV entry by R. Liu; M. Tewari; R. Kong; R. Zhang; P. Ingravallo; R. Ralston (pp. 172-179).
The HCV envelope proteins E1 and E2 are required for virus binding to cellular receptors and pH-dependent fusion with endosomal membranes. Envelope protein interactions within this multistep process may provide novel targets for development of antiviral agents. To identify E1 and E2 regions involved in critical steps of HCV entry, we screened an E1E2 overlapping peptide library for inhibition of infection using a lentiviral reporter vector pseudotyped with E1E2 envelope proteins. A 16-residue polypeptide containing a portion of the E2 transmembrane domain (Peptide 75) inhibited HCV pseudoparticle infection with an IC50 of approximately 0.3μM and did not inhibit infection by VSV-g pseudoparticles at concentrations up to 50μM. Structure–activity analysis of Peptide 75 showed that antiviral activity was dependent upon L-configuration and hydrophobic character, and that the native sequence was required for maximal activity. Peptide 75 did not show virocidal activity against HCV pseudoparticles or other viruses. Temperature-shift experiments showed that the peptide acted at a post-binding step and that inhibition was further increased when used in combination with an anti-CD81 antibody previously shown to inhibit pseudoparticle entry at a post-binding step. These data suggest that interactions involving the C terminal region of E2 may play an important role in the HCV entry process.
Keywords: E2; Peptide; Entry
Reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against H5N1 infection in mice by Mookkan Prabakaran; Selvaraj Madhan; Nayana Prabhu; Grace Yuhong Geng; Roger New; Jimmy Kwang (pp. 180-187).
Induction of mucosal immunity through oral immunization is an effective way to control influenza infection. In this study, baculovirus displaying influenza hemagglutinin was encapsulated within a reverse micelle structure of phosphatidylcholine and delivered into the gastrointestinal tract of mice to study its efficacy as an oral vaccine against cross-clade H5N1 infection. Mice vaccinated with encapsulated baculovirus displaying HA (En-BacHA) showed significantly enhanced HA specific serum IgG and mucosal IgA antibodies, and higher hemagglutination inhibition (HI) titers, when compared to its non-encapsulated form (BacHA). Estimation of serum neutralizing antibodies also indicated that En-BacHA formulation was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, clade 4.0 and clade 8.0). Further, mice vaccinated with En-BacHA alone were able to confer 100% protection against 5MLD50 of HPAI heterologous H5N1 strain (clade 1). Inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant in the vaccine formulation did not show any significant effect in both systemic and mucosal immune responses. Oral delivery of encapsulated recombinant H5 HA expressed on baculovirus surface is an effective way to prime the immune system against H5N1 infection in mice and will have no biosafety concerns associated with their production or administration.
Keywords: Reverse micelle; Baculovirus; Oral; H5N1; Surface display
Recurrent vaginal shedding of herpes simplex type 2 virus in the mouse and effects of antiviral therapy by Nicholas Farley; David I. Bernstein; Fernando J. Bravo; Julie Earwood; Nancy Sawtell; Rhonda D. Cardin (pp. 188-195).
A mouse model of recurrent herpes simplex type 2 (HSV-2) would improve our understanding of the immunobiology of recurrent disease and provide a useful model for evaluating antiviral treatments. We developed a model to evaluate recurrent vaginal HSV-2 shedding using high-dose acyclovir (ACV) therapy beginning at 3 days post infection (dpi). Treatment with 150mg/kg of ACV for 10 days increased survival to 80% following vaginal challenge with HSV-2 strain 186 and to 100% after challenge with strain MS. We then evaluated recurrent vaginal HSV-2 shedding in surviving mice. Although infectious virus was not detected in vaginal samples after 21dpi, viral DNA was detectable by PCR in 80% of mice (47/59) on at least 1 day, while no animal was positive for virus on every day. ACV therapy administered from day 21 to 31 significantly reduced recurrent virus shedding during this period from 7.3% (8/109 swabs) to 0.8% (1/126 swabs) ( p=0.013). Lastly, ACV-rescued HSV-2-infected mice treated with cyclophosphamide at 35 and 38dpi rapidly succumbed, indicating that this model can be used to study immune control of the persistent infection. Thus, this model provides an inexpensive model for evaluating therapeutic strategies and immune control of persistent HSV.
Keywords: Herpes simplex virus; Recurrent HSV shedding; Genital herpes; Animal model
A highly lipophilic sulfated tetrasaccharide glycoside related to muparfostat (PI-88) exhibits virucidal activity against herpes simplex virus by Maria Ekblad; Beata Adamiak; Tomas Bergstrom; Ken D. Johnstone; Tomislav Karoli; Ligong Liu; Vito Ferro; Edward Trybala (pp. 196-203).
Although sulfated polysaccharides potently inhibit the infectivity of herpes simplex virus (HSV) and human immunodeficiency virus in cultured cells, these compounds fail to show protective effects in humans, most likely due to their poor virucidal activity. Herein we report on sulfated oligosaccharide glycosides related to muparfostat (formerly known as PI-88) and their assessment for anti-HSV activity. Chemical modifications based on the introduction of specific hydrophobic groups at the reducing end of a sulfated oligosaccharide chain enhanced the compound's capability to inhibit the infection of cells by HSV-1 and HSV-2 and abrogated the cell-to-cell transmission of HSV-2. Furthermore, modification with a highly lipophilic cholestanyl group provided a compound with virucidal activity against HSV. This glycoside targeted the viral particle and, to a lesser degree, the cell, and exhibited an antiviral mode of action typical for sulfated polysaccharides and virucides, i.e., interference with the virus attachment to cells and irreversible inactivation of virus infectivity, respectively. The virucidal activity was decreased in the presence of human cervical secretions suggesting that higher doses of this glycoside might be needed for in vivo application. Altogether, the sulfated oligosaccharide-cholestanyl glycoside exhibits potent anti-HSV activity and is, therefore, a good candidate for development as a virucide.
Keywords: Herpes simplex virus; Sulfated oligosaccharide glycosides; Virucidal activity
Optimization of shRNA inhibitors by variation of the terminal loop sequence by Nick C.T. Schopman; Ying Poi Liu; Pavlina Konstantinova; Olivier ter Brake; Ben Berkhout (pp. 204-211).
Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. We were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1.
Keywords: Gene silencing; shRNA; siRNA; Hairpin loop; HIV-1 replication
In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor by H. Javanbakht; R.G. Ptak; E. Chow; J.M. Yan; J.D. Russell; M.K. Mankowski; P.A. Hogan; J.H. Hogg; H. Vora; J.Q. Hang; Y. Li; G. Su; A. Paul; N. Cammack; K. Klumpp; G. Heilek (pp. 212-219).
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.
Keywords: HIV-1; Nonnucleoside reverse transcriptase inhibitor; Resistance
Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection by Zhilei Chen; Rudo L. Simeon; Karuppiah Chockalingam; Charles M. Rice (pp. 220-223).
The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20-fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2-fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors.
Keywords: Abbreviations; HCV; hepatitis C virus; HCVcc; HCV cell culture; shRNA; small hairpin RNA; Gluc; Gaussia; luciferase; 2′-CMA; 2′-C-methyladenosineCytopathic effect; Genetic selection; Enrichment; Plaque assay; n4mBid (hepatoma cell line)
Clinical outcome in resistant HIV-2 infection treated with raltegravir and maraviroc by Darius Armstrong-James; Justin Stebbing; Andrew Scourfield; Erasmus Smit; Bridget Ferns; Deenan Pillay; Mark Nelson (pp. 224-226).
Therapy for infection with HIV-2 remains limited. We report an HIV-2-infected patient in whom genotyping demonstrated PI, NRTI and NNRTI resistance, with a subsequent response to raltegravir- and maraviroc-based therapy. Further studies are required to assess the clinical efficacy of maraviroc in HIV-2 infection.
Keywords: HIV-2; Raltegravir; Maraviroc; Outcome; Genotyping
The frequency and reasons for antiretroviral switching with specific antiretroviral associations: The SWITCH study by I. Davidson; H. Beardsell; B. Smith; S. Mandalia; M. Bower; B. Gazzard; M. Nelson; J. Stebbing (pp. 227-229).
We investigated the reasons for switching antiretroviral regimens, an issue rarely addressed in cohort studies.An observed toxicity switch rate (OTSR) was calculated by Poisson regression using the number of days individuals received each individual antiretroviral drug.Of 3333 individuals receiving HAART, a total of 14% of regimens were switched, the majority occurring after 6 months of therapy. Toxicity was the major reason for switching (61%) and there were no major statistically significant differences in OTSR between the protease inhibitor (OTSR 26.4, 95% CI 18.3–37) and non-nucleoside reverse transcriptase inhibitor (OTSR 22.2, 95% CI 13.6–34.4) based regimes. For individual antiretrovirals, stavudine and zidovudine had significantly higher “switch” scores than all other drugs.There were no differences between the major HAART classes in OTSR. We suggest that newer antiretrovirals will require differentiation in terms of longer-term toxicity, as this is the major reason for switching.
Keywords: Adherence; Compliance; Toxicity; Switching; Antiretroviral; HAART