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Antiviral Research (v.91, #2)

Prelim II: Editorial Board (pp. ii).

Inside Back Cover: Notes to Authors (pp. ibc).

Assessment of the efficacy of the neuraminidase inhibitor oseltamivir against 2009 pandemic H1N1 influenza virus in ferrets by Elena A. Govorkova; Bindumadhav M. Marathe; Ashley Prevost; Jerold E. Rehg; Robert G. Webster (pp. 81-88).

Pandemic 2009 influenza A (H1N1) virus (H1N1pdm) is different from contemporary seasonal human viruses in that it can cause infection deep in the lungs of critical care patients. Here we establish a mammalian animal model and assessed the efficacy of the neuraminidase (NA) inhibitor oseltamivir treatment against H1N1pdm virus infection. Oseltamivir (25mg/kg/day twice daily for 5days) was orally administered to groups of ferrets, starting either 2 or 24h after inoculation with 10⁶PFU of A/California/04/2009 (H1N1) influenza virus. We determined that virus replication was restricted to 1 or 2 of 4 lung lobes in oseltamivir-treated animals, while virus was consistently isolated from 4 of 4 lung lobes in control animals (1.5–3.8log₁₀PFU/g). Analysis of arterial blood oxygenation revealed less pronounced changes in partial oxygen and carbon dioxide pressure in oseltamivir-treated ferrets, and histologic examination confirmed reduced pneumonia. Treated animals had significantly decreased inflammatory responses in the upper respiratory tract ( P<0.05), less fever and weight loss, and less reduction of activity. Virus titers in the nasal washes of treated and control ferrets did not differ significantly. NA sequencing and fluorescence-based phenotypic assays identified no oseltamivir-resistant variants. Overall, oseltamivir treatment decreases the signs of infection and reduced the spread of H1N1pdm influenza virus in the lungs of ferrets and therefore impeded the development of viral pneumonia.

Keywords: Pandemic 2009 H1N1 virus; Neuraminidase inhibitor; Oseltamivir; Ferret model

Depletion of GTP pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on Lassa virus by Stephan Ölschläger; Johan Neyts; Stephan Günther (pp. 89-93).

Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is the standard treatment for Lassa fever, though its mode of action is unknown. One possibility is depletion of the intracellular GTP pool via inhibition of the cellular enzyme inosine monophosphate dehydrogenase (IMPDH). This study compared the anti-arenaviral effect of ribavirin with that of two other IMPDH inhibitors, mycophenolic acid (MPA) and 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR). All three compounds were able to inhibit Lassa virus replication by ⩾2 log units in cell culture. Restoring the intracellular GTP pool by exogenous addition of guanosine reversed the inhibitory effects of MPA and EICAR, while ribavirin remained fully active. Analogous experiments performed with Zaire Ebola virus showed that IMPDH inhibitors are also active against this virus, although to a lesser extent than against Lassa virus. In conclusion, the experiments with MPA and EICAR indicate that replication of Lassa and Ebola virus is sensitive to depletion of the GTP pool mediated via inhibition of IMPDH. However, this is not the predominant mechanism by which ribavirin exerts its in-vitro antiviral effect on Lassa virus.

Keywords: Abbreviations; EICAR; 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide; IMPDH; inosine monophosphate dehydrogenase; LCMV; lymphocytic choriomeningitis virus; MOI; multiplicity of infection; MPA; mycophenolic acid; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide; RdRp; RNA-dependent RNA polymerase; Ribavirin; 1-β-; d; -ribofuranosyl-1,2,4-triazole-3-carboxamideLassa virus; Mopeia virus; Arenavirus; Ebola virus; Ribavirin; Inosine monophosphate dehydrogenase

Identification of low-molecular weight inhibitors of HIV-1 reverse transcriptase using a cell-based high-throughput screening system by Oyebisi Jegede; Ana Khodyakova; Mikhail Chernov; Jan Weber; Luis Menéndez-Arias; Andrei Gudkov; Miguel E. Quiñones-Mateu (pp. 94-98).

A cell-based drug screening system that utilizes a green fluorescent protein (GFP)-tagged recombinant lentiviral vector has been used to screen a chemical library of 34,000 small molecules for antiretroviral compounds. Thirty-three initial hits were analyzed and four compounds were selected based on their anti-human immunodeficiency virus type 1 (HIV-1) activity (EC₅₀ values ranging from 0.17 to 1.9μM) and low cellular toxicity (CC₅₀ values >50μM). The four compounds blocked reverse transcription and were able to inhibit the replication of a panel of different HIV-1 strains, including non-B subtype and viruses resistant to different drug classes. Serial in vitro passages of HIV-1_B-HXB2 in the presence of increasing drug concentrations selected for viruses with reduced susceptibility. Mutations previously associated with resistance to non-nucleoside reverse transcriptase (RT) inhibitors (L100I and Y181C for CBL-17 and CBL-21, respectively) or linked to nucleoside analogue resistance (A62V for CBL-4.0 and CBL-4.1) were identified. Viruses with reduced susceptibility to CBL-17 and CBL-21 but not the ones resistant to CBL-4.0 or CBL-4.1 showed a decrease in replicative fitness. Interestingly, two of the small molecules (CBL-4.0 and CBL-4.1) are indolopyridinones that were previously described as nucleotide-competing RT inhibitors.

Keywords: HIV; Drug screening; Reverse transcriptase inhibitors; Drug resistance; Fitness

Antiviral activity of type I and type III interferons against porcine reproductive and respiratory syndrome virus (PRRSV) by Rui Luo; Liurong Fang; Hui Jin; Yunbo Jiang; Dang Wang; Huanchun Chen; Shaobo Xiao (pp. 99-101).

The newly identified type III interferons (IFNs), also known as IFN-λ1/IL-29, IFN-λ2/IL-28A and IFN-λ3/IL-28B, like type I IFNs, have antiviral activity against a broad spectrum of viruses. We therefore examined whether type III IFNs, as well as type I IFNs, has the ability to inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in MARC-145 cells. We found that replication of PRRSV in MARC-145 cells was significantly reduced following treatment with IFN-λ1, IFN-λ2 and IFN-λ3, respectively, and such inhibition was dose-dependent. However, type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) was less effective than type I IFNs (IFN-α and IFN-β) in antiviral activity against PRRSV. Mixture of two types of IFNs could not improve the antiviral activity of each type alone. In addition, all types of IFNs in our study were able to induce the expression of ISG56, 2′,5′-OAS and MxA in MARC-145 cells. These data demonstrate that type III IFNs had antiviral activity against PRRSV and may serve as useful antiviral agents against infectious swine diseases.

Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV); Type I IFNs; Type III IFNs; Antiviral activity

Development of hepatitis C virus chimeric replicons for identifying broad spectrum NS3 protease inhibitors by Joseph Binder; Selwyna Tetangco; Megan Weinshank; Karen Maegley; Laura Lingardo; Wade Diehl; Robert Love; Amy K. Patick; George J. Smith III (pp. 102-111).

Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy.

Keywords: HCV; NS3 protease; Chimeric replicon

Effects of nevirapine and efavirenz on human adipocyte differentiation, gene expression, and release of adipokines and cytokines by Julieta Díaz-Delfín; M. del Mar Gutiérrez; José M. Gallego-Escuredo; Joan C. Domingo; M. Gracia Mateo; Francesc Villarroya; Pere Domingo; Marta Giralt (pp. 112-119).

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz are drugs of choice for initial antiretroviral treatment for HIV-1 infection. Although NNRTIs have not traditionally been associated with the appearance of adipose alterations, recent data suggest that efavirenz may contribute to adipose tissue alterations in antiretroviral-treated patients, consistent with its ability to impair differentiation of adipocytes in cell cultures. No such effects have been reported for nevirapine, the other most commonly used NNRTI. In this study, we determined the effects of nevirapine on differentiation, gene expression and release of regulatory proteins (adipokines and cytokines) in differentiating human adipocytes, and compared them with those of efavirenz. Efavirenz caused a dose-dependent repression of adipocyte differentiation that was associated with down-regulation of the master adipogenesis regulator genes SREBP-1, PPARγ and C/EBPα, and their target genes encoding lipoprotein lipase, leptin and adiponectin, which are key proteins in adipocyte function. In contrast, nevirapine does not affect adipogenesis and causes a modest but significant coordinate increase in the expression of SREBP-1, PPARγ and C/EBPα and their target genes only at a concentration of 20μM. Whereas efavirenz caused a significant increase in the release of pro-inflammatory cytokines (interleukin [IL]-8, IL-6, monocyte chemoattractant protein-1), plasminogen activator inhibitor type-1 and hepatocyte growth factor (HGF), nevirapine either had no effect on these factors or decreased their release (IL-6 and HGF). Nevirapine significantly increased adiponectin release, whereas efavirenz strongly repressed it. Moreover, nevirapine inhibited preadipocyte endogenous reverse transcriptase activity, whereas efavirenz did not alter it. It is concluded that, in contrast with the profound anti-adipogenic and pro-inflammatory response elicited by efavirenz, nevirapine does not impair adipogenesis.

Keywords: Lipodystrophy; Adipocyte; Nevirapine; Efavirenz; Non-nucleoside analog reverse transcriptase inhibitor; Adipokine

Activity and the metabolic activation pathway of the potent and selective hepatitis C virus pronucleotide inhibitor PSI-353661 by Phillip A. Furman; Eisuke Murakami; Congrong Niu; Angela M. Lam; Christine Espiritu; Shalini Bansal; Haiying Bao; Tatiana Tolstykh; Holly Micolochick Steuer; Meg Keilman; Veronique Zennou; Nigel Bourne; Ronald L. Veselenak; Wonsuk Chang; Bruce S. Ross; Jinfa Du; Michael J. Otto; Michael J. Sofia (pp. 120-132).

PSI-353661, a phosphoramidate prodrug of 2′-deoxy-2′-fluoro-2′- C-methylguanosine-5′-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5′-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O⁶-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2′-deoxy-2′-fluoro-2′- C-methylguanosine-5′-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.

Keywords: Abbreviations; HCV; hepatitis C virus; HPLC; high pressure liquid chromatography; PCR; polymerase chain reaction; ddC; dideoxycytidine; GMP; guanosine-5′-monophosphate; GDP; guanosine-5′-diphosphate; RdRp; HCV RNA-dependent RNA polymerase; NS5B; HCV non-structural protein 5B; Cat A; cathepsin A; CES1; carboxylesterase 1; Hint 1; histidine triad nucleotide-binding protein 1; ADAL1; adenosine deaminase-like protein 1; hGUK1; human guanylate kinase; NDPK; human nucleoside diphosphate kinase; PK; pyruvate kinase; 3-PGK; 3-phosphoglycerate kinase; K; _m; Michaelis constant; k; _cat; turnover number; k; _cat; /; K; _m; catalytic efficiencyHepatitis C virus; Antiviral; Nucleotide analog; Prodrug; Phosphoramidate; 2′-Deoxy-2′-fluoro-2′-; C; -methylguanosine-5′-monophosphate

Acridine derivatives as anti-BVDV agents by Michele Tonelli; Gerolamo Vettoretti; Bruno Tasso; Federica Novelli; Vito Boido; Fabio Sparatore; Bernardetta Busonera; Aicha Ouhtit; Pamela Farci; Sylvain Blois; Gabriele Giliberti; Paolo La Colla (pp. 133-141).

Twenty-six 9-aminoacridine derivatives were evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. While seven compounds (9,10,14,19,21,22,24) did not affect any virus and two (6,11) were moderately active against CVB-5 or Reo-1, 17 compounds exhibited a marked specific activity against BVDV, prototype of pestiviruses which are responsible for severe diseases of livestock. Most anti-BVDV agents showed EC₅₀ values in the range 0.1–8μM, thus comparing favorably with the reference drugs ribavirine and NM 108. Some compounds, particularly those bearing a quinolizidinylalkyl side chain, displayed pronounced cytotoxicity. Further studies are warranted in order to achieve still better anti-BVDV agents, and to explore the potential antiproliferative activity of this kind of compounds.

Keywords: Abbreviations; ACG; acyclovir; Ar; aromatic ring; ATCC; American type culture collection; AZT; 3′-azidothymidine; BHK; baby hamster kidney; BVDV; bovine viral diarrhea virus; CC; column chromatography; CCDI; ₅₀; cell culture infectious dose 50%; CVB-5; coxsackie virus, type 5; DMEM; Dulbecco’s modified Eagle medium; DMF; dimethylformamide; DMSO; dimethylsulfoxide; ds-RNA; double-stranded RNA virus; EMCV; encephalomyocarditis virus; FBS; fetal bovine serum; HIV-1; human immunodeficiency virus, type 1; HCV; hepatitis C virus; HSV-1; herpes simplex virus, type 1; HTLV-1; human T-cell leukemia virus type 1; MDBK; Madin Darby bovine kidney; MEM-E; Minimum Essential Medium Eagle; m.o.i.; multiplicity of infection; MT-4; CD4; ⁺; human T-cells containing an integrated HTLV-1 genome; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS; phosphate buffer solution; PFU; Plaque Forming Unit; RdRp; RNA-dependent RNA-polymerase; Reo-1; Reovirus, type 1; RPMI; Roswell Park Memorial Institut (medium); RSV; respiratory syncytial virus; Sb-1; poliovirus type 1-Sabin strain; SI; selectivity index; ssRNA; ⁺; single-stranded positive RNA virus; ssRNA; ⁻; single-stranded negative RNA virus; Tris/HCl; tris(hydroxymethyl)aminomethane hydrochloride; Vero-76; monkey kidney; VSV; vesicular stomatitis virus; VV; vaccinia virus; YFV; yellow fever virus9-Aminoacridine derivatives; RNA and DNA viruses; Anti-BVDV activity

Expression of herpes simplex virus type 1 recombinant thymidine kinase and its application to a rapid antiviral sensitivity assay by Tomoyuki Shiota; Wang Lixin; Mutsuyo Takayama-Ito; Itoe Iizuka; Momoko Ogata; Masanori Tsuji; Hidekazu Nishimura; Shuichi Taniguchi; Shigeru Morikawa; Ichiro Kurane; Masashi Mizuguchi; Masayuki Saijo (pp. 142-149).

Antiviral-resistant herpesvirus infection has become a great concern for immunocompromised patients. Herpes simplex virus type 1 (HSV-1) infections are treated with viral thymidine kinase (vTK)-associated drugs such as acyclovir (ACV), and most ACV-resistance (ACV^r) is due to mutations in the vTK. The standard drug sensitivity test is usually carried out by the plaque reduction assay-based method, which requires over 10days. To shorten the time required, a novel system was developed by the concept, in which 293T cells transiently expressing recombinant vTK derived from the test sample by transfection of the cells with an expression vector were infected with vTK-deficient and ACV^r HSV-1 (TAR), and then cultured in a maintenance medium with or without designated concentrations of ACV, ganciclovir (GCV) and brivudine (BVdU). The replication of TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressing recombinant vTK of the ACV-sensitive HSV-1, whereas replication was not or slightly inhibited in cells expressing the recombinant vTK of highly resistant or intermediately resistant HSV-1, respectively. An inverse correlation was demonstrated in the 50% effective concentrations (EC₅₀s) and inhibitory effects of these compounds on the replication of TAR among ACV^s and ACV^r HSV-1 clones. These results indicate that the EC₅₀s of the vTK-associated drugs including ACV can be assumed by measuring the inhibitory effect of drugs in 293T cells expressing recombinant vTK of the target virus. The newly developed antiviral sensitivity assay system for HSV-1 makes it possible to estimate EC₅₀ for vTK-associated drugs, when whole vTK gene is available for use by gene amplification directly from lesion’s samples or from virus isolates.

Keywords: Herpes simplex virus type 1; Acyclovir; Drug-resistance; Rapid antiviral sensitivity assay; Thymidine kinase

Transmission of HIV drug resistance and non-B subtype distribution in the Spanish cohort of antiretroviral treatment naïve HIV-infected individuals (CoRIS) by Federico García; Santiago Pérez-Cachafeiro; Vicente Guillot; Marta Alvarez; Pilar Pérez-Romero; María Jesús Pérez-Elías; Isabel Viciana; Jose Ramón Blanco; Maria López-Dieguez; Carmen de Mendoza (pp. 150-153).

CoRIS is an open multicentre cohort of HIV seroprevalent ARV-naïve subjects who began treatment at 32 Spanish healthcare centres from January 2004. Up to November 2008, a total of 683 FASTA format sequences, encoding the HIV protease and reverse transcriptase (RT) derived from plasma samples at entry into the cohort, had been obtained for examination of transmitted drug resistance (TDR) and HIV clade. TDR was found in 8.5% of the patients (4.4% NRTIs, 4% NNRTIs, 2.2% PIs). The most prevalent resistance mutations were: T215 revertants (3.8%), D67NG (1.3%), K219QENR (1.2%) and M41L (1%), for NRTIs; K103N (3.2%), for NNRTIs; I54VLMSAT, M46I and L90M (0.7%), for PIs. Non-B subtypes were recognized in 104 patients (15.2%) and were more common in Sub-Saharan Africans (15/17, 88.2%), Eastern Europeans (7/12, 58.3%) and Northern Africans (8/16, 50%) than among Spaniards (53/479, 11%) ( p<0.001). The most prevalent non-B subtype was CRF02_AG (4.4%), followed by subtype D (1.9%), CRF03_AB (1.5%), CRF07_BC and subtype F1 (1%). A trend was observed for the transmission of non-B subtypes to increase and for TDR to decrease.

Keywords: HIV; Transmitted drug resistance; Non-B subtypes; CoRIS

Topical cream-based oxyresveratrol in the treatment of cutaneous HSV-1 infection in mice by Vimolmas Lipipun; Pattaraporn Sasivimolphan; Yoshihiro Yoshida; Tohru Daikoku; Boonchoo Sritularak; Garnpimol Ritthidej; Kittisak Likhitwitayawuid; Pornpen Pramyothin; Masao Hattori; Kimiyasu Shiraki (pp. 154-160).

Anti-herpes simplex virus (HSV) activities of oxyresveratrol in vitro and topical administration in cutaneous HSV-1 infection in mice were examined. The inhibitory concentrations for 50% plaque formation (IC₅₀) of oxyresveratrol against HSV-1 clinical isolates and HSV-2 clinical isolates were 20.9–29.5 and 22.2–27.5μg/ml, respectively. In topical administration in cutaneous HSV-1 infection in mice, 2.5%, 5%, 10% and 20% oxyresveratrol in cream vehicle applied three times daily for 7days after infection were evaluated and 10% and 20% oxyresveratrol cream were significantly effective in delaying the development of skin lesions and protection from death ( P<0.01). The concentration of 10% oxyresveratrol in cream was significantly more effective than that of 30% oxyresveratrol in vaseline applied three times daily ( P<0.01). Oxyresveratrol cream at 20% was as effective as 5% ACV cream applied three times daily ( P<0.01). Both 10% and 20% oxyresveratrol cream were as effective as that of 5% ACV cream applied two times daily ( P>0.05). Therapeutic efficacy of oxyresveratrol in cream vehicle was dose-dependent and the maximum efficacy observed on day 6 after infection was shown at 10% oxyresveratrol in cream applied three times daily. The frequency of application of 10% oxyresveratrol cream at three, four and five times daily was as effective as that of 5% ACV cream applied five times daily ( P>0.05). These results demonstrated that topical administration of oxyresveratrol in novel cream vehicle reduced the concentration of oxyresveratrol to 10% and was suitable for cutaneous HSV infection.

Keywords: Oxyresveratrol; Cream; Herpes simplex virus; Therapeutic efficacy; Cutaneous infection in mice

Lopinavir shows greater specificity than zinc finger ejecting compounds as a potential treatment for human papillomavirus-related lesions by Ingeborg Zehbe; Christina Richard; Kyle F. Lee; Michael Campbell; Lynne Hampson; Ian N. Hampson (pp. 161-166).

Non-surgical, antiviral treatment options are desirable for HPV-related lesions within the genitourinary and upper digestive tract. We compared the toxicity of three zinc finger-ejecting (ZFE) compounds (4,4-dithiodimorpholine, azodicarbonamide, and diamide) to the HIV protease inhibitor lopinavir using HPV-positive SiHa, CaSki, HeLa, ME180, and HPV-negative C33A cervical carcinoma cell lines as well as primary human foreskin keratinocytes (PHFKs). Colorimetric growth assays revealed selective toxicity when treated with lopinavir. All carcinoma cell lines, except CaSki, were sensitive to 20μM lopinavir whereas primary PHFKs were highly resistant. In contrast, 4,4-dithiodimorpholine was uniformly toxic to all cells tested while azodicarbonamide and diamide showed no effect at all. It is concluded that lopinavir may be an attractive candidate to treat pre-cancerous and cancerous HPV-positive lesions.

Keywords: Abbreviations; HPV; human papillomavirus; PHFK; primary human foreskin keratinocyteNon-surgical treatment; Zinc finger-ejecting compound; Protease-inhibitor lopinavir; HPV-related lesion

Resistance to raltegravir highlights integrase mutations at codon 148 in conferring cross-resistance to a second-generation HIV-1 integrase inhibitor by Olivia Goethals; Marcia Van Ginderen; Ann Vos; Maxwell D. Cummings; Koen Van Der Borght; Liesbeth Van Wesenbeeck; Maxim Feyaerts; Ann Verheyen; Veerle Smits; Marnix Van Loock; Kurt Hertogs; Dominique Schols; Reginald F. Clayton (pp. 167-176).

Raltegravir is the first integrase strand-transfer inhibitor (INSTI) approved for use in highly active antiretroviral therapy (HAART) for the management of HIV infection. Resistance to antiretrovirals can compromise the efficacy of HAART regimens. Therefore it is important to understand the emergence of resistance to RAL and cross-resistance to other INSTIs including potential second-generation INSTIs such as MK-2048.We have now studied the question of whether in vitro resistance selection (IVRS) with RAL initiated with viruses derived from clinical isolates would result in selection of resistance mutations consistent with those arising during treatment regimens with HAART containing RAL. Some correlation was observed between the primary mutations selected in vitro and during therapy, initiated with viruses with identical IN sequences. Additionally, phenotypic cross-resistance conferred by specific mutations to RAL and MK-2048 was quantified. N155H, a RAL-associated primary resistance mutation, was selected after IVRS with MK-2048, suggesting similar mechanisms of resistance to RAL and MK-2048. This was confirmed by phenotypic analysis of 766 clonal viruses harboring IN sequences isolated at the point of virological failure from 106 patients on HAART (including RAL), where mutation Q148H/K/R together with additional secondary mutations conferred reduced susceptibility to both RAL and MK-2048. A homology model of full length HIV-1 integrase complexed with viral DNA and RAL or MK-2048, based on an X-ray structure of the prototype foamy virus integrase–DNA complex, was used to explain resistance to RAL and cross-resistance to MK-2048. These findings will be important for the further discovery and profiling of next-generation INSTIs.

Keywords: HIV-1; Integrase; Raltegravir; Resistance; MK-2048

HPV episome levels are potently decreased by pyrrole–imidazole polyamides by Terri G. Edwards; Kevin J. Koeller; Urszula Slomczynska; Kam Fok; Michael Helmus; James K. Bashkin; Chris Fisher (pp. 177-186).

Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole–imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4–18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication ( ori), four polyamides were identified that possessed apparent IC₅₀s⩽150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.

Keywords: Papillomavirus; Antiviral; Polyamide; Episome; Cervical cancer

G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates by Yang Wang; Kirk J. Rowley; Brian J. Booth; Susan E. Sloan; Donna M. Ambrosino; Gregory J. Babcock (pp. 187-194).

Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization.

Keywords: Rabies virus; Monoclonal antibody; Neutralization; G glycoprotein; Pseudovirus

Minigenomes, transcription and replication competent virus-like particles and beyond: Reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses by Thomas Hoenen; Allison Groseth; Fabian de Kok-Mercado; Jens H. Kuhn; Victoria Wahl-Jensen (pp. 195-208).

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research.

Keywords: Reverse genetics; Virus-like particles; Minigenome; Minireplicon; Viral hemorrhagic fever; Filoviruses

In vitro inhibition of CSFV replication by multiple siRNA expression by Jiangnan Li; Yajuan Dai; Shuai Liu; Huancheng Guo; Tiedong Wang; Hongsheng Ouyang; Changchun Tu (pp. 209-216).

Classical swine fever (CSF) is a highly contagious viral disease of pigs which causes major economic losses worldwide. No specific drug is currently available for the effective treatment of CSFV infection; however, RNA interference (RNAi) has been applied successfully to inhibit the replication of human and other animal viruses. In this study, three effective siRNAs targeting NS3 of CSFV were selected. siNS3-2 targeting NS3 gene was chosen for further experimentation, while siN1 and siN2 targetingN ^pro gene, and siNS5B targeting NS5B gene describe previously. Single, double and quadruple anti-CSFV siRNA expression plasmids, with loxp sites at each end of the selectable marker genes, were constructed and analyzed using the same promoters or four different promoters, targetingN ^pro, NS3 and NS5B genes of CSFV. Results indicate that single or multiple siRNA expression plasmids can efficiently inhibit CSFV replication and that inhibition was markedly stronger when multiple siRNAs were expressed targeting different genes of CSFV. Since RNAi applied to anti-CSFV research, this study provides anti-CSFV methods by single and multiple siRNA expression which can target most viral isolates of different subtypes and prevent viral escape. It also provides a basis for development of CSFV-resistant transgenic pigs.

Keywords: CSFV; RNAi; Multiple siRNA expression; Inhibition; Viral escape

Early identification of availability issues for poorly water-soluble microbicide candidates in biorelevant media: A case study with saquinavir by Joachim Brouwers; Kurt Vermeire; Carolien Grammen; Dominique Schols; Patrick Augustijns (pp. 217-223).

In the search for a successful HIV microbicide, many poorly water-soluble antiviral agents are currently being investigated. Unfortunately, solubility and precipitation issues may limit intravaginal concentrations and thus availability of these agents upon application of an aqueous gel formulation. In the present study, we evaluated the in vitro precipitation behavior of the HIV protease inhibitor saquinavir in vaginal and seminal fluid simulants (VFS and SFS). Despite its limited solubility, the mesylate salt of saquinavir enables formulation of sufficiently high concentrations (2.5mM, i.e. ca. 10⁵-fold in vitro IC₅₀ values) in a standard aqueous vehicle. While saquinavir stays in solution upon dilution with VFS, SFS induces precipitation of saquinavir, resulting in a 5-fold reduced availability and antiviral potency. Inclusion of the solubilizing excipients polyethylene glycol 1000 (12%) and hydroxypropyl-β-cyclodextrin (2.5%) was required to avoid saquinavir precipitation in SFS and to restore the antiviral potency of the formulation. This study illustrates the importance of identifying solubility and precipitation issues of microbicide candidates in biorelevant media and provides a simple in vitro procedure to implement this evaluation in early microbicide development.

Keywords: HIV microbicide; Biopharmaceutical profiling; Saquinavir; Vaginal gel; Solubility; Precipitation

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Antiviral Research (v.91, #2)

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Antiviral Research (v.91, #2)