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Antiviral Research (v.92, #2)

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

Prelim II: Editorial Board (pp. ii).

Lymphocytic choriomeningitis virus (LCMV) infection of macaques: A model for Lassa fever by Juan C. Zapata; C. David Pauza; Mahmoud M. Djavani; Juan D. Rodas; Dmitry Moshkoff; Joseph Bryant; Eugene Ateh; Cybele Garcia; Igor S. Lukashevich; Maria S. Salvato (pp. 125-138).

► History of LCMV infections of primates (from mice to monkeys and man). ► Comparison of outcomes in murine and primate infections. ► Description of findings from LCMV-infected rhesus macaques. ► Utility of the model for testing host-response antivirals over virus-specific antivirals.Arenaviruses such as Lassa fever virus (LASV) and lymphocytic choriomeningitis virus (LCMV) are benign in their natural reservoir hosts, and can occasionally cause severe viral hemorrhagic fever (VHF) in non-human primates and in human beings. LCMV is considerably more benign for human beings than Lassa virus, however certain strains, like the LCMV-WE strain, can cause severe disease when the virus is delivered as a high-dose inoculum. Here we describe a rhesus macaque model for Lassa fever that employs a virulent strain of LCMV. Since LASV must be studied within Biosafety Level-4 (BSL-4) facilities, the LCMV-infected macaque model has the advantage that it can be used at BSL-3. LCMV-induced disease is rarely as severe as other VHF, but it is similar in cases where vascular leakage leads to lethal systemic failure. The LCMV-infected macaque has been valuable for describing the course of disease with differing viral strains, doses and routes of infection. By monitoring system-wide changes in physiology and gene expression in a controlled experimental setting, it is possible to identify events that are pathognomonic for developing VHF and potential treatment targets.

Keywords: Abbreviations; AST/ALT; ratio of the enzymes, alanine aminotransferase and aspartate aminotransferase; pfu; infectious units or plaque forming units; ffu; focus forming units; VHF; Viral hemorrhagic fever; LCMV-WE; Lymphocytic choriomeningitis virus (virulent) strain WE; LCMV-ARM; lymphocytic choriomeningitis (benign) strain Armstrong; PICV; Pichinde virus; TACV; Tacaribe virus; LASV; Lassa fever virus; MOPV; Mopeia Virus; ML29; a Mopeia/Lassa reassortant vaccine candidate; iv; intravenous; sc; subcutaneous; ig; intragastric; im; intramuscular; IL-6; Interleukin-6; sIL6R; soluble interleukin-6 receptor; sTnfRI; soluble TNF-α receptor 1; sTNFRII; soluble TNF-α receptor 2; PMN; polymorphonuclear leukocytesViral hemorrhagic fever; Monkey models; Rhesus macaques; Arenaviruses; LCMV; Lassa fever virus

HIV-1 reverse transcriptase connection subdomain mutations involved in resistance to approved non-nucleoside inhibitors by Luis Menéndez-Arias; Gilberto Betancor; Tania Matamoros (pp. 139-149).

► HIV-1 reverse transcriptase (RT) is a major target of antiretroviral intervention. ► Non-nucleoside RT inhibitors are nevirapine, delavirdine, efavirenz, etravirine and rilpivirine. ► Mutations away from their binding pocket have been associated with NNRTI resistance. ► RT connection subdomain mutations (e.g. N348I, A376S, etc.) modulate NNRTI resistance. ► The mechanisms by which N348I and related mutations mediate resistance are discussed.The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target of antiretroviral intervention. Non-nucleoside RT inhibitors (NNRTIs) bind to a hydrophobic pocket located away from the DNA polymerase catalytic site of the RT. Approved NNRTIs are nevirapine, delavirdine, efavirenz, etravirine and rilpivirine. This review describes how these inhibitors affect RT function, the structural basis of NNRTI binding, and the role of specific amino acid substitutions at the NNRTI binding pocket in the acquisition of high-level drug resistance. However, two or more amino acid substitutions are required to achieve >20-fold decreased susceptibility to recently developed NNRTIs such as etravirine or rilpivirine, in phenotypic assays. While genotypic analysis of HIV-1 isolates in infected patients is usually restricted to residues 1–250 of the RT, recent reports indicate that several residues in the connection subdomain of the RT (comprising residues 319–426) could also modulate NNRTI resistance. Examples are Y318F or W, N348I, A376S and T369I or V. Tyr-318 participates in NNRTI binding, but other amino acid substitutions in the connection subdomain may affect resistance through an indirect mechanism. Studies on the effects of N348I and A376S on NNRTI resistance indicate that these changes could affect inhibitor binding by altering the interaction between RT subunits or between the RT and the template-primer. Moreover, those mutations could also modulate RNase H activity not only during DNA strand elongation, but also at the initiation of plus strand DNA synthesis as demonstrated for the N348I mutation.

Keywords: HIV; Reverse transcriptase; Drug resistance; Non-nucleoside RT inhibitors

Zoonotic Brazilian Vaccinia virus: From field to therapy by Erna Geessien Kroon; Bruno Eduardo F. Mota; Jônatas Santos Abrahão; Flávio Guimarães da Fonseca; Giliane de Souza Trindade (pp. 150-163).

► Vaccinia virus, an orthopoxvirus, causes an occupational zoonotic disease in Brazil. ► Brazilian vaccinia virus constitute a risk to people and livestock. ► Brazilian vaccinia virus show genetic and phenotypic diversity forming at least two clades. ► Brazilian vaccinia virus were detected in wildlife and peri-domestic species. ► Co-infection and co-circulation of viruses was detected in bovine vaccinia.Vaccinia virus (VACV), the prototype species of the Orthopoxvirus (OPV) genus, causes an occupational zoonotic disease in Brazil that is primarily associated with the handling of infected dairy cattle. Cattle and human outbreaks have been described in southeastern Brazil since 1999 and have now occurred in almost half of the territory. Phylogenetic studies have shown high levels of polymorphisms among isolated VACVs, which indicate the existence of at least two genetically divergent clades; this has also been proven in virulence assays in a mouse model system. In humans, VACV infection is characterized by skin lesions, primarily on the hands, accompanied by systemic symptoms such as fever, myalgia, headache and lymphadenopathy. In this review, we will discuss the virological, epidemiological, ecological and clinical aspects of VACV infection, its diagnosis and compounds that potentially could be used for the treatment of severe cases.

Keywords: Orthopoxvirus; Vaccinia virus (VACV); Bovine vaccinia (BV); Antiviral drugs; Zoonosis; Occupational disease

Camelpox: Target for eradication? by Mike Bray; Shawn Babiuk (pp. 164-166).

Camelpox virus by Sophie Duraffour; Hermann Meyer; Graciela Andrei; Robert Snoeck (pp. 167-186).

► Camelpox virus induces a smallpox-like illness in the natural host, camels. ► Camelpox virus encodes multiple genes counteracting the host immune response. ► Camelpox disease is enzootic in countries practicing camel husbandry. ► Arthropods are suspected in the transmission of camelpox. ► Vaccines exist for prevention of camelpox and antivirals may be available.Camelpox virus (CMLV) causes a smallpox-like illness in a unique host, the camel. The disease is enzootic in almost all regions where camel husbandry is practiced, and is responsible for severe economic losses. Although it is genetically the closest known virus to variola virus, the etiologic agent of smallpox, CMLV remains poorly studied. It is characterized by a narrow host range, the capacity to induce giant cells in culture and to counteract host immune defenses; however, the genetic bases associated with these features are not understood. Also, it still needs to be demonstrated whether CMLV strains of variable virulence circulate and how arthropod vectors might be involved in virus transmission. Current evidence indicates that, under certain circumstances, CMLV can be mildly pathogenic in humans. A reservoir host other than camels is unlikely to exist. We review here current knowledge of CMLV, including clinical and laboratory aspects of the disease. We also discuss prevention and therapy by use of vaccines and antiviral treatments, as well as the possibility of camelpox eradication.

Keywords: Poxvirus; Camelpox virus; Animal models; Zoonosis; Camelpox vaccines; Antivirals

Inhibitors of SARS-CoV entry – Identification using an internally-controlled dual envelope pseudovirion assay by Yanchen Zhou; Juliet Agudelo; Kai Lu; David H. Goetz; Elizabeth Hansell; Yen Ting Chen; William R. Roush; James McKerrow; Charles S. Craik; Sean M. Amberg; Graham Simmons (pp. 187-194).

► We describe a novel assay for screening inhibitors of viral entry. ► The inclusion of an internal control envelope controls for specificity. ► This assay system can be adapted for most enveloped viruses. ► Examples of screens for inhibitors of SARS-CoV entry were performed. ► A number of compounds were identified that inhibit SARS-CoV entry and replication.Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.

Keywords: Inhibitors of SARS-CoV entry; Antiviral; HTS; High-throughput screening; Dual envelope pseudovirion assay; Pseudovirus

Antiviral activity of the MEK-inhibitor U0126 against pandemic H1N1v and highly pathogenic avian influenza virus in vitro and in vivo by Karoline Droebner; Stephan Pleschka; Stephan Ludwig; Oliver Planz (pp. 195-203).

► Antiviral activity of MEK-inhibtors. ► Aerosol treatment against influenza virus. ► No adverse effects after treatment with MEK-inhibitors.The emergence of the 2009 H1N1 pandemic swine influenza A virus is a good example of how this viral infection can impact health systems around the world in a very short time. The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. In the present work we investigated whether the MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, is able to suppress propagation of the 2009 pandemic IV H1N1v (v=variant) as well as highly pathogenic avian influenza viruses (HPAIV) in cell culture and also in vivo in the mouse lung. U0126 showed antiviral activity in cell culture against all tested IAV strains including oseltamivir resistant variants. Furthermore, we were able to demonstrate that treatment of mice with U0126 via the aerosol route led to (i) inhibition of MEK activation in the lung (ii) reduction of progeny IAV titers compared to untreated controls (iii) protection of IAV infected mice against a 100× lethal viral challenge. Moreover, no adverse effects of U0126 were found in cell culture or in the mouse. Thus, we conclude that U0126, by inhibiting the cellular target MEK, has an antiviral potential not only in vitro in cell culture, but also in vivo in the mouse model.

Keywords: Influenza virus; MEK-inhibitor; U0126; Mouse model

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease by Lili Zhu; Shyla George; Marco F. Schmidt; Samer I. Al-Gharabli; Jörg Rademann; Rolf Hilgenfeld (pp. 204-212).

► We present crystal structures of 6 peptide aldehydes in complex with SARS-CoV M^pro. ► The aldehydes form thiohemiacetals with the catalytic cysteine of the enzyme. ► Two different configurations of the thiohemiacetal can be observed for one aldehyde. ► Asp and Ser side-chains of the aldehydes bind to the hydrophobic S2 pocket. ► P2-Asp interacts with two methionines of the enzyme via O...S nonbonded interactions.SARS coronavirus main protease (SARS-CoV M^pro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M^pro. Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M^pro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M^pro in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M^pro, withK _i values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2=Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M^pro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M^pro, withK _i=2.24±0.58μM. These results show that the stringent substrate specificity of the SARS-CoV M^pro with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.

Keywords: Abbreviations; SARS-CoV M; ^pro; SARS coronavirus main protease; Cm-FF-H; Cinnamoyl-Phe-Phe-H; Boc; tert-butyl oxycarbonyl; IBX; 2-iodoxybenzoic acid; FRET; fluorescence resonance energy transfer; PEG; polyethylene glycol; MPD; 2-methyl-2,4-pentanediol; MES; 2-(; N; -morpholino)ethanesulfonic acid; DMSO; dimethylsulfoxideAldehyde inhibitor; Antiviral drug design; Cysteine protease; Methionine–aspartic acid interaction; X-ray crystallography

Prediction of drug-resistance in HIV-1 subtype C based on protease sequences from ART naive and first-line treatment failures in North India using genotypic and docking analysis by Jaideep S. Toor; Aman Sharma; Rajender Kumar; Pawan Gupta; Prabha Garg; Sunil K. Arora (pp. 213-218).

► DR genotyping of HIV isolates from therapy-naive and treatment-failures conducted. ► Docking of various PI drugs attempted on computational model of HIV subtype C protease. ► Binding energies calculated after introducing detected mutations. ► A prediction model proposed for calculating the susceptibility levels for each drug. ► The model is based on HIV subtype C protease sequences for screening of PI drugs.Genotyping reveal emergence of drug resistance (DR)-related mutations in HIV-1 protease (PR) gene in the first-line treatment failure patients as per Stanford DR database. In order to have a subtype C specific prediction model, a three dimensional structure of local wild type C variant is created and the identified mutations were introduced to assess the mutational effects on protease inhibitors (PI) in a homology model.We estimated viral load, CD4 count and conducted DR genotyping in HIV isolates from 129 therapy naive and 20 first-line treatment failure individuals. Several genotypic variations, as compared to subtype B sequence in the Stanford gene database were detected in HIV-1 subtype C isolates from treatment naive individuals. Among these, nine mutations (12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, 93L) occurred in more than 60% of the isolates and were considered as local wild type for molecular modelling studies. No major mutations were seen in the PR sequences in isolates from treatment-naive individuals, although isolates from two patients had T74S mutation, known to be associated with reduced susceptibility to nelfinavir (NFV) and a combination of M36I, H69K and L89M mutations found in isolates from 77 patients (59.7%), considered to be conferring resistance to tipranavir (TPV) according to ANRS algorithm. Among the first-line treatment failures, an isolate from one patient showed L33F, I47T, M46G, and G48E mutations conferring intermediate resistance to saquinavir (SQV) and lopinavir (LPV). Though the docking energy scores are in agreement with this interpretation for SQV, it, however, indicated these mutations to be causing intermediate to high level resistance to atazanavir (ATV) and tipranavir (TPV) but making it susceptible to LPV. The patient finally responded to a second-line regimen containing 3TC, AZT and LPV with significant viral suppression.All the DR genotyping studies analyse the results using available databases which are all based on subtype B specific sequences. The proposed homology model in this study is unique, as it may predict subtype C specific susceptibility criteria for the available PIs.

Keywords: First-line treatment failure; HIV-1 subtype C; Drug resistance; Protease inhibitors; Molecular modelling; Stanford drug resistance database

Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle by Alejandra V. Capozzo; Maximiliano Wilda; Danilo Bucafusco; María de los Ángeles Lavoria; Olga L. Franco-Mahecha; Florencia C. Mansilla; Daniel M. Pérez-Filgueira; Pablo R. Grigera (pp. 219-227).

► A chimeric antigen was designed to enhance FMD-peptides’ immunogenicity in cattle. ► It contains the FMDV antigenic site A in the context of VSV glycoprotein G. ► The chimeric construct was evaluated as a peptide and DNA vaccine in cattle. ► Antibodies elicited by the chimera recognized the epitope in the native virus. ► High neutralizing titers were developed after two doses in vaccinated calves.Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139–149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an “expected percentage of protection” above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.

Keywords: Cattle; Peptide vaccines; DNA vaccines; Foot and Mouth Disease Virus; Vesicular Stomatitis Virus glycoprotein G

Use of plethysmography in assessing the efficacy of antivirals in a mouse model of pandemic influenza A virus by Justin G. Julander; Joe Hagloch; Scott Latimer; Neil Motter; Ashley Dagley; Dale L. Barnard; Donald F. Smee; John D. Morrey (pp. 228-236).

► Lung function (plethysmography) is useful in evaluating anti-influenza therapies. ► Mean breath volume (MBV) is used to show early reduction in lung function. ► Mean breath time demonstrates long-lasting effects of lung disease. ► Treatment with oseltamivir or ribavirin resulted in significant improvement of MBV. ► Superior improvement in lung function was observed with combination therapy.The recently emerged swine-origin H1N1 influenza A virus (IAV) caused a pandemic outbreak in 2009 with higher risk of severe disease among children and pregnant women in their third trimester (), and is continuing to be important seasonal IAV strain. Mice are commonly used in antiviral studies as models of influenza disease, which utilize morbidity and mortality to assess the efficacy of a test compound. Here, we investigated the utility of unrestrained plethysomography to quantify the lung function of IAV-infected BALB/c mice. Administration of a lethal dose (∼30X LD₅₀) of pandemic H1N1 IAV resulted in a rapid decline in breath volume, as determined by a significant ( P<0.001) decrease in the pressure associated with inspiration and expiration detected as early as 2days after virus challenge. Severe disease was also accompanied by a significant ( P<0.05) increase in breath time on 8dpi. Plethysmography parameters correlated with weight loss and other parameters of disease such as gross pathology and the weight of the lung. Breath time was reduced in surviving mice challenged with a sublethal dose of virus as compared with normal controls, and is a predictive indicator of outcome in these mice. In antiviral studies, the use of plethysmography resulted in the detection of a clear and rapid treatment response, which was similar to other non-invasive parameters, such as weight change. Oseltamivir and ribavirin significantly ( P<0.001) improved parameters of lung function, particularly mean breath volume, as early as 2dpi and in a dose-dependent manner. Moreover, a combination of these two drugs further improved these parameters. Plethysmography provides a sensitive evaluation of lung function in IAV-infected mice in response to antiviral therapy.

Keywords: Influenza; Mouse; Plethysmography; Lung function; Antiviral; Combination

In vitro inhibitory effect of carrageenan oligosaccharide on influenza A H1N1 virus by Wei Wang; Pan Zhang; Cui Hao; Xian-En Zhang; Zong-Qiang Cui; Hua-Shi Guan (pp. 237-246).

► Carrageenan oligosaccharide CO-1 effectively inhibits IAV replication in vitro. ► Oligosaccharide CO-1 does not interfere with IAV adsorption and internalization. ► CO-1 mainly blocks an early step in IAV replication after virus internalization. ► CO-1 inhibits IAV mRNA and protein expression after its internalization into cells.Carrageenan polysaccharide has been reported to be able to inhibit the infection and replication of many different kinds of viruses. Here, we demonstrated that a 2kDa κ-carrageenan oligosaccharide (CO-1) derived from the carrageenan polysaccharide, effectively inhibited influenza A (H1N1) virus replication in MDCK cells (selectivity index >25.0). Moreover, the 2kDa CO-1 inhibited influenza A virus (IAV) replication better than that of 3kDa and 5kDa κ-carrageenan oligosaccharides (CO-2 and CO-3). IAV multiplication was suppressed by carrageenan oligosaccharide treatment in a dose-dependent manner. Carrageenan oligosaccharide CO-1 did not bind to the cell surface of MDCK cells but inactivated virus particles after pretreatment. Different to the actions of carrageenan polysaccharide, CO-1 could enter into MDCK cells and did not interfere with IAV adsorption. CO-1 also inhibited IAV mRNA and protein expression after its internalization into cells. Moreover, carrageenan oligosaccharide CO-1 had an antiviral effect on IAV replication subsequent to viral internalization but prior to virus release in one replication cycle. Therefore, inhibition of IAV intracellular replication by carrageenan oligosaccharide might be an alternative approach for anti-influenza A virus therapy.

Keywords: Influenza A virus; Carrageenan oligosaccharide; Adsorption; Internalization; Cell surface

The effect of cytokine profiles on the viral response to re-treatment in antiviral-experienced patients with chronic hepatitis C virus infection by Yonghong Zhang; Dandan Guo; Yan Zhao; Xiaoyun Chen; Lina Ma; Yi Jin; Huiping Yan; Hao Wu; Lai Wei; Tao Dong; Xinyue Chen (pp. 247-254).

► A distinct virological response was found between antiviral naïve and experienced. ► Antiviral naïve and experienced demonstrated difference of immune mediators profiling. ► IFN-γ was predictor of viral response and sign to evaluate status of immune tolerance. ► Dominant Th1-polar cytokines were associated with favorable viral response.There have been few studies on the potential immunological factors associated with viral controls in antiviral-experienced patients on a second round of combination therapy. In this study, we evaluated the level of systemic cytokines and potential impact on combination therapy in both antiviral-naïve and -experienced patients chronically infected with hepatitis C virus.Longitudinal analysis of 27 cytokines and chemokines was performed using the multiplex Biorad 27 plex assay in 37 antiviral-naïve and 24 experienced chronically HCV-1b-infected patients during combination therapy with peginterferon-alfa and ribavirin. A group of healthy donors was included as the control ( n=11).Fifty percent of antiviral-experienced chronically HCV-patients could achieve a delayed and slow virologic response after 48weeks combination therapy, comparing with an early and fast virologic response in antiviral-naïve patients. A distinction of immune mediators profiling before and during antiviral therapy between antiviral-naïve and -experienced patients was identified, IL-4, IFN-γ and CCL-3 (MIP-1a) were significantly higher in naïve patients than those in experienced patients ( P=0.005, 0.047 and 0.017, respectively) while G-CSF in naïve was lower than in experienced patients ( P<0.05). Notably, higher Th1 type cytokine IFN-γ and lower Th2 type cytokine IL-4 at baseline and week 4 were associated with HCV clearance in naïve patients, and a similar trend appeared at week 12 in experienced patients.We found a successful second round therapy in antiviral-experienced patients appears to be associated with the host immune response. Dominant Th1-polar cytokines, especially IFN-γ, is a potential predictor of viral responsiveness.

Keywords: Abbreviations; HCV; hepatitis C virus; RVR; rapid virologic response; EVR; early virologic response; ETVR; end-of-treatment virologic responseHepatitis C virus (HCV); Cytokines; Combination therapy; End of treatment virological response (ETVR)

Comparative antiviral activity of integrase inhibitors in human monocyte-derived macrophages and lymphocytes by Fernanda Scopelliti; Michela Pollicita; Francesca Ceccherini-Silberstein; Fabiola Di Santo; Matteo Surdo; Stefano Aquaro; Carlo-Federico Perno (pp. 255-261).

► We investigated the anti HIV activity of integrase inhibitors (INIs) in macrophages, PBMC and C8166 T lymphocytes. ► Raltegravir and the other INIs showed similar or slightly higher efficacy in macrophages compared to PBMC and T cells. ► Raltegravir (and other INIs) may represent a therapy of choice to prevent and control the replication of HIV in the brain.The activity of raltegravir and 4 other integrase inhibitors (MK-2048, L870,810, IN2, and IN5) was investigated in primary human macrophages, PBMC and C8166-lymphocytic T cells, in order to determine their relative potency and efficacy in different cellular systems of HIV infection. Raltegravir showed better protective efficacy in all cell types; MK-2048, L870,810 and IN5 showed a potent anti-HIV-1 activity in macrophages, while in lymphocytes only MK-2048 and L870,810 showed an inhibitory effect comparable to raltegravir. IN2 was a poorly effective anti-HIV-1 compound in all cellular systems. All effective integrase inhibitors exhibited a potent antiviral activity against both X4 and R5 HIV-1 strains. In general, raltegravir, MK-2048, L870,810 and IN5 showed anti HIV activity similar or slightly higher in macrophages compared to PBMC and C8166 T cells: for MK-2048, the EC₅₀ was 0.4, 0.9, 11.5nM in macrophages, in PBMCs and T cells, respectively; for L870,810, the EC₅₀ was 1.5, 14.3, and 10.6nM, respectively; for IN5 the EC₅₀ was 0.5, 13.7, and 5.7nM, respectively.

Keywords: HIV; Integrase inhibitors; Macrophages; PBMC; Lymphocytes

Role of macrophages in early protective immune responses induced by two vaccines against foot and mouth disease by V. Quattrocchi; C. Langellotti; J.S. Pappalardo; V. Olivera; S. Di Giacomo; N. van Rooijen; C. Mongini; C. Waldner; P.I. Zamorano (pp. 262-270).

► We studied two vaccines in their ability to achieve early protection against FMDV. ► Both vaccines yielded protection against the virus at 4 and 7dpv in a mouse model. ► Both vaccines induced FMDV-specific, but non neutralizing, antibody titers. ► Macrophages play a central role in the response elicited by the vaccines. ► Opsonization and phagocytosis play a main role in early protection against FMDV.Foot and Mouth Disease (FMD) is an acute disease of cloven-hoofed species. We studied the protection and early immune response induced in the murine model by vaccines formulated with inactivated virus and two different adjuvants.The presence of IMS12802PR or ISA206VG adjuvants yielded protection against viral challenge at early times post vaccination and induced FMDV-specific, but non neutralizing, antibody titers. In vivo macrophage depletion in vaccinated mice severely decreased the protection levels after virus challenge, indicating a central role of this cell population in the response elicited by the vaccines. Accordingly, opsonophagocytosis of FITC-labelled virus was augmented in 802-FMDVi and 206-FMDVi vaccinated mice. These results demonstrate the ability of the studied adjuvants to enhance the protective responses of these inactivated vaccines without the increase in seroneutralizing antibodies and the main role of opsonization and phagocytosis in the early protective immune responses against FMD infection in the murine model.

Keywords: FMDV; Early protection; Macrophages

The main Hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication by Gaëtan Billioud; Christian Pichoud; Gerhard Puerstinger; Johan Neyts; Fabien Zoulim (pp. 271-276).

► Non-nucleosidic compounds inhibit the main resistant mutants replication. ► Non-nucleosidic compounds acting against capsid morphogenesis are not cytotoxic. ► Combination of non-nucleosidic compounds results in inhibition of resistant mutant replication. ► Compounds targeting nucleocapsid assembly could be relevant to decrease resistance to polymerase inhibitors.Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively. The sensitivity of the main nucleos(t)ide analog- resistant mutants to these inhibitors was evaluated in vitro. HepG2 stable cell lines permanently expressing wild type (WT) HBV or the main HBV mutants resistant to lamivudine and/or adefovir (rtL180M+rtM204V, rtV173L+rtL180M+rtM204V, rtM204I, rtL180M+rtM204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T, rtA181T+rtN236T) were treated with AT-61, AT-130 or BAY-41 4109. Analysis of intracellular encapsidated viral DNA showed that all mutants were almost as sensitive to these molecules as WT HBV; indeed, the fold-resistance ranged between 0.7 and 2.3. Furthermore, the effect of a combination of either AT-61 or AT-130 with BAY41-4109, and the combination of these compounds with tenofovir was studied on wild type HBV as well as on a lamivudine and an adefovir-resistant mutant (rtL180M+M204V and rtN236T, respectively). These combinations of compounds resulted in inhibition of viral replication but showed slight antagonistic effects on the three HBV species. Based on this in vitro study, BAY-41 4109, AT-61 and AT-130 molecules that interfere with capsid morphogenesis are active against the main lamivudine- and adefovir-resistant mutants. These results suggest that targeting nucleocapsid functions may represent an interesting approach to the development of novel HBV inhibitors to prevent and combat drug resistance.

Keywords: Abbreviations; HBV; hepatitis B virus; NA; nucleos(t)ide; HAP; heteroaryldihydropyrimidineHBV; Nucleos(t)ide analog; Drug resistance; Non-nucleos(t)idic inhibitors; Nucleocapsid

Application of kinase bypass strategies to nucleoside antivirals by Adrian S. Ray; Karl Y. Hostetler (pp. 277-291).

► Nucleoside and nucleotides are the cornerstones of antiviral therapy. ► Requirement for activation and side-effects limit the efficacy of the current drugs. ► Kinase bypass strategies could further advance the field of antiviral therapy. ► At least 10 prodrugs, from seven different classes, are in clinical trials.Nucleoside and nucleotide analogs have served as the cornerstones of antiviral therapy for many viruses. However, the requirement for intracellular activation and side-effects caused by distribution to off-target sites of toxicity still limit the efficacy of the current generation of drugs. Kinase bypass strategies, where phosphorylated nucleosides are delivered directly into cells, thereby, removing the requirement for enzyme catalyzed phosphorylation steps, have already changed the face of antiviral therapy in the form of the acyclic nucleoside phosphonates, cidofovir, adefovir (given orally as its dipivoxil prodrug) and tenofovir (given orally as its disoproxil prodrug), currently used clinically. These strategies hold further promise to advance the field of antiviral therapy with at least 10 kinase bypass and tissue targeted prodrugs, representing seven distinct prodrug classes, currently in clinical trials. This article reviews the history of kinase bypass strategies applied to nucleoside antivirals and the evolution of different tissue targeted prodrug strategies, highlighting clinically relevant examples.

Keywords: Nucleotide; Phosphonate; Prodrug; Antiviral; HCV; HIV

An attenuated EIAV vaccine strain induces significantly different immune responses from its pathogenic parental strain although with similar in vivo replication pattern by Yue-Zhi Lin; Rong-Xian Shen; Zhen-Ying Zhu; Xi-Lin Deng; Xue-Zhi Cao; Xue-Feng Wang; Jian Ma; Cheng-Gang Jiang; Li-Ping Zhao; Xiao-Ling Lv; Yi-Ming Shao; Jian-Hua Zhou (pp. 292-304).

► We compared immune responses elicited by a vaccine EIAV_DLV121 or virulent EIAV_LN40 strain. ► Infection of EIAV_DLV121 and sublethal dose of EIAV_LN40 resulted in similar plasma viral loads. ► EIAV_DLV121 stimulated significantly higher level of conformational dependent env and neutralizing antibodies. ► EIAV_DLV121 significantly enhanced CD4⁺ T cell responses especially in the early stage. ► p26 Capsid-specific antibody appeared to be a marker of protective immunity to EIAV.The EIAV (equine infectious anemia virus) multi-species attenuated vaccine EIAV_DLV121 successfully prevented the spread of equine infectious anemia (EIA) in China in the 1970s and provided an excellent model for the study of protective immunity to lentiviruses. In this study, we compared immune responses induced by EIAV_DLV121 to immunity elicited by the virulent EIAV_LN40 strain and correlated immune responses to protection from infection. Horses were randomly grouped and inoculated with either EIAV_DLV121 (Vaccinees, Vac) or a sublethal dose of EIAV_LN40 (asymptomatic carriers, Car). Car horses became EIAV_LN40 carriers without disease symptoms. Two of the four Vac horses were protected against infection and the other two had delayed onset or reduced severity of EIA with a lethal EIAV_LN40 challenge 5.5months post initial inoculation. In contrast, all three Car animals developed acute EIA and two succumbed to death. Specific humoral and cellular immune responses in both Vac and Car groups were evaluated for potential correlations with protection. These analyses revealed that although plasma viral loads remained between 10³ and 10⁵copies/ml for both groups before EIAV_LN40 challenge, Vac-treated animals developed significantly higher levels of conformational dependent, Env-specific antibody, neutralizing antibody as well as significantly elevated CD4⁺ T cell proliferation and IFN-γ-secreting CD8⁺ T cells than those observed in EIAV_LN40 asymptomatic carriers. Further analysis of protected and unprotected cases in vaccinated horses identified that cellular response parameters and the reciprocal anti-p26-specific antibody titers closely correlated with protection against infection with the pathogenic EIAV_LN40. These data provide a better understanding of protective immunity to lentiviruses.

Keywords: EIAV; Vaccine; Immune response

Virostatic potential of micro–nano filopodia-like ZnO structures against herpes simplex virus-1 by Yogendra Kumar Mishra; Rainer Adelung; Claudia Röhl; Deepak Shukla; Frank Spors; Vaibhav Tiwari (pp. 305-312).

► Discovery of ZnO-based micro–nanostructures (MNSs) as an efficient anti-HSV agent. ► Easy to synthesize particles with MNSs to block HSV-1 infection. ► Filopodia-like assembly of these MNSs provides multivalent binding sites for HSV-1. ► MNSs block infection both in vitro and in vivo. ► MNSs show the promise of development as prophylactic as well as therapeutic anti-HSV-1 agents.Herpes simplex virus type-1 (HSV-1) entry into target cell is initiated by the ionic interactions between positively charged viral envelop glycoproteins and a negatively charged cell surface heparan sulfate (HS). This first step involves the induction of HS-rich filopodia-like structures on the cell surface that facilitate viral transport during cell entry. Targeting this initial first step in HSV-1 pathogenesis, we generated different zinc oxide (ZnO) micro–nano structures (MNSs) that were capped with multiple nanoscopic spikes mimicking cell induced filopodia. These MNSs were predicted to target the virus to compete for its binding to cellular HS through their partially negatively charged oxygen vacancies on their nanoscopic spikes, to affect viral entry and subsequent spread. Our results demonstrate that the partially negatively charged ZnO-MNSs efficiently trap the virions via a novel virostatic mechanism rendering them unable to enter into human corneal fibroblasts – a natural target cell for HSV-1 infection. The anti-HSV-1 activity of ZnO MNSs was drastically enhanced after creating additional oxygen vacancies under UV-light illumination. Our results provide a novel insight into the significance of ZnO MNSs as the potent HSV-1 inhibitor and rationalize their development as a novel topical agent for the prevention of HSV-1 infection.

Keywords: Zinc oxide structures; Herpes simplex virus type-1 (HSV-1); Virus-cell interaction

Resistance to maribavir is associated with the exclusion of pUL27 from nucleoli during human cytomegalovirus infection by Morgan Hakki; Coyne Drummond; Benjamin Houser; Gail Marousek; Sunwen Chou (pp. 313-318).

► We studied the localization of the HCMV protein pUL27 in maribavir resistant mutants. ► Maribavir resistant UL27 mutations result in exclusion of pUL27 from nucleoli. ► A carboxy-terminal domain of pUL27 is also necessary for nucleolar localization. ► Loss of a nucleolar function of pUL27 may compensate for lack pUL97 kinase activity.Select mutations in the human cytomegalovirus (HCMV) gene UL27 confer low-grade resistance to the HCMV UL97 kinase inhibitor maribavir (MBV). It has been reported that the 608-amino acid UL27 gene product (pUL27) normally localizes to cell nuclei and nucleoli, whereas its truncation at codon 415, as found in a MBV-resistant mutant, results in cytoplasmic localization. We now show that in the context of full-length pUL27, diverse single amino acid substitutions associated with MBV resistance result in loss of its nucleolar localization when visualized after transient transfection, whereas substitutions representing normal interstrain polymorphism had no such effect. The same differences in localization were observed during a complete infection cycle with recombinant HCMV strains over-expressing full-length fluorescent pUL27 variants. Nested UL27 C-terminal truncation expression plasmids showed that amino acids 596–599 were required for the nucleolar localization of pUL27. These results indicate that the loss of a nucleolar function of pUL27 may contribute to MBV resistance, and that the nucleolar localization of pUL27 during HCMV infection depends not only on a carboxy-terminal domain but also on a property of pUL27 that is affected by MBV-resistant mutations, such as an interaction with component(s) of the nucleolus.

Keywords: Cytomegalovirus; Maribavir; UL27; Nucleolus; Antiviral; Drug resistance

GM-CSF modulates pulmonary resistance to influenza A infection by Zvjezdana Sever-Chroneos; Aditi Murthy; Jeremy Davis; Jon Matthew Florence; Anna Kurdowska; Agnieszka Krupa; Jay W. Tichelaar; Mitchell R. White; Kevan L. Hartshorn; Lester Kobzik; Jeffrey A. Whitsett; Zissis C. Chroneos (pp. 319-328).

► Early GM-CSF expression in conditional mice protects against influenza A virus. ► GM-CSF links innate and adaptive immunity against influenza. ► Unregulated GM-CSF causes lung immunopathology secondary to influenza infection. ► GM-CSF enhances expression of SP-R210 and MARCO on alveolar macrophages. ► MARCO decreases resistance to influenza infection.Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9×10⁴ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient ( GM^−/−) mice compared to GM^−/− mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO^−/− alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza.

Keywords: Abbreviations; SFTPC; or; SP-C; surfactant protein C gene promoter; SCG1A1; secretoglobin 1A1 gene promoter; SR-A; scavenger receptor class A; MARCO; macrophage receptor with collagenous structure; tet-GM; ⁺; ^/; ⁺; GM-CSF; ^−/−; ,; SCGB1A1-rtTA; /(; teto; ); ₇; CMV-GM-CSF; GM; GM-CSF; SP-R210; surfactant protein receptor 210; pfu; plaque forming unit; ffc; fluorescent focus countsGM-CSF; Influenza; Epithelial cells; Alveolar macrophages; SP-A; Scavenger receptors

In vitro and in vivo efficacy of fluorodeoxycytidine analogs against highly pathogenic avian influenza H5N1, seasonal, and pandemic H1N1 virus infections by Yohichi Kumaki; Craig W. Day; Donald F. Smee; John D. Morrey; Dale L. Barnard (pp. 329-340).

► 2′-Deoxy-2′-fluorocytidine potently inhibited H5N1 influenza viruses. ► 2′-FdC protected H5N1 influenza-virus infected BALB/c mice from death. ► 2′-FdC was administered 72h post virus exposure and still significantly protect mice from death. ► 2-FdC also significantly enhanced survival in mice infected with a pandemic H1N1 virus.Various fluorodeoxyribonucleosides were evaluated for their antiviral activities against influenza virus infections in vitro and in vivo. Among the most potent inhibitors was 2′-deoxy-2′-fluorocytidine (2′-FdC). It inhibited various strains of low and highly pathogenic avian influenza H5N1 viruses, pandemic H1N1 viruses, an oseltamivir-resistant pandemic H1N1 virus, and seasonal influenza viruses (H3N2, H1N1, influenza B) in MDCK cells, with the 90% inhibitory concentrations ranging from 0.13 to 4.6μM, as determined by a virus yield reduction assay. 2′-FdC was then tested for efficacy in BALB/c mice infected with a lethal dose of highly pathogenic influenza A/Vietnam/1203/2004 H5N1 virus. 2′FdC (60mg/kg/d) administered intraperitoneally (i.p.) twice a day beginning 24h after virus exposure significantly promoted survival (80% survival) of infected mice ( p=0.0001). Equally efficacious were the treatment regimens in which mice were treated with 2′-FdC at 30 or 60mg/kg/day (bid X 8) beginning 24h before virus exposure. At these doses, 70–80% of the mice were protected from death due to virus infection ( p=0.0005, p=0.0001; respectively). The lungs harvested from treated mice at day four of the infection displayed little surface pathology or histopathology, lung weights were lower, and the 60mg/kg dose reduced lung virus titers, although not significantly compared to the placebo controls. All doses were well tolerated in uninfected mice. 2′-FdC could also be administered as late as 72h post virus exposure and still significantly protect 60% mice from the lethal effects of the H5N1 virus infection ( p=0.019). Other fluorodeoxyribonucleosides tested in the H5N1 mouse model, 2′-deoxy-5-fluorocytidine and 2′-deoxy-2′,2′-difluorocytidine, were very toxic at higher doses and not inhibitory at lower doses. Finally, 2′-FdC, which was active in the H5N1 mouse model, was also active in a pandemic H1N1 influenza A infection model in mice. When given at 30mg/kg/d (bid X 5) beginning 24h before virus exposure), 2′-FdC also significantly enhanced survival of H1N1-infected mice (50%, p=0.038) similar to the results obtained in the H5N1 infection model using a similar dosing regimen (50%, p<0.05). Given the demonstrated in vitro and in vivo inhibition of avian influenza virus replication, 2′FdC may qualify as a lead compound for the development of agents treating influenza virus infections.

Keywords: 2′-Deoxy-2′-fluorocytidine; Fluorodeoxyribonucleosides; H5N1 avian influenza virus; BALB/c mouse; Pandemic H1N1 virus

Houttuynia cordata blocks HSV infection through inhibition of NF-κB activation by Xiaoqing Chen; Zhongxia Wang; Ziying Yang; Jingjing Wang; Yunxia Xu; Ren-xiang Tan; Erguang Li (pp. 341-345).

► Houttuynia cordata Thunb. exhibits strong activity against herpes simplex virus. ► H. cordata blocks HSV-2 infection through inhibition of NF-κB, not MAPK. ► Flavonoids like quercetin, quercitrin and isoquercitrin as major active components. Houttuynia cordata Thunb. is a medicinal plant widely used in folk medicine in several Asian countries. It has been reported that a water extract of H. cordata exhibits activity against herpes simplex virus (HSV) and the virus of severe acute respiratory syndrome (SARS), although the mechanisms are not fully understood yet. Previous studies have demonstrated absolute requirement of NF-κB activation for efficient replication of HSV-1 and HSV-2 and inhibition of NF-κB activation has been shown to suppress HSV infection. Here we show that a hot water extract of H. cordata (HCWE) inhibits HSV-2 infection through inhibition of NF-κB activation. The IC₅₀ was estimated at 50μg/ml of lyophilized HCWE powder. At 150 and 450μg/ml, HCWE blocked infectious HSV-2 production by more than 3 and 4 logs, respectively. The inhibitory activity was concomitant with an inhibition of NF-κB activation by HSV-2 infection. Although activation of NF-κB and Erk MAPK has been implicated for HSV replication and growth, HCWE showed no effect on HSV-2-induced Erk activation. Furthermore, we show that treatment with quercetin, quercitrin or isoquercitrin, major water extractable flavonoids from H. cordata, significantly blocked HSV-2 infection. These results together demonstrated that H. cordata blocks HSV-2 infection through inhibition of NF-κB activation.

Keywords: Houttuynia cordata; Herpes simplex virus; NF-κB; Flavonoid; AntiviralAbbreviations; HCWE; Houttuynia cordata; water extract; ACV; acyclovir

FimH, a TLR4 ligand, induces innate antiviral responses in the lung leading to protection against lethal influenza infection in mice by Mohamed F. Abdul-Careem; M. Firoz Mian; Amy E. Gillgrass; Meghan J. Chenoweth; Nicole G. Barra; Tiffany Chan; Amal A. Al-Garawi; Marianne V. Chew; Geoffry Yue; Nico van Roojen; Zhou Xing; Ali A. Ashkar (pp. 346-355).

► Local delivery of FimH provides innate antiviral protection against influenza. ► Innate antiviral activity of FimH is TLR4 dependent. ► Alveolar macrophages play a role in the FimH-mediated antiviral responses. ► Delivery of FimH into lung induces production of pro-inflammatory cytokines.Fimbriae H protein (FimH) is a novel TLR4 ligand that has been shown to stimulate the innate immune system and elicits protective responses against bacterial and viral infections. Here, we evaluated the protective role of local delivery of FimH against influenza A infection in a mouse model. We show that intranasal delivery of FimH prior to lethal challenge with influenza A virus, resulted in decreased morbidity and mortality in wild-type, but not TLR4^−/−, mice. Importantly, FimH was able to reduce the early viral burden in the lung leading to minimal cell infiltration into the airway lumen and reduced pulmonary pathology following infection in wild type mice compared to TLR4^−/− mice. Local delivery of FimH to C57BL/6, not TLR4^−/−, mice in a prophylactic manner increased the IL-12 and RANTES responses as well as neutrophil recruitment into the airway lumen. These effects correlate to the course of influenza infection. The FimH-mediated antiviral response against influenza virus appears to be partially dependent on alveolar macrophages. The antiviral effects are likely mediated by the innate mediators (TNF-α, IL-12 or RANTES) and/or by activation of a feedback inhibition loop to curtail the pulmonary inflammation possibly be the potential mechanisms involved in FimH-mediated protection. FimH thus holds promise to be a possible prophylactic mean of control against influenza viral infection.

Keywords: Influenza; TLR4; FimH; Innate immunity; Lung pathology

Virological failure of intralesional cidofovir therapy in recurrent respiratory papillomatosis is not associated with genetic or epigenetic changes of HPV11: Complete genome comparison of sequential isolates by Tamás Gáll; Andrea Kis; Enikő Fehér; Lajos Gergely; Krisztina Szarka (pp. 356-358).

Five sequential human papillomavirus type 11 (HPV11) positive samples collected from an aggressive juvenile onset recurrent respiratory papillomatosis before, during and after intralesional cidofovir therapy leading to virological failure after initial response were analyzed. Sequencing of the complete genome as well as methylation analysis by bisulfate modification and sequencing of the long control region (LCR) were performed to seek for genetic and epigenetic changes as a possible background for therapy failure. Single-strand conformation polymorphism of E1, E2, E6, E7 and LCR was used to exclude the presence of multiple HPV11 infection. All five complete genomes were identical and all four E2 binding sites in the LCR were uniformly unmethylated in all five genomes. Thus the virological failure was not due to virological factors suggesting that cidofovir action may depend more heavily on the host.

Keywords: Laryngeal papillomatosis; Therapeutic response; Antiviral resistance; Complete genome; LCR methylation

A DNA vaccine encoding foot-and-mouth disease virus B and T-cell epitopes targeted to class II swine leukocyte antigens protects pigs against viral challenge by Belén Borrego; Jordi M. Argilaguet; Eva Pérez-Martín; Javier Dominguez; Mariano Pérez-Filgueira; José M. Escribano; Francisco Sobrino; Fernando Rodriguez (pp. 359-363).

► DNA vaccination in pigs can be improved by SLA II targeting. ► Fusion of B and T-cell epitopes to the APCH1 molecule improve the protection afforded by DNA vaccines. ► Both humoral and cellular responses are essential to confer full protection against FMDV.Development of efficient and safer vaccines against foot-and-mouth disease virus (FMDV) is a must. Previous results obtained in our laboratory have demonstrated that DNA vaccines encoding B and T cell epitopes from type C FMDV, efficiently controlled virus replication in mice, while they did not protect against FMDV challenge in pigs, one of the FMDV natural hosts. The main finding of this work is the ability to improve the protection afforded in swine using a new DNA-vaccine prototype (pCMV-APCH1BTT), encoding FMDV B and T-cell epitopes fused to the single-chain variable fragment of the 1F12 mouse monoclonal antibody that recognizes Class-II Swine Leukocyte antigens. Half of the DNA-immunized pigs were fully protected upon viral challenge, while the remaining animals were partially protected, showing a delayed, shorter and milder disease than control pigs. Full protection in a given vaccinated-pig correlated with the induction of specific IFNγ-secreting T-cells, detectable prior to FMDV-challenge, together with a rapid development of neutralizing antibodies after viral challenge, pointing towards the relevance that both arms of the immune response can play in protection. Our results open new avenues for developing future FMDV subunit vaccines.

Keywords: DNA vaccine; FMDV; ScFv; T-cell; Neutralizing antibody

The unique antiviral activity of artesunate is broadly effective against human cytomegaloviruses including therapy-resistant mutants by Sunwen Chou; Gail Marousek; Sabrina Auerochs; Thomas Stamminger; Jens Milbradt; Manfred Marschall (pp. 364-368).

► We present novel data about the broad anti-cytomegaloviral activity of artesunate. ► The drug is similarly active against viral strains and therapy-resistant mutants. ► Viral replication is blocked at a very early stage. ► In vitro efficacy is optimized when the drug is applied as fractional doses. ► A synergistic effect occurs with artesunate-maribavir combination treatment.Current therapy options to treat infections with human cytomegalovirus face severe limitations leading to a continued search for novel drug candidates. Here, we describe novel characteristics of the strong antiviral potency of the drug artesunate. In vitro virus replication systems were applied to analyze a number of laboratory and clinically relevant strains of human cytomegalovirus. An inhibitory block at a very early stage of infection was demonstrated. Time-of-addition experiments indicated that the antiviral efficacy could be optimized when artesunate was applied as fractional doses consecutively added post-infection. Artesunate showed a clearly higher anti-cytomegaloviral activity than its parental drug artemisinin (approximately 10-fold) or other artesunate-related compounds. Mean IC₅₀ values of artesunate for a variety of standard therapy-resistant virus mutants were within a 2-fold range compared to wild-type virus. Furthermore, a synergistic effect was identified when artesunate was combined with the mechanistically distinct antiviral compound maribavir. These findings point to unique antiviral properties of artesunate which may offer an advantage over standard antiviral therapy particularly in cases of drug resistance.

Keywords: Human cytomegalovirus; Antiviral activity of artesunate; Artesunate-related drugs; Therapy-resistant virus mutants; Synergistic effect

Competitive inhibitor of cellular α-glucosidases protects mice from lethal dengue virus infection by Jinhong Chang; Wouter Schul; Andy Yip; Xiaodong Xu; Ju-Tao Guo; Timothy M. Block (pp. 369-371).

► Dengue virus infection causes Dengue fever and life-threatening Hemorrhagic Fever/ Shock Syndrome. ► Previously we reported that a host -glucosidase inhibitor CM-10-18 reduced viremia in dengue virus infected AG129 mice. ► Here we show that CM-10-18 significantly protects mice from death in two mouse models of lethal dengue virus infection. ► Our results provide support for the development of CM-10-18 or its derivatives as antiviral agents against dengue virus.Dengue virus infection causes diseases in people, ranging from the acute febrile illness dengue fever, to life-threatening dengue hemorrhagic fever/dengue shock syndrome. We previously reported that a host cellular α-glucosidases I and II inhibitor, imino sugar CM-10–18, potently inhibited dengue virus replication in cultured cells, and significantly reduced viremia in dengue virus infected AG129 mice. In this report we show that CM-10–18 also significantly protects mice from death and/or disease progress in two mouse models of lethal dengue virus infection. Our results thus provide a strong support for the development of CM-10–18 or its derivatives as antiviral agents to treat servere dengue virus infections.

Keywords: Glucosidase inhibitor; Dengue virus; Lethal infection mouse model

In silico study supports the efficacy of a reduced dose regimen for stavudine by Selwyn J. Hurwitz; Raymond F. Schinazi (pp. 372-377).

► Stavudine (d4T) is widely used in resource poor settings despite toxicity. ► WHO recommended reducing d4T dose from 40 to 30mg bid when replacement is not feasible. ► A population PK/biochemical model was used to simulate cellular d4T-TP versus dose. ► d4T-TP concentrations for 30mg bid exceeded wt HIV-1 EC₅₀ in most virtual subjects. ► This suggests that this low dose could maintain antiviral efficacy.Stavudine (d4T) is used extensively as part of HAART in resource poor settings, despite its toxicities. The revised WHO guidelines specify replacement of d4T with less toxic but more expensive drugs when feasible, and that d4T doses be standardized to 30mg twice daily (bid) (irrespective of body-weight), from the approved 40mg bid in adults (body-weight ⩾60kg). Therefore, an in silico population pharmacokinetic and biochemical model was utilized to compare relative efficacies of the two doses in humans. Assessment of predicted quartile ranges of simulated concentrations of the triphosphate of d4T suggested sufficient trough concentrations to inhibit wild type HIV-1 reverse transcriptase at the reduced dose, lending support to the revised WHO recommendations.

Keywords: Antiretroviral; Nucleosides; NONMEM; Simulation; HIV; d4T

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants by Josef Špak; Ivan Votruba; Daniela Pavingerová; Antonín Holý; Vlastimila Špaková; Karel Petrzik (pp. 378-381).

► We study antiviral effect of tenofovir on CaMV in Brassica pekinensis grown in vitro. ► Tenofovir (25–50mg/l) reduced CaMV concentration in plants within 6–9weeks. ► Virus-free plants were obtained after 3-month cultivation on medium with tenofovir. ► We proved metabolism of tenofovir in B. pekinensis leaves but not in roots. ► Mono- and diphosphate analogs of NDP and/or NTP are the only metabolites formed.The antiviral effect of the acyclic nucleoside phosphonate tenofovir ( R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. ( R)-PMPA at concentrations of 25 and 50mg/l significantly reduced CaMV titers in plants within 6–9weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50mg/l ( R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of ( R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward ( R)-PMPA as substrate. The extent of phosphorylation in the plant’s leaves represents only 4.5% of applied labeled ( R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of ( R)-PMPAp or ( R)-PMPApp.

Keywords: Abbreviations; (; R; )-PMPAp; N; ⁶; -methyl-(; R; )-9-[2-(phosphonomethoxy)propyl]adenine monophosphate; (; R; )-mePMPA; N; ⁶; -methyl-(; R; )-9-[2-(phosphonomethoxy)propyl]adenine; (; R; )-me; ₂; PMPA; N; ⁶; -dimethyl-(; R; )-9-[2-(phosphonomethoxy)propyl]adenine; (; R; )-PMPHx; (; R; )-9-[2-(phosphonomethoxy)propyl]hypoxanthineCaulimovirus; dsDNA; Pararetrovirus; Chemotherapy; Virus eradication; Brassica

Characterization of drug-resistance mutations in HBV D-genotype chronically infected patients, naïve to antiviral drugs by R. Salpini; V. Svicher; V. Cento; C. Gori; A. Bertoli; F. Scopelliti; V. Micheli; T. Cappiello; A. Spanò; G. Rizzardini; G.M. De Sanctis; C. Sarrecchia; M. Angelico; C.F. Perno (pp. 382-385).

► Our study defined prevalence of drug-resistance in 140 drug-naïve patients. ► No YMDD mutations, rtM204V/I, were found. ► 1.4% of patients carried primary drug-resistance, 2.1% harboured secondary mutations. ► Five polymorphic mutations, with a suggested role in drug resistance, were detected. ► Our study suggests potential full efficacy of antiviral drugs used as 1st line therapy.Presence of drug-resistance mutations in drug-naïve hepatitis B virus (HBV) infected patients can seriously compromise response to antiviral treatment. Therefore, our study was aimed at defining the prevalence of HBV drug-resistance in a population of 140 patients, all infected with HBV-D-genotype (the most common HBV-genotype in Eastern Europe, Mediterranean countries and Middle East) and naïve to antiviral therapy. HBV reverse-transcriptase (RT) region was sequenced and analyzed for 20 mutations, confirmed by in vitro studies as associated with resistance to nucleos(t)ide HBV-RT inhibitors (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C/G/I-rtM204V/I-rtN236T-rtM250V). Amino acid changes at other six RT positions, potentially associated with resistance, were also analyzed (rtV84M-rtV191I-rtV207L-rtV214A-rtQ215S-rtI233V). Overall, only 2/140 (1.4%) patients carried primary drug-resistance mutations [rtA181V (0.7%), and rtA194T (0.7%)], while 3/140 (2.1%) patients harbored the secondary mutations rtV173L (1.4%) and rtL180M (0.7%). Additionally, five polymorphic mutations, with a suggested role in drug resistance, were detected [rtQ215S (12.8%), rtI233V (4.3%), rtV214A (3.6%), rtV191I (0.7%), rtV207L (0.7%)]. Notably, no YMDD mutations, namely rtM204V/I, were found. Taken together, the rate of important drug resistance mutations in naïve HBV D-genotype infected patients is today very low, and suggests the potential full efficacy of new-generation antiviral drugs used in first line therapy. Whether such low rate can be extrapolated to non HBV-D subtypes, requires a detailed investigation to be performed in a different cohort of patients.

Keywords: HBV infection; Drug resistance; D genotype; Drug-naïve

N-Methanocarbathymidine is more effective than acyclovir for treating neonatal herpes simplex virus infection in guinea pigs by David I. Bernstein; Fernando J. Bravo; Jennifer R. Clark; Julie D. Earwood; Aquilur Rahman; Robert Glazer; Rhonda D. Cardin (pp. 386-388).

► We compared ( N)-MCT to ACV for the treatment of guinea pig neonatal HSV infections. ► ( N)-MCT was effective at lower doses than ACV. ► Both ACV and ( N)-MCT improved survival when begun on day one after infection. ► Only ( N)-MCT reduced the number of animals with symptoms when begun on day one. ► Only ( N)-MCT was effective when begun on day 2 or 3 after infection.The outcome of neonatal herpes simplex (HSV) infection, even after therapy with high dose acyclovir (ACV), is not optimum. We therefore evaluated N-Methanocarbathymidine (( N)-MCT) using the guinea pig model of neonatal herpes. Treatment with ACV (60mg/kg/day) was compared to doses of 1, 5, and 25mg/kg/day of ( N)-MCT initiated 1, 2, or 3days postinoculation (dpi). Both ACV and ( N)-MCT significantly improved survival, but only ( N)-MCT significantly reduced the number of animals with symptoms when begun at 1dpi. When therapy was begun at 2dpi, only ( N)-MCT (1, 5, or 25mg/kg/day) significantly increased survival. In fact, ( N)-MCT improved survival up to 3dpi, the last time point evaluated. ( N)-MCT was highly effective and superior to high dose ACV therapy for the treatment of neonatal herpes in the guinea pig model.

Keywords: Herpes simplex virus; Neonatal herpes; (; N; )-MCT; N; -Methanocarbathymidine

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Antiviral Research (v.92, #2)