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Antiviral Research (v.76, #2)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

NNRTI-selected mutations at codon 190 of human immunodeficiency virus type 1 reverse transcriptase decrease susceptibility to stavudine and zidovudine by Stefania Paolucci; Fausto Baldanti; Giulia Campanini; Reynel Cancio; Amalia Belfiore; Giovanni Maga; Giuseppe Gerna (pp. 99-103).

The non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI)-binding pocket of HIV-1 RT spans codons 100–110, 180–190 and 220–240 and mutations in these domains are responsible for HIV-1 NNRTI resistance. Recombinant HIV-1 strains carrying G190S/A/E, G190S+T215Y, T215Y and K103N mutations were constructed to evaluate susceptibility to both NNRTIs and nucleoside RT inhibitors (NRTIs). In addition, purified recombinant RT enzymes were obtained to determine the degree of in vitro inhibition by drugs of both classes. High-level resistance to nevirapine and moderate level resistance to both stavudine and zidovudine were associated with G190S/A/E substitutions. The simultaneous presence of G190S and T215Y decreased stavudine and zidovudine susceptibility more than T215Y alone. On the other hand, G190S was associated with a marked decrease in RT catalytic efficiency, while T215Y showed a more limited effect. Interestingly, the simultaneous presence of G190S and T215Y was associated with a reduction in the impairment of the G190S-mutated enzyme. Mutations in the HIV-1 RT NNRTI binding pocket may be associated with cross-resistance to NRTI. Selection of double mutants, with further decrease in NRTI susceptibility, might be favoured by the compensatory effect of T215Y on the reduction of RT catalytic efficiency associated with G190S.

Keywords: HIV-1; RT mutations; NNRTI; NRTI; Drug resistance

A model of human cytomegalovirus infection in severe combined immunodeficient mice by Fernando J. Bravo; Rhonda D. Cardin; David I. Bernstein (pp. 104-110).

Animal models for the evaluation of therapies against human cytomegalovirus (HCMV) are limited due to the species-specific replication of CMV. Models utilizing human fetal tissues implanted into SCID mice have been used but tend to be labor intensive and require human tissues. We therefore developed a model using HCMV-infected human foreskin fibroblasts (HFF) seeded onto a biodegradable gelatin matrix (Gelfoam). Infected HFFs are then implanted subcutaneously into SCID mice. We next evaluated two antivirals in this model. Treatment from days 0 to 5 with ganciclovir (GCV) produced a marginally significant reduction in viral titer while treatment from days 0 to 14 resulted in a more significant reduction in viral titers of 1.47log₁₀pfu/ml ( P<0.0001). Viral titers were similarly reduced in a group receiving GCV treatment from days 7 to 14 post-implantation (1.50log₁₀pfu/ml, P<0.0001). Cidofovir therapy from days 7 to 14 reduced viral titers by almost 2log₁₀pfu/ml (from 3.51±0.31log₁₀pfu/ml in untreated animals to 1.56±0.40log₁₀pfu/ml in treated animals, P<0.0001). These results indicate that the Gelfoam-HCMV SCID mouse model is suitable for the in vivo evaluation of new antivirals against HCMV and is simpler and more convenient than previous models.

Keywords: Human cytomegalovirus (HCMV); SCID mice; Gelfoam; Antiviral; Ganciclovir; Cidofovir

Dual antiviral activity of human alpha-defensin-1 against viral haemorrhagic septicaemia rhabdovirus (VHSV): Inactivation of virus particles and induction of a type I interferon-related response by A. Falco; V. Mas; C. Tafalla; L. Perez; J.M. Coll; A. Estepa (pp. 111-123).

It is well known that human alpha-defensin-1, also designated as human neutrophil peptide 1 (HNP1), is a potent inhibitor towards several enveloped virus infecting mammals. In this report, we analyzed the mechanism of the antiviral action of this antimicrobial peptide (AMP) on viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Against VHSV, synthetic HNP1 possesses two antiviral activities. The inactivation of VHSV particles probably through interfering with VHSV-G protein-dependent fusion and the inhibition of VHSV replication in target cells by up-regulating genes related to the type I interferon (IFN) response, such as Mx. Neither induction of IFN-stimulated genes (ISGs) by HNP1 nor their antiviral activity against fish rhabovirus has been previously reported. Therefore, we can conclude that besides to acting as direct effector, HNP1 acts across species and can elicit one of the strongest antiviral responses mediated by innate immune system. Since the application of vaccine-based immunization strategies is very limited, the used of chemicals is restricted because of their potential harmful impact on the environment and no antimicrobial peptides from fish that exhibit both antiviral and immunoenhancing capabilities have been described so far, HNP1 could be a model molecule for the development of antiviral agents for fish. In addition, these results further confirm that molecules that mediate the innate resistance of animals to virus may prove useful as templates for new antivirals in both human and animal health.

Keywords: HNP1; VHSV; IFN; Mx; AMP; Rhabdovirus; Alpha-defensin

Efficacy of N-methanocarbathymidine in treating mice infected intranasally with the IHD and WR strains of vaccinia virus by Donald F. Smee; Brett L. Hurst; Min-Hui Wong; Robert I. Glazer; Aquilur Rahman; Robert W. Sidwell (pp. 124-129).

N-Methanocarbathymidine [( N)-MCT] is a newly identified inhibitor of orthopoxvirus replication in cell culture and in mice. Limited published animal studies indicated the compound is effective by intraperitoneal (i.p.) route at 10–100mg/(kgday). More extensive studies using different treatment regimens in intranasally infected mice were conducted in order to further explore the potential of this compound compared to cidofovir in treating vaccinia virus infections. ( N)-MCT was given twice a day for 7 days, whereas cidofovir was administered once a day for 2 days, each starting 24h after virus exposure for most experiments. ( N)-MCT was not toxic up to 1000mg/(kgday) by the i.p. treatment route. Oral and i.p. treatment regimens with ( N)-MCT were directly compared during a vaccinia virus (IHD strain) infection, indicating that the nucleoside has good oral bioavailability in mice. Treatments by i.p. route with ( N)-MCT (100mg/(kgday)) reduced lung, nasal, and brain virus titers during an IHD virus infection, but not nearly to the same extent as i.p. cidofovir (100mg/(kgday)). Treatment with both compounds decreased liver, spleen, and kidney virus titers, as well as reduced lung consolidation scores and lung weights. Onset of treatment could be delayed by 2 days with ( N)-MCT and by 3 days with cidofovir, providing significant survival benefit during the IHD virus infection. Against a vaccinia virus (WR strain) infection in mice, i.p. ( N)-MCT treatment prevented death at 500mg/(kgday), which was comparable in activity to i.p. cidofovir (100mg/(kgday)). Significant reductions in tissue virus titers occurred with both treatment regimens. ( N)-MCT could be further pursued for its potential to treat orthopoxvirus infections in humans.

Keywords: Antiviral; Orthopoxvirus; Vaccinia virus; (; N; )-MCT; Cidofovir; Thymidine

Efficient conversion of tetrapeptide-based TSAO prodrugs to the parent drug by dipeptidyl-peptidase IV (DPPIV/CD26) by Carlos García-Aparicio; Alberto Diez-Torrubia; Jan Balzarini; Anne-Marie Lambeir; Sonsoles Velázquez; María-José Camarasa (pp. 130-139).

A novel prodrug approach has been evaluated using the anti-HIV-active TSAO molecule as the prototype drug to prove the kinetics with purified enzyme and the principles of conversion to the parent compound in sera and cell culture. When a variety of tetrapeptidyl amide prodrugs of NAP-TSAO were synthesized and exposed to purified dipeptidyl-peptidase IV (DPPIV/CD26) as well as human and bovine sera, they are converted to the parent NAP-TSAO drug in two successive steps by both purified CD26 and human and bovine serum. The efficiency of conversion strongly depends on the nature of the amino acid that has to be cleaved-off from the prodrug molecule. The tetrapeptidyl prodrug20 showed a more than 10-fold improved water-solubility in comparison to that of the parent compound NAP-TSAO. The antiviral activity of the prototype NAP-TSAO could also be modulated by introducing different tetrapeptide moieties on the molecule resulting, in some cases, in a superior antiviral potential in cell culture than the parent drug.

Keywords: Dipepeptidyl-peptidase IV (DPPIV); CD-26; Prodrugs; TSAO conjugates; TSAO-T compounds

Coimmunization with RANTES plasmid polarized Th1 immune response against hepatitis B virus envelope via recruitment of dendritic cells by Ke Ma; Wei Xu; Xianan Shao; YanYue; Linkun Hu; Huanbin Xu; Zhigang Yuan; Xiujuan Zheng; Sidong Xiong (pp. 140-149).

Induction of T help cell type 1 (Th1) response seems to be a prerequisite of HBV clearance. DNA vaccines have shown its potential to elicit Th1-biased immune response. However, its immunogenicity needs to be improved. Regulated upon activation normal T cell expressed and secreted (RANTES) is an inflammatory chemokine that promotes the accumulation and activation of CD4+, CD8+ T cells, and dendritic cells (DCs), which would favor antiviral immunity. In this study, the efficacy of a DNA vaccine encoding hepatitis B virus (HBV) preS2 plus S protein was enhanced through co-injection of a plasmid encoding RANTES in a BALB/c model. Co-injection of RANTES gene resulted in a moderate increase in the HBV specific humoral and cellular immune responses and a significant increase following an HBsAg booster vaccination compared to DNA encoding HBsAg alone. This enhancement was due to an enrichment of DCs in the draining lymph node and an up-regulation of DCs maturation by RANTES. More importantly, RANTES polarized the specific immunity towards a dominant Th1 profile and even converted an established Th2 response to a Th1 phenotype. Our study suggested the feasibility of using a plasmid-encoded RANTES as a modulatory Th1 adjuvant in genetic vaccination.

Keywords: HBV; DNA vaccine; RANTES; DC; Th1

The human fatty acid synthase: A new therapeutic target for coxsackievirus B3-induced diseases? by Alexander Rassmann; Andreas Henke; Nadine Jarasch; Friedrich Lottspeich; Hans-Peter Saluz; Thomas Munder (pp. 150-158).

Coxsackievirus is linked to a large variety of severe human and animal diseases such as myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. However, host proteins which may play a role in coxsackievirus-induced diseases are ill-defined. Two-dimensional gel electrophoresis of protein extracts obtained from coxsackievirus B3 (CVB3)-infected and uninfected HeLa or HepG2 cells combined with spot analysis revealed several proteins which are exclusively up-regulated in infected cells. One of these proteins was identified as the fatty acid synthase (FAS). By using cerulenin and C75, two known inhibitors of FAS we were able to significantly block CVB3 replication. FAS appears to be directly involved in CVB3-caused pathology and is therefore suitable as a therapeutic target in CVB3-induced diseases.

Keywords: Coxsackievirus; Fatty acid synthase (FAS); Cerulenin; c75; TOFA

Expression of IL-27 p28 by Theiler's virus-infected macrophages depends on TLR3 and TLR7 activation of JNK-MAP-kinases by Lara Hause; Fahd M. Al-Salleeh; Thomas M. Petro (pp. 159-167).

Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating disease (DD) due to infection of macrophages, stimulation of macrophage Toll-like receptor (TLR)3 and TLR7 pathways, activation of Mitogen-activated protein kinases (MAPK)s, and production of macrophages cytokines. Because expression of IL-27, a macrophage cytokine composed of p28 and EBI3 subunits, has been implicated in DD, we examined IL-27 subunit mRNA expression during TMEV infection of RAW264.7 cells, a macrophage cell line. TMEV infection of RAW264.7 cells did not affect cell viability, resulted in viral RNA replication, as well as p28 and EBI3 expression. Expression of p28 in TMEV-infected RAW264.7 cells depended on TLR3 and TLR7, as well as JNK but not p38 or ERK MAPKs. Since TMEV causes DD in SJL/J but not B10.S mice we determined the difference in expression of IL-27 subunit mRNA in SJL/J compared to B10.S macrophages. SJL/J macrophages expressed significantly more p28 mRNA after TMEV infection and after stimulation with TLR3 and TLR7 agonists compared with B10.S macrophages. Therefore, macrophages expression of IL-27 p28 mRNA in response to TMEV is due to activation of TLR3, TLR7, and JNK MAPKs pathways.

Keywords: IL-27; TMEV; JNK; TLR3; TLR7; Macrophages; RAW264.7 cells

The phenylpropenamide derivative AT-130 blocks HBV replication at the level of viral RNA packaging by J.J. Feld; D. Colledge; V. Sozzi; R. Edwards; M. Littlejohn; S.A. Locarnini (pp. 168-177).

Nucleos(t)ide analogue antiviral therapy for chronic hepatitis B has proven to be effective in the short term but the frequent development of resistance limits its clinical utility. Agents targeting other stages of viral replication are needed in order to develop improved combination therapies. The phenylpropenamide derivatives AT-61 and AT-130 have been shown to inhibit HBV replication in vitro, but the mechanism of action of these compounds remains undefined. The aim of this study was to determine the mechanism of action of AT-130, a non-nucleoside inhibitor of HBV in several in vitro models of replication. These studies found that AT-130 inhibited HBV DNA replication in hepatoma cells but had no effect on viral DNA polymerase activity or core protein translation. Total HBV RNA production was also unaffected in the presence of the drug whilst the amount of encapsidated RNA was significantly reduced, thereby inhibiting subsequent viral reverse transcription. These studies have established that the inhibition of HBV genome replication by a non-nucleoside analogue acting at the level of viral encapsidation and packaging is a potentially useful strategy for future therapeutic drug development in the management of chronic hepatitis B.

Keywords: Hepatitis B virus; Drug resistance; Phenylpropenamide; Viral RNA packaging

Biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives by Jae Min Song; Ki Duk Park; Kwang Hee Lee; Young Ho Byun; Ju Hee Park; Sung Han Kim; Jae Hong Kim; Baik Lin Seong (pp. 178-185).

Catechin derivatives with different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group were synthesized from epigallocatechin (EGC) and (+)-catechin (C) and their anti-influenza viral activity were evaluated in vitro and in ovo. Pronounced antiviral activity was observed for derivatives carrying moderate chain length (7–9 carbons) as compared to those with aromatic rings, whereas the 5′-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The derivatives exerted inhibitory effects for all six influenza subtypes tested including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The compounds strongly inhibited adsorption of the viruses on red blood cell (RBC). They also restricted the growth of avian influenza virus in ovo with minimum inhibition concentration (MIC) of 5–10μM far exceeding the neuraminidase (NA) inhibitor oseltamivir or M2 proton channel inhibitor amantadine. The antiviral activity appears to be mediated by interaction with hemagglutinin (HA)/viral membrane rendering HA less fusogenic at the initial stage of infection. The broad spectrum activity against various subtypes of influenza viruses may complement the limitations of current antivirals and contribute for managing potentially emerging influenza pandemic. The structure-activity data of catechin derivatives may usefully guideline future research endeavors for applying green tea catechins as alternative anti-viral agents.

Keywords: Influenza virus; Catechin derivatives; Alkyl chain; Hemagglutinin

Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice by Hongbo Zhou; Meilin Jin; Zhengjun Yu; Xiaojuan Xu; Yaping Peng; Haiya Wu; Jinlin Liu; Hu Liu; Shengbo Cao; Huanchun Chen (pp. 186-193).

RNA interference (RNAi) is a powerful tool to silence gene expression. Small interfering RNA (siRNA)-induced RNA degradation has been recently used as an antivirus agent to inhibit specific virus replication. Here, we showed that several siRNAs specific for conserved regions of influenza virus matrix (M2) and nucleocapsid protein (NP) genes could effectively inhibit expression of the corresponding viral protein. We also evaluated the antiviral potential of these siRNAs targeting M2 and NP of H5N1 avian influenza virus (AIV), which are essential to viral replication. We investigated the inhibitory effect of M2-specific siRNAs and NP-specific siRNAs on influenza A virus (H5N1, H1N1 and H9N2) replication in Madin-Darby canine kidney (MDCK) cells and BALB/c mice. The results showed that treatment with these siRNAs could specifically inhibit influenza A virus replication in MDCK cells (0.51–1.63 TCID₅₀ reduction in virus titers), and delivery of pS-M48 and pS-NP1383 significantly reduced lung virus titers in the infected mice (16–50-fold reduction in lung virus titers) and partially protected the mice from lethal influenza virus challenge (a survival rate of 4/8 for H1N1 virus-infected mice and 2/8 for H5N1 virus infected mice). Moreover, the treatment of pS-M48 and pS-NP1383 could suppress replication of different subtypes of influenza A viruses, including a H5N1 highly pathogenic avian isolate strain. The results provided a basis for further development of siRNA for prophylaxis and therapy of influenza virus infection in humans and animals.

Keywords: RNA interference; Influenza virus; NP; M2; Cells; Mice

HCV structural proteins interfere with interferon-alpha Jak/STAT signalling pathway by Esther Luquin; Esther Larrea; Maria P. Civeira; Jesús Prieto; Rafael Aldabe (pp. 194-197).

Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection. The current treatment with IFN-α given alone or in combination with ribavirin is ineffective in eliminating the virus in a large proportion of individuals with chronic hepatitis C. Recent data suggest that HCV blocks IFN-α signalling, an effect that facilitates viral persistence. We have used the HCV genomic and subgenomic replicon system to analyze the effect of structural and non-structural viral proteins on the activation of the Jak/STAT pathway and induction of antiviral activity by IFN-α. Our results show that IFN-α-mediated STAT activation (but not IFN-γ-stimulated STAT phosphorylation) is blocked in Huh7 cell line containing the genomic replicon, while this is not observed in cells with the subgenomic replicon. In agreement with these findings, the transcriptional activity and the antiviral effect of IFN-α were significantly lower in cells harboring the genomic replicon than in cells with the subgenomic replicon. These results indicate that HCV structural proteins play an important role in the escape of HCV from the interferon system.

Keywords: HCV; Replicon; Interferon; STAT phosphorylation; Interferon-stimulated genes

Comparative evaluation of the activity of antivirals towards feline immunodeficiency virus in different cell culture systems by F.J.U.M. van der Meer; N.M.P. Schuurman; J. Balzarini; H.F. Egberink (pp. 198-201).

Influences of the cell system on observed EC₅₀ values of different agents against feline immunodeficiency virus (FIV) were assessed. The activity of various nucleoside reverse transcriptase inhibitors (NRTI) against a lymphotropic FIV strain was evaluated using monocultured thymocytes and a DC–thymocyte coculture. In the second set of experiments activity of carbohydrate binding agents (CBA) towards FIV strains derived from different cell lines (e.g. Crandall feline kidney cells (CRFK) and thymocytes) was compared. We examined three different FIV-based antiviral evaluation systems and obtained marked differences in EC₅₀ values, especially for CBA entry inhibitors. Our study confirms and extends earlier observed differences between cell systems used for the evaluation of the activity of antivirals towards FIV.

Keywords: Antiviral testing; NRTI; Plant lectins; FIV; In vitro assay

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Antiviral Research (v.76, #2)