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Antiviral Research (v.75, #1)

Editorial Board (pp. ii).
Notes to Authors (pp. co3).

The acyclic nucleoside phosphonates from inception to clinical use: Historical perspective by Erik De Clercq (pp. 1-13).
The collaboration between Antonín Holý [Institute of Organic Chemistry and Biochemistry (IOCB, Prague, Czech Republic)] and Erik De Clercq (Rega Institute for Medical Research, K.U. Leuven, Belgium) started exactly 30 years ago. It led to the discovery of a (rather small) series of acyclic nucleoside analogues (prototype: DHPA), followed by the discovery of a large number of nucleotide analogues [acyclic nucleoside phosphonates (ANPs)] (prototype: HPMPA). From HPMPA originated three compounds, which have been approved by regulatory agencies worldwide for clinical use: (i) HPMPC [cidofovir (Vistide®)] for the treatment of cytomegalovirus (CMV) retinitis in AIDS patients, and “off label” for the treatment of polyoma-, papilloma-, adeno-, herpes- and poxvirus infections; (ii) PMEA [adefovir (in its oral prodrug form, adefovir dipivoxil (Hepsera®)] for the treatment of chronic HBV (hepatitis B virus) infections, and (iii) PMPA [tenofovir (in its oral prodrug form, tenofovir disoproxil fumarate (Viread®)] for the treatment of HIV infections (AIDS). The latter has also been approved, in combination with emtricitabine (Truvada®), and in combination with emtricitabine and efavirenz (Atripla®) for the treatment of HIV infections. Many other ANPs such as the DAP derivatives HPMPDAP, PMEDAP and PMPDAP, and the DAPy derivatives HPMPO-DAPy, PMEO-DAPy, and PMPO-DAPy, were found to exhibit an antiviral activity spectrum and potency comparable to that of the parent compounds HPMPA (and HPMPC), PMEA and PMPA, respectively.

Keywords: Abbreviations; ANPs; acyclic nucleoside phosphonates; DHPA; [(; S; )-9-(2,3-dihydroxypropyl)adenine]; HPMPA; [(; S; )-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine]; HPMPC; [(; S; )-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine]; PMEA; [9-(2-phosphonylmethoxyethyl)adenine]; FPMPA; [(; S; )-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine]; PMPA; [(; R; )-9-(2-phosphonylmethoxypropyl)adenine]; HPMPDAP; [(; S; )-9-(3-hydroxyl-2-phosphonylmethoxypropyl)-2,6-diaminopurine]; PMEDAP; [9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine]; PMPDAP; [(; R; )-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine]; HPMPO-DAPy; [(; R; )-6-(3-hydroxy-2-(phosphonylmethoxy)propoxy)-2,4-diaminopyrimidine]; PMEO-DAPy; [6-(2-(phosphonylmethoxy)ethoxy)-2,4-diaminopyrimidine]; PMPO-DAPy; [(; R; )-6-(2-(phosphonylmethoxy)propoxy)-2,4-diaminopyrimidine]Acyclic nucleoside phosphonates (ANPs); HPMPA; Cidofovir (HPMPC); Adefovir (PMEA); Tenofovir (PMPA)


Prophylactic treatment with recombinant Eimeria protein, alone or in combination with an agonist cocktail, protects mice from Banzi virus infection by Justin G. Julander; John W. Judge; Aaron L. Olsen; Barnett Rosenberg; Kristiina Schafer; Robert W. Sidwell (pp. 14-19).
A recombinant Eimeria protozoan protein antigen (rEA) has been shown to have antitumor and antiviral activity. The purpose of this study was to determine the effect of rEA treatment alone or in combination with an agonist cocktail consisting of granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ), interleukin 4 (IL-4), and anti CD-40 antibody, in the treatment of Banzi virus (BV) disease in BALB/c mice. Treatment with rEA resulted in a significant increase in survival, weight gain, and mean day to death in BV-infected mice and resulted in a significant decrease in brain virus titer. Treatment with rEA, in combination with a 4-agonist cocktail, improved disease parameters to a greater degree than rEA treatment alone. The effect of treatment with a reduced concentration of agonist cocktail or fewer components of the agonist cocktail, in combination with rEA, on disease outcome in BV-infected mice was also investigated. Treatment with rEA, alone or in combination with agonist cocktail, 24h after virus challenge did not improve disease. Treatment with rEA, alone or in combination with an agonist cocktail, is efficacious for the prophylaxis of BV infection in mice.

Keywords: Banzi virus; Eimeria; Cytokine; Treatment; Mice; rEA; Barrogen; Immune modulation


Inhibition of multiple strains of Venezuelan equine encephalitis virus by a pool of four short interfering RNAs by Lyn O’Brien (pp. 20-29).
RNA interference, mediated by short interfering RNAs (siRNAs), has been shown to have activity against a wide range of viruses and is a promising new antiviral therapy. Using multiple siRNAs that target conserved areas of the genome allows for increased chances of antiviral activity against different viral strains and also helps to prevent the emergence of escape mutants. In this study, four siRNAs were designed to target areas of conserved sequence between divergent strains of Venezuelan equine encephalitis virus (VEEV). A pool of these siRNAs inhibited the replication of all six strains of VEEV tested. A single nucleotide mismatch at the extreme 3′ end of one of the siRNA sense strands did not affect antiviral activity but other mutations were not tolerated. Two strains of VEEV were tested for their abilities to overcome the inhibitory effects of RNA interference following 10 consecutive incubations in the presence of siRNAs. One strain remained susceptible throughout the course of the experiment but the other strain became resistant to the activity of siRNAs. Sequence analysis of the siRNA target sites in this strain showed that no mutations had been generated, indicating that the virus may had become resistant in some other manner. In the absence of effective antiviral drugs and vaccines to combat VEEV infection, these siRNAs offer a potential new therapeutic approach but, as with all antimicrobial agents, caution needs to be exercised with respect to the generation of resistance.

Keywords: RNA interference; Short interfering RNA (si RNA); Venezuelan equine encephalitis virus


The helicase primase inhibitor, BAY 57-1293 shows potent therapeutic antiviral activity superior to famciclovir in BALB/c mice infected with herpes simplex virus type 1 by Subhajit Biswas; Lyn Jennens; Hugh J. Field (pp. 30-35).
BAY 57-1293 represents a new class of potent inhibitors of herpes simplex virus (HSV) that target the virus helicase primase complex. The present study was conducted using the zosteriform infection model in BALB/c mice. The helicase primase inhibitor, BAY 57-1293 was shown to be highly efficacious in this model. The beneficial effects of therapy were obtained rapidly (within 2 days) although the onset of treatment was delayed for 1 day after virus inoculation. The compound given orally, or intraperitoneally once per day at a dose of 15mg/kg for 4 successive days was equally effective or superior to a much higher dose of famciclovir (1mg/ml, i.e. approximately 140–200mg/kg/day) given in the drinking water for 7 consecutive days, which, in our hands, is the most effective method for administering famciclovir to mice. In contrast to the vehicle-treated infected mice, all mice that received antiviral therapy looked normal and active with no mortality, no detectable loss of weight and no marked change in ear thickness. BAY 57-1293 and famciclovir reduced the virus titers in the skin to below the level of detection by days 3 and 7 post infection, respectively. In both BAY 57-1293 and famciclovir-treated mice, infectious virus titers in the ear pinna and brainstem remained below the level of detection. Consistent with these findings, BAY 57-1293 also showed a potent antiviral effect in an experiment involving a small number of severely immunocompromised athymic-nude BALB/c mice.

Keywords: HSV; Helicase primase; BAY 57-1293; Famciclovir; Antiviral


Direct evidence for immunomodulatory properties of ribavirin on T-cell reactivity to hepatitis C virus by Eirini I. Rigopoulou; William G.H. Abbott; Roger Williams; Nikolai V. Naoumov (pp. 36-42).
An understanding of ribavirin's beneficial effects on treatment outcome in chronic hepatitis C (CH-C) may help to develop new treatment approaches. Here we investigated whether ribavirin directly affects HCV-specific reactivity of CD4+T-lymphocytes from patients with CH-C.Peripheral blood mononuclear cells from forty HCV RNA positive patients were cultured ex vivo with HCV core, NS3, NS4 alone, and with different concentrations of ribavirin. Virus-specific CD4+ T-cell reactivity was analysed by a proliferation assay; quantitation of cytokine (interferon-gamma, IL-10, IL-5, IL-12p35, IL-12p40) mRNA levels; measurement of interferon-gamma and IL-10 production (by ELISA) and enumeration of interferon-gamma and IL-10 producing T-cells by Elispot assays.At 2–5μM ribavirin induced de novo or enhanced T-cell proliferation to HCV antigens in a proportion of patients. Increased T-cell proliferation was associated with decreased IL-10 production in response to HCV core and reduced frequency of IL-10 producing CD4+ T-cells, while interferon-gamma levels remained unchanged. At 20μM ribavirin markedly suppressed T-cell proliferation, and interferon-gamma mRNA expression to HCV antigens.Ribavirin, at clinically achievable plasma levels, modulates directly the T-cell responses to HCV antigens in some CH-C patients. Suppression of IL-10 production may represent a useful strategy to induce/augment T-cell reactivity to HCV.

Keywords: Ribavirin; Chronic hepatitis C; IL-10; IFN-γ


CD81 down-regulation on B cells is associated with the response to interferon-alpha-based treatment for chronic hepatitis C virus infection by Li-Ling Chang; Pin-Nan Cheng; Jiann-Shiuh Chen; Kung-Chia Young (pp. 43-51).
The lymphocytic CD81 molecule, capable of modulating type-1/-2 T-helper responses and serving as a putative receptor for hepatitis C virus (HCV), might influence the outcome of anti-HCV treatment. This study characterized the interferon-alpha-induced alteration of lymphocytic CD81. The CD81 levels in healthy subjects and naïve chronic HCV patients were compared, with the results showing that the two groups had comparable surface CD81 levels for total peripheral blood lymphocytes, subpopulation-B, -T, and -NK cells. In vitro interferon-alpha treatment could suppress the CD81 expression from both groups. Subsequently, we compared the in vitro interferon-alpha modulatory effects on lymphocytic CD81 from patients having received anti-HCV therapy with either sustained virological response (SVR) or without SVR. There was a significant down-regulation of the B-cell's CD81 only in the SVR group. The CD81 modulation was further investigated using Daudi lymphoid cell line, showing declined surface CD81 levels following treatment with interferon-alpha, interferon-beta or polyI:C. Thus, interferons could directly decrease CD81 expression. The interferon-alpha effect could be restored by 2-aminopurine, suggesting that double-stranded RNA activated kinase might be involved in the suppression of CD81. In conclusion, CD81 down-regulation is a primary host response to interferon-alpha-based therapy and an immunophenotype associated with anti-HCV SVR.

Keywords: Abbreviations; 2-AP; 2-aminopurine; ALT; alanine transaminase; E; envelope; FACS; fluorescence-activated cell scan; HCV; hepatitis C virus; IFN; interferon; IL; interleukin; MFI; mean fluorescence intensity; NK; natural killer; PBLs; peripheral blood lymphocytes; PKR; double-stranded RNA activated kinase; S.D.; standard deviation; SVR; sustained virological response; TSA; trichostatin ACD81; Interferon-alpha; Hepatitis C virus


In vitro evaluation of the anti-orf virus activity of alkoxyalkyl esters of CDV, cCDV and ( S)-HPMPA by Fabiana Dal Pozzo; Graciela Andrei; Ilya Lebeau; James R. Beadle; Karl Y. Hostetler; Erik De Clercq; Robert Snoeck (pp. 52-57).
Acyclic nucleoside phosphonates (ANPs) and in particular ( S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide®) and its adenine counterpart ( S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [( S)-HPMPA] are highly active against orf virus infections. This parapoxvirus commonly causes infection in sheep, goats, but also humans. Alkoxyalkyl esters of CDV have an increased oral bioavailability and are more active against orthopoxviruses than the parent compounds. In the present study, the potency of several alkoxyalkyl esters of CDV, cyclic cidofovir (cCDV) and ( S)-HPMPA was evaluated against different orf virus isolates in two cell types, human embryonic lung (HEL) fibroblast and primary lamb keratinocytes. Each prodrug was at least 10-fold more active than its parent compound in both cell types. Of all the compounds tested, the ( S)-HPMPA alkoxyalkyl esters showed the highest activity and selectivity against orf virus. Our results support the development of alkoxyalkyl esters of ANPs as antivirals not only for the treatment of complicated human orf lesions, but also in the therapy and prophylaxis of contagious ecthyma in sheep and goats.

Keywords: Orf virus; Contagious ecthyma; Acyclic nucleoside phosphonates; Oral bioavailability; Prodrugs


Assay of HIV gp41 amino acid sequence to identify baseline variation and mutation development in patients with virologic failure on enfuvirtide by M.R. Loutfy; J.M. Raboud; J.S.G. Montaner; T. Antoniou; B. Wynhoven; F. Smaill; D. Rouleau; J. Gill; W. Schlech; Z.L. Brumme; T. Mo; K. Gough; A. Rachlis; P.R. Harrigan; S.L. Walmsley (pp. 58-63).
In this study, we retrospectively assessed a gp41 genotypic assay in 404 enfuvirtide-naïve individuals (340 clade B, 64 non-B clade) to determine the prevalence of baseline polymorphisms and in 41 patients virologically failing enfuvirtide to determine correlates of resistance to this agent. Conserved and polymorphic regions of gp41 were identified in clade B isolates, with 127 of 328 codons (38.7%) being highly conserved (<1.0% variation) and 74 of 328 codons (22.6%) being partially conserved (1.0–5.0% variation). Polymorphisms were observed throughout gp41 in non-B clade virus sequences compared to the clade B reference strain, ranging from 53 natural substitutions in clade D to 76 in clade A. Insertions were common at positions 3, 105, 215 and 276. In the patients failing enfuvirtide, mutations were detected in the 10 amino acid region at positions 36–45 in all plasma virus sequences. Six additional mutations were selected outside of the common region which may be clinically significant at positions 33, 73, 75, 126, and 138. Two or three mutations at positions 36–45 were observed in the majority of plasma virus sequences from patients with virologic failure following the use of enfuvirtide. Further study is required to determine the clinical relevance of the clade related polymorphisms and the new mutations identified in the patients with virologic failure.

Keywords: HIV; Enfuvirtide; gp41; Genotyping assay; Polymorphism; Resistance


SEQHEPB: A sequence analysis program and relational database system for chronic hepatitis B by Lilly K.W. Yuen; Anna Ayres; Margaret Littlejohn; Danielle Colledge; Andrew Edgely; William J. Maskill; Stephen A. Locarnini; Angeline Bartholomeusz (pp. 64-74).
SeqHepB is a combination of a HBV genome sequence analysis program and a relational database that houses data collected from multiple data sources. Registered users can access the sequence analysis component of SeqHepB online for rapid and detailed interrogation of HBV genomic sequences. Its main function is to determine the HBV genotype, identify key mutations associated with antiviral resistance, and identify clinically important HBV mutants. All information generated is uploaded into a database and integrated with patient medical records, pathology laboratory tests, and supplemental virology results such as in vitro drug cross-resistance values. Combined with structured query language (SQL) queries developed in the database, it is possible to extract and correlate clinical, virological, and in vitro phenotypic data rapidly and efficiently. An important component of SeqHepB is its ability to integrate mutations detected within the reverse transcriptase (RT) and locate them onto a three-dimensional (3D) model of the HBV RT that can be viewed at any angle with known antiviral drug molecules in the catalytic pocket of the enzyme. SeqHepB will enable virologists and physicians to individualise patient management, cope with the explosion of antiviral associated HBV mutations, and to conduct cross-sectional retrospective or prospective studies on HBV-infected individuals during therapy.

Keywords: Hepatitis B virus; Drug resistance; Relational database; Data mining


Drug-resistant HIV-1 prevalence in patients newly diagnosed with HIV/AIDS in Japan by Hiroyuki Gatanaga; Shiro Ibe; Masakazu Matsuda; Shigeru Yoshida; Tsukasa Asagi; Makiko Kondo; Kenji Sadamasu; Hiroki Tsukada; Aki Masakane; Haruyo Mori; Noboru Takata; Rumi Minami; Masao Tateyama; Takao Koike; Toshihiro Itoh; Mitsunobu Imai; Mami Nagashima; Fumitake Gejyo; Mikio Ueda; Motohiro Hamaguchi; Yoko Kojima; Takuma Shirasaka; Akiro Kimura; Masahiro Yamamoto; Jiro Fujita; Shinichi Oka; Wataru Sugiura (pp. 75-82).
The increasing prevalence of drug-resistant HIV transmission has become a critical epidemic in the world today. Studies in developed countries reported 8–27% of newly diagnosed HIV/AIDS patients are infected by drug-resistant strains. To determine the prevalence of drug-resistant HIV-1 among newly diagnosed cases in Japan, eight HIV/AIDS clinical centers, three public health laboratories and the National Institute of Infectious Diseases conducted a nationwide survey. Between January 2003 and December 2004, 575 newly diagnosed HIV/AIDS patients with both acute and chronic infections were enrolled in the study. Twenty-three cases, including three recently infected patients, were infected with HIV-1 having major drug-resistance mutations, including M41L, D67N, L100I, K103N, V106A, M184I, M184V, L210W, and revertant mutations at the 215 codon in reverse transcriptase and M46I in protease encoding regions. In this newly diagnosed population, we also clarified the prevalence of hepatitis virus coinfection, which was 8.8% for HBV and 4.3% for HCV. In conclusion, the drug-resistant transmission rate was 4.0% in Japan. Although this rate is significantly lower than that of other developed countries, this rate almost reaches the threshold at which baseline genotypic resistance testing would be cost-effective for all infected persons before initiating therapy.

Keywords: HIV-1; Drug resistance; Newly infected; Japan


Herpes glycoprotein gL is distantly related to chemokine receptor ligands by Lucjan S. Wyrwicz; Leszek Rychlewski (pp. 83-86).
Glycoprotein L (gL) is one of the critical proteins involved in transmission of Herpesviridae. We applied the methodology of protein structure prediction to shed a light on the so far unknown molecular mechanism of its action. Here we show that gL forms a chemokine-like protein. Alphaherpesvirinae gL as well as CMV functional homolog (UL130) create a novel CX chemokine-like protein, while Gammaherpesvirinae gL (HHV8 and EBV) adopt a regular CC beta-chemokine fold.We conclude that gL may interact with specific cellular chemokine receptors during the invasion of Herpesviridae. The proposed mechanism has a potential impact on future development of novel therapeutic and prophylactic strategies.

Keywords: Herpes; Cellular receptor; Cell invasion


Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir by Jacqueline C. Ruiz; James R. Beadle; Kathy A. Aldern; Kathy A. Keith; Caroll B. Hartline; Earl R. Kern; Karl Y. Hostetler (pp. 87-90).
Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40–100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6–40 times more active against HCMV and 7–30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.

Keywords: Cidofovir; Adefovir; Prodrugs; Antivirals
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