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Antiviral Research (v.90, #3)

Prelim II: Editorial Board (pp. ii).

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

In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant by Ilenia Delogu; Boris Pastorino; Cécile Baronti; Antoine Nougairède; Emilie Bonnet; Xavier de Lamballerie (pp. 99-107).

Arbidol (ARB) is an antiviral drug originally licensed in Russia for use against influenza and other respiratory viral infections. Although a broad-spectrum antiviral activity has been reported for this drug, there is until now no data regarding its effects against alphavirus infection. Here, the in vitro antiviral effect of ARB on Chikungunya virus (CHIKV) replication was investigated and this compound was found to present potent inhibitory activity against the virus propagated onto immortalized Vero cells or primary human fibroblasts (MRC-5 lung cells) (IC₅₀<10μg/ml). A CHIKV resistant mutant was then selected and adapted to growth in the presence of 30μg/ml ARB in MRC5 cells; its complete sequence analysis revealed a single amino acid substitution (G407R) localized in the E2 envelope protein. To confirm the G407R role in the molecular mechanism of ARB resistance, a CHIKV infectious clone harboring the same substitution was engineered, tested, and was found to display a similar level of resistance. Finally, our results demonstrated the effective in vitro antiviral activity of ARB against CHIKV and gave some tracks to understand the molecular basis of ARB activity.

Keywords: Chikungunya virus; Arbidol; Antiviral; Alphavirus; Arbidol resistance; Mutant

Effect of propolis and caffeic acid phenethyl ester (CAPE) on NFκB activation by HTLV-1 Tax by Jenny Shvarzbeyn; Mahmoud Huleihel (pp. 108-115).

HTLV-1 is the etiological agent of aggressive malignancy of the CD4⁺ T-cells, adult T-cell leukemia (ATL), and other severe clinical disorders. The viral Tax protein is a key factor in HTLV-1 pathogenicity. A major part of Tax oncogenic potential is accounted for by its capacity of inducing the transcriptional activity of the NFκB factors, which regulate the expression of numerous cellular genes. Propolis (PE), a natural product produced by honeybees, has been used for a long time in folk medicine. One of PE active components, caffeic acid phenylethyl ester (CAPE), was well characterized and found to be a potent inhibitor of NFκB activation. Therefore, the aim of this study was to pursue the possibility of blocking Tax oncogenic effects by treatment with these natural products. Human T-cell lines were used in this study since these cells are the main targets of HTLV-1 infections. We tried to determine which step of Tax -induced NFκB activation is blocked by these products. Our results showed that both tested products substantially inhibited the activation of NFκB-dependent promoter by Tax. However, only PE could efficiently inhibit also the Tax-induced activation of SRF- and CREB-dependent promoters. Our results showed also that PE and CAPE strongly prevented both Tax binding to IκBα and its induced degradation by Tax. However, both products did not interfere in the nuclear transport of Tax or NFκB proteins.

Keywords: Tax; HTLV-1; NFκB; Propolis; Caffeic acid phenethyl ester (CAPE)

Evolutionary pattern of full hepatitis B virus genome during sequential nucleos(t)ide analog therapy by Ying-Zi Tang; Lin Liu; Mei-Min Pan; Yu-Ming Wang; Guo-Hong Deng (pp. 116-125).

The evolutionary and mutational pattern of full hepatitis B virus (HBV) quasispecies during sequential nucleos(t)ide analog (NUC) therapy remains unclear. In this study, full-length HBV clones were generated from serial serum samples of five chronic hepatitis B patients who received sequential NUC therapies (treated patients) and two untreated patients with acute flares. The evolutionary and mutational patterns of full HBV quasispecies were studied. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence all decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed a distinct change in divergence during adefovir–telbivudine sequential therapies. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at ALT flare. Four of the treated patients presented amino acid changes in the “a” determinant during NUC therapy. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in reverse transcriptase (RT) region. Co-variations in the core promoter, the preC region and in the known epitopes of the preS gene accompanied by RT mutations, were common. In untreated patients, most of these co-variations located in the preC/C gene. In conclusion, the distribution of genetic variability of HBV shows remarkably different patterns between the treated and untreated subjects and the quasispecies divergence of different regions of HBV may vary remarkably even within a single host.

Keywords: Abbreviations; CHB; chronic hepatitis B; HBV; hepatitis B virus; NUC; nucleos(t)ide analog; RT; reverse transcriptase; LAM; lamivudine; ADV; adefovir dipivoxil; LDT; telbivudine; ETV; entecavir; ALT; alanine amino transferase; ORF; open reading frame; HBeAg; hepatitis B e antigen; anti-HBe; antibody to HBeAg; HBsAg; hepatitis B surface antigen; PCR; polymerase chain reaction; preC; precore; BCP; basic core promoterHepatitis B virus; Full genome; Chronic hepatitis B; Mutations

Evaluation of imiquimod for topical treatment of vaccinia virus cutaneous infections in immunosuppressed hairless mice by E. Bart Tarbet; Deanna Larson; Bentley J. Anderson; Kevin W. Bailey; Min-Hui Wong; Donald F. Smee (pp. 126-133).

Imiquimod is an immune response modifier prescribed as a topical medication for a number of viral and neoplastic conditions. We evaluated the antiviral activity of imiquimod against vaccinia virus (WR strain) cutaneous infections in immunosuppressed (with cyclophosphamide) hairless mice when administered after virus exposure. Primary lesions progressed in severity, satellite lesions developed, and infection eventually killed the mice. Once daily topical treatment with 1% imiquimod cream for 3, 4, or 5days were compared to twice daily topical treatment with 1% cidofovir cream for 7days. Survival time of mice in all treated groups was significantly prolonged compared to placebo controls. The mean day of death for the placebo group, 3-day imiquimod, 4-day imiquimod, 5-day imiquimod, and cidofovir groups were 15.5, 20.0, 20.5, 19.5, and 20.5days post-infection, respectively. All treatment groups showed significant reductions in primary lesion size and in the number of satellite lesions. The cidofovir and 4-day imiquimod treatments delayed the appearance of lung virus titers by 3 and 6days, respectively, although cutaneous lesion and snout virus titers were not as affected by treatment. Benefits in survival and lesion reduction were observed when imiquimod treatment was delayed from 24, 48, and 72h post-infection. However, increasing the treatment dose of imiquimod from 1% to 5% led to a significant decrease in antiviral efficacy. These results demonstrate the protective effects of topically administered imiquimod against a disseminated vaccinia virus infection in this mouse model.

Keywords: Poxvirus; Imiquimod; Immunomodulator; Cidofovir; Antiviral; Cyclophosphamide

Induction of specific cytotoxic activity for bovine herpesvirus-1 by DNA immunization with different adjuvants by C.A. Langellotti; J.S. Pappalardo; V. Quattrocchi; C. Mongini; P. Zamorano (pp. 134-142).

It is well documented that adjuvants improve the immune response generated by traditional viral vaccines; however, less is known about their effects on the immune response elicited by DNA vaccines.In this study, we have investigated the use of adjuvants, and have analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the BoHV-1 glycoprotein D (secreted version) in pCIneo vector with and without Montanide ISA25 (O/W), ISA206 VG (SEPPIC) and Cliptox™ (natural microparticles of clinoptilolite).The comparison of the immune response induced in mice by pCIgD formulated with or without adjuvants showed that the immunomodulators affect the total specific humoral and cellular response. The isotypes induced by these adjuvants were of the type Th1/Th2. A significant increase in the mac-3+ and F4/80+ populations of the groups receiving pCIneo with ISA25, ISA206; and an increase in CD4+ populations of the group receiving pCIneo ISA25, in comparison with the pCIneo group was observed.On the other hand, mice vaccinated with pCIgD/ISA25, pCIgD/ISA206, or pCIgD/Cliptox developed a significantly higher specific cytotoxic activity against BoHV-1 than the pCIgD and pCIneo groups. In this report we propose the use of ISA25, ISA206 or Cliptox as adjuvants in a DNA vaccine since they are able to induce not only a specific humoral immune response but also a specific cellular immune response.

Keywords: BoHV-1 DNA vaccine; Adjuvants; Cytotoxic response; Mice model

An immunotoxin targeting the gH glycoprotein of KSHV for selective killing of cells in the lytic phase of infection by Yingyun Cai; Edward A. Berger (pp. 143-150).

Amongst the pathologies associated with infection by Kaposi’s sarcoma-associated herpesvirus (KSHV), multicentric Castleman’s disease is distinctive for involvement of the lytic phase of the virus replication cycle. This B cell lymphoproliferative disorder has shown clinical responsiveness not only to generalized immunotherapy and cytotoxic chemotherapy, but also to inhibitors of herpesvirus DNA replication, consistent with the involvement of lytic phase of replication. These findings suggest that selective killing of virus-producing cells might represent a novel therapeutic strategy. We designed an immunotoxin, YC15-PE38, containing a single chain variable region fragment of a monoclonal antibody against KSHV glycoprotein H (gH) linked to the effector domains of Pseudomonas aeruginosa exotoxin A. Purified YC15-PE38 displayed highly selective and potent killing of a gH-expressing transfectant cell line (subnanomolar IC₅₀). The immunotoxin also strongly inhibited production of infectious KSHV virions from an induced chronically infected cell line, by virtue of selective killing of the virus-producing cells. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir. These results provide support for the development of anti-KSHV strategies based on targeted killing of infected cells expressing lytic phase genes.

Keywords: Abbreviations; KSHV; Kaposi’s sarcoma-associated herpesvirus; KS; Kaposi’s sarcoma; MCD; multicentric Castleman’s disease; PEL; primary effusion lymphoma; HAART; highly active antiretroviral therapy; mAb; monoclonal antibody; FBS; fetal bovine serum; scFv; single chain variable fragment; IPTG; isopropyl-1-thio-β-; d; -galactopyranosideKaposi’s sarcoma-associated herpesvirus; Ganciclovir; Immunotoxin; Glycoprotein H; Multicentric Castleman’s disease; Lytic infection

Cytomegalovirus reactivation in critically ill immunocompetent hosts: A decade of progress and remaining challenges by Charles H. Cook; Joanne Trgovcich (pp. 151-159).

Human cytomegalovirus (HCMV) is an undisputed pathogen in humans with severe immune compromise, which has historically been thought to carry little consequence in immunocompetent hosts. During the past decade, however, accumulating data suggest that significant numbers of immunocompetent humans reactivate HCMV during critical illness, and that these reactivation episodes are associated with worsened outcomes. Because most people are infected with this ubiquitous virus by adulthood, confirming pathogenicity has now become a clinical priority. In this article, we will review the incidence and implications of reactivation, the relevant immune responses and reactivation triggers relevant to the immunocompetent host. We will summarize the progress made during the past ten years, outline the work ongoing in this field, and identify the major gaps remaining in our emerging understanding of this phenomenon.

Keywords: Cytomegalovirus; Reactivation; Critical illness; Immunocompetent host; Murine models

Replication inhibition activity of carbocycles related to oseltamivir on influenza A virus in vitro by Masahiro Niikura; Nicole Bance; Sankar Mohan; B. Mario Pinto (pp. 160-163).

We have recently demonstrated that newly synthesized oseltamivir derivatives that contain a substituted triazole ring at the C-5 amino group interact with the 150 cavity found specifically in the group-1 neuraminidase (NA) subtypes of influenza A virus. These compounds exhibited in vitro inhibition activity of a group-1 NA enzyme incorporated in virus-like particles (VLPs). In the current study, we tested these nine triazole-containing carbocycles as well as an amino- and a guanidino-substituted derivative in virus replication inhibitory assays in vitro. None of the triazole-containing carbocycles significantly inhibited influenza A virus replication in MDCK cells with either a virus strain containing a group-1 or a group-2 subtype NA. In contrast, the amino- and guanidino-substituted derivatives clearly inhibited the cytopathic effect or spread of virus infection detected by immunostaining in MDCK monolayers as well as progeny virus release; these compounds were also reported to have shown the highest inhibition of group-1 NA in the context of VLPs. These results, together with the structures of these compounds, suggest that hydrogen-bonding interactions between the polar amino or guanidino functions and complementary groups in the neuraminidase active site ( e.g. Asp151, Glu 119) may be essential for strong inhibition of the neuraminidase enzyme and, in turn, the inhibition of influenza A virus replication.

Keywords: Influenza A virus; Replication inhibition; Neuraminidase inhibitors; Group-1 neuraminidase; The 150 cavity; Oseltamiver derivatives

Resistance associated mutations to dolutegravir (S/GSK1349572) in HIV-infected patients – Impact of HIV subtypes and prior raltegravir experience by Carolina Garrido; Vincent Soriano; Anna Maria Geretti; Natalia Zahonero; Silvia Garcia; Clare Booth; Felix Gutierrez; Isabel Viciana; Carmen de Mendoza (pp. 164-167).

Dolutegravir (S/GSK1349572) is a second-generation HIV-1 integrase inhibitor (INI) in advanced clinical development. It has shown good antiviral activity in most patients with prior raltegravir failure, although changes at the integrase codon 148, particularly when combined with other mutations, confer reduced susceptibility and may impair dolutegravir activity.Mutations believed to be associated with dolutegravir resistance at positions 92, 101, 124, 148, 153, and 193 were assessed in patients either INI-naïve or experiencing failure to raltegravir-based regimens. The integrase coding region was sequenced using an in-house nested-PCR protocol. HIV-1 subtyping was carried out using the Stanford algorithm.A total of 638 plasma samples were analyzed from 535 INI-naïve and 103 raltegravir-experienced patients. Non-B subtypes were recognized in 20.8% patients. Mutations L101I and T124A were significantly more prevalent in patients with non-B subtypes (66.9% vs. 45.7% for L101I; 61.7% vs. 25.9% for T124A; and 39.1% vs. 12.7% for L101I+T124A; p<0.001 in all cases). E92Q and Q148H/R were only seen in raltegravir-experienced patients and exclusively infected with subtype B (1.9% vs. 0%, p=0.026, for E92Q and 12.6% vs. 0%, p<0.001, for Q148H/R). On the contrary, T124A was more frequent in INI-naïve than raltegravir-experienced patients (35.1% vs. 24.3%, p=0.040). S153Y/F was absent in this dataset.Polymorphic changes L101I and T124A were more frequent in HIV-1 non-B than B subtypes. T124A was more frequent in INI-naïve patients but E92Q and Q148H/R were only seen in raltegravir-experienced individuals. Thus, both HIV-1 subtype and raltegravir exposure may influence the antiviral activity of dolutegravir.

Keywords: Dolutegravir; S/GSK1349572; Raltegravir; Integrase; Drug resistance; HIV subtypes

The rise and fall of polyanionic inhibitors of the human immunodeficiency virus type 1 by Vanessa Pirrone; Brian Wigdahl; Fred C. Krebs (pp. 168-182).

Infection by the human immunodeficiency virus type 1 (HIV-1) is an ordered, multistep process involving binding and entry, reverse transcription, integration, viral gene transcription, translation, processing, and finally assembly. Numerous therapeutic and preventive compounds, which are currently available for clinical use or are under preclinical and clinical development, act on at least one of these steps. Polyanionic HIV-1 inhibitors comprise a family of compounds that are generally considered entry inhibitors. The main mechanism of anti-HIV-1 activity associated with these compounds involves electrostatic interactions with HIV-1 glycoprotein 120 that ultimately prevent binding of the virus to target cells. A number of these compounds have been considered for systemic use and for use as microbicides, which are products designed to prevent sexual HIV-1 transmission. These compounds have been studied extensively using in vitro assays of activity, cytotoxicity, and mechanism of action, ex vivo models of HIV-1 transmission, and animal models of in vivo efficacy and toxicity. Three of these polyanionic compounds – cellulose sulfate, carrageenan, and PRO 2000 – were advanced into clinical trials of microbicide safety and efficacy. Although phase I and phase II clinical trials showed these compounds to be safe and well tolerated, none of the phase III trials provided any evidence that these compounds were effective against heterosexual HIV-1 transmission. Furthermore, clinical and in vitro results suggest enhancement of HIV-1 infection in the presence of polyanionic compounds. We discuss the preclinical development of polyanionic HIV-1 inhibitors, the clinical trials of polyanionic compounds used systemically and as topical vaginal microbicides, and the prospects for the future development of these compounds as inhibitors of HIV-1 infection.

Keywords: HIV-1; Polyanionic; Inhibitors; Microbicides; Safety; Efficacy

Human cytomegalovirus kinetics following institution of artesunate after hematopoietic stem cell transplantation by Dana G. Wolf; Avichai Shimoni; Igor B. Resnick; Thomas Stamminger; Avidan U. Neumann; Sunwen Chou; Thomas Efferth; Orit Caplan; Jessica Rose; Arnon Nagler; Manfred Marschall (pp. 183-186).

The anti-malaria drug artesunate has been shown to be an effective inhibitor of cytomegalovirus (CMV) in vitro, in an experimental animal model, and in a recent single-case clinical use. In this first case-series of 6 stem cell transplant recipients who received preemptive artesunate treatment for CMV infection, we have examined the viral kinetics following institution of artesunate, and employed first-phase viral kinetics studies to calculate its antiviral effectiveness. Two patients demonstrated a rapid 0.8–2.1 log viral load decline by 7days, with a viral decay half-live of 0.9–1.9days. Four patients demonstrated a continued yet stalled viral growth slope during treatment. No adverse events were noted in treatment courses of up to 28days. Overall, a divergent antiviral efficacy was revealed, ranging from 43% to 90%, which appeared to be primarily dependent on the virus baseline growth dynamics. Further dose escalation studies are needed to examine the role of artesunate in the treatment of CMV infection in the transplantation setting.

Keywords: Cytomegalovirus; Artesunate; Antiviral drugs; Viral kinetics

Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV) by Harry Bohle; Niels Lorenzen; Brian Dall Schyth (pp. 187-194).

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use.

Keywords: Gene silencing; Target control; DsiRNA; Rhabdovirus; VHSV; IHNV

Virucidal activity of the dendrimer microbicide SPL7013 against HIV-1 by Sushama Telwatte; Katie Moore; Adam Johnson; David Tyssen; Jasminka Sterjovski; Muriel Aldunate; Paul R. Gorry; Paul A. Ramsland; Gareth R. Lewis; Jeremy R.A. Paull; Secondo Sonza; Gilda Tachedjian (pp. 195-199).

Topical microbicides for use by women to prevent the transmission of human immunodeficiency virus (HIV) and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. SPL7013 is a dendrimer with broad-spectrum activity against HIV type I (HIV-1) and -2 (HIV-2), herpes simplex viruses type-1 (HSV-1) and -2 (HSV-2) and human papillomavirus. SPL7013 [3% (w/w)] has been formulated in a mucoadhesive carbopol gel (VivaGel®) for use as a topical microbicide. Previous studies showed that SPL7013 has similar potency against CXCR4-(X4) and CCR5-using (R5) strains of HIV-1 and that it blocks viral entry. However, the ability of SPL7013 to directly inactivate HIV-1 is unknown. We examined whether SPL7013 demonstrates virucidal activity against X4 (NL4.3, MBC200, CMU02 clade EA and 92UG046 clade D), R5 (Ba-L, NB25 and 92RW016 clade A) and dual-tropic (R5X4; MACS1-spln) HIV-1 using a modified HLA-DR viral capture method and by polyethylene glycol precipitation. Evaluation of virion integrity was determined by ultracentrifugation through a sucrose cushion and detection of viral proteins by Western blot analysis. SPL7013 demonstrated potent virucidal activity against X4 and R5X4 strains, although virucidal activity was less potent for the 92UG046 X4 clade D isolate. Where potent virucidal activity was observed, the 50% virucidal concentrations were similar to the 50% effective concentrations previously reported in drug susceptibility assays, indicating that the main mode of action of SPL7013 is by direct viral inactivation for these strains. In contrast, SPL7013 lacked potent virucidal activity against R5 HIV-1 strains. Evaluation of the virucidal mechanism showed that SPL7013-treated NL4.3, 92UG046 and MACS1-spln virions were intact with no significant decrease in gp120 surface protein with respect to p24 capsid content compared to the corresponding untreated virus. These studies demonstrate that SPL7013 is virucidal against HIV-1 strains that utilize the CXCR4 coreceptor but not viruses tested in this study that solely use CCR5 by a mechanism that is distinct from virion disruption or loss of gp120. In addition, the mode of action by which SPL7013 prevents infection of cells with X4 and R5X4 strains is likely to differ from R5 strains of HIV-1.

Keywords: Dendrimer; Microbicide; SPL7013; HIV; Virucidal activityAbbreviations; X4; CXCR4-using; R5; CCR5-using; EC; ₅₀; 50% effective concentration; VC; ₅₀; 50% virucidal concentration

Synergistic in vitro anti-HIV type 1 activity of tenofovir with carbohydrate-binding agents (CBAs) by Geoffrey Férir; Kurt Vermeire; Dana Huskens; Jan Balzarini; Els J.M. Van Damme; Jan-Christoph Kehr; Elke Dittmann; Michael D. Swanson; David M. Markovitz; Dominique Schols (pp. 200-204).

Tenofovir, a well-known and highly prescribed anti-HIV-1 drug for the treatment of HIV/AIDS infections, has recently also shown its effectiveness as a potential microbicide drug in the prevention of HIV transmission.Here, we evaluated the combination of tenofovir with various members of the class of carbohydrate-binding agents (CBAs) targeting the glycans on the viral envelope gp120 for their anti-HIV efficacy. The tenofovir/CBA combinations predominantly showed synergistic antiviral activity using the median effect principle.These findings illustrate that combination of tenofovir with CBAs may increase the antiviral potency of the individual drugs and reducing the risk on potential side-effects.

Keywords: Tenofovir; Carbohydrate-binding agents; HIV; Synergy; Microbicide

Evaluation of the antiviral drug susceptibility of influenza viruses in Italy from 2004/05 to 2009/10 epidemics and from the recent 2009 pandemic by Simona Puzelli; Marzia Facchini; Angela Di Martino; Concetta Fabiani; Angie Lackenby; Maria Zambon; Isabella Donatelli (pp. 205-212).

Antiviral monitoring of influenza viruses circulating in Italy has been carried out since 2007 by the National Influenza Centre (NIC), using both phenotypic and sequence-based assays. Here, we report results of the susceptibility evaluation to neuraminidase (NA) inhibitors (NAIs, zanamivir and oseltamivir) and adamantanes of nearly 300 influenza type A and B seasonal viruses isolated in Italy during six recent seasons, together with over 30 pandemic (H1N1) 2009 virus strains. The present work is the first such study conducted in Italy, aimed to develop national data on antiviral drug profile and to establish a nationwide surveillance programme on antiviral susceptibility. Sequencing of the NA gene was undertaken either to confirm the phenotypic findings or to identify any NA change, in potentially resistant viruses (outliers), which might be associated with reduced susceptibility to NAIs. The 50% inhibitory concentration values (IC₅₀s) showed slightly different sensitivities of the seasonal Italian isolates to the two NAI drugs, depending on the specific NA subtype. We found mean zanamivir IC₅₀s of 0.74, 1.33 and 7nM, and oseltamivir IC₅₀s of 0.67, 2.34 and 30.1nM for the N2, N1 and B NAs, respectively. The pandemic (H1N1) 2009 viruses showed IC₅₀values overall comparable to the seasonal N1 viruses from previous years, showing mean zanamivir IC₅₀s of 1.02nM and mean oseltamivir IC₅₀s of 2.82nM. Oseltamivir resistance was found in a total of 19 seasonal N1viruses of 2007/2008 and 2008/2009, and in three pandemic (H1N1) 2009 strains. A gradual increase of resistance to adamantanes was observed among the N2 viruses isolated in recent seasons; no resistant viruses were found among the seasonal N1 strains, whereas all the pandemic (H1N1) 2009 isolates analysed were resistant to the M2 blockers.

Keywords: Influenza; Antiviral resistance; Oseltamivir; Zanamivir; Adamantanes; Italy

Towards the design of combination therapy for the treatment of enterovirus infections by Hendrik Jan Thibaut; Pieter Leyssen; Gerhard Puerstinger; Alexandra Muigg; Johan Neyts; Armando Mirko De Palma (pp. 213-217).

We report here on a comparative study of the activity of 10 enterovirus inhibitors against poliovirus 1, enterovirus 71 and human rhinovirus 14. Three of the selected molecules (Pleconaril, BTA-798 and V-073) are in clinical development. The in vitro antiviral activity of pairwise combinations of inhibitors indicated that most combinations resulted in an additive to slightly synergistic antiviral activity. However, the combination of ribavirin with a nucleoside polymerase inhibitor resulted in a pronounced antagonistic effect.

Keywords: Enterovirus; Poliovirus; Rhinovirus; Antiviral; Combination therapy; Virus

Interferon-γ influences immunity elicited by vaccines against very virulent Marek’s disease virus by Kamran Haq; Inas Elawadli; Payvand Parvizi; Amirul I. Mallick; Shahriar Behboudi; Shayan Sharif (pp. 218-226).

Vaccination of chickens with herpesvirus of turkey (HVT) confers only partial protection against challenge with a very virulent Marek’s disease virus (MDV). Here, we evaluated the ability of recombinant chicken interferon-gamma (rChIFN-γ) to enhance protective efficacy of HVT against the very virulent MDV strain, RB1B. The bioactivity of IFN-γ expressed by a plasmid expression vector was confirmed by its ability to stimulate a chicken macrophage cell line (HD11) to produce nitric oxide (NO) in vitro. The administration of HVT with 5μg of pcDNA:chIFN-γ plasmid reduced the incidence of tumor development significantly when compared to vaccinated birds (77.7% in the HVT+empty vector group and 80% in HVT group versus 33.3% in the HVT+chIFN-γ group) and significantly increased IFN-γ expression in the splenocytes of the protected group, suggesting that rChIFN-γ increases the potency of HVT against MDV. Further analysis demonstrated that the protected birds that received HVT vaccine and/or plasmid had lower MDV genome load and lower amounts of transcripts for meq and vIL-8 than in the birds without lesions. Similarly, lower expression of IL-10, IL-18 and IL-6 was observed in the chickens without lesions compared to the chickens that had lesions, suggesting an inverse association between up-regulation of these cytokines and vaccine-induced immunity. In conclusion, IFN-γ can positively influence immunity conferred by HVT vaccination against challenge with a very virulent Marek’s disease virus (vvMDV) in chickens.

Keywords: Chicken; Immunology; Vaccine; Interferon-γ; Marek’s disease; Adjuvant

Breaking B and T cell tolerance using cationic lipid–DNA complexes (CLDC) as a vaccine adjuvant with hepatitis B virus (HBV) surface antigen in transgenic mice expressing HBV by John D. Morrey; Neil E. Motter; Stella Chang; Jeffery Fairman (pp. 227-230).

Cationic lipid DNA complexes (CLDC), referred to here as JVRS-100, were evaluated as an adjuvant for hepatitis B surface antigen (HBsAg) for eliciting B and T cell responses in transgenic mice expressing hepatitis B virus (HBV). To confirm the immunogenicity of HBsAg+JVRS-1000, a study was conducted in C57BL/6 mice, the genetic background of the HBV transgenic mice used in the study. HBsAg+JVRS-100 elicited a T cell response and B cell response as evidenced by interferon-gamma (IFN-γ) secretion by re-stimulated splenocytes and anti-HBsAg IgG induction, respectively, whereas, HBsAg only elicited a B cell response. In HBV transgenic mice, HBsAg did not elicit either T or B cell responses, unlike the HBsAg+JVRS-100 that elicited both. Energix-B vaccine did perform better than the HBsAg by eliciting a B cell response in the transgenic mice, but it did not perform as HBsAg+JVRS-100 since it did not elicit a T cell response. The response by HBsAg+JVRS-100 was not sufficient to cause destruction of infected liver cells, but it did suppress HBV DNA non-cytolytically. From these results, JVRS-100 might be considered for further development as an adjuvant for HBV therapeutic vaccines.

Keywords: Hepatitis B virus; Transgenic mice; Therapeutic vaccine

Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja saponaria Molina by Ka Ian Tam; Michael R. Roner (pp. 231-241).

Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide with approximately 300,000 pre-adolescent deaths each year. Quillaja saponins are a natural aqueous extract obtained from the Chilean soapbark tree. The extract is approved for use in humans by the FDA for use in beverages as a food addictive. We have demonstrated that Quillaja extracts have strong antiviral activities in vitro against six different viruses. In this study, we evaluated the in vivo antiviral activity of these extracts against rhesus rotavirus (RRV) using a mouse model. We established that at a dosage of 0.015mg/mouse of saponin extract, RRV induced diarrhea can be significantly reduced from 79% to 11% when mice are exposed to 500 plaque-forming-units (PFU) for each of five consecutive days. Additionally, while a reduction of RRV induced diarrhea depended both on the concentration of virus introduced and on the amount of Quillaja extract given to each mouse, the severity and interval of diarrhea under a variety of conditions tested, in all the treated mice were greatly reduced when compared to those that did not receive the Quillaja extracts. Mechanistically, there is strong evidence that the Quillaja extracts are able to “block” rotavirus infection by inhibiting virus–host attachment through disruption of cellular membrane proteins and/or virus receptors. We believe that Quillaja extracts have promise as antivirals to reduce rotavirus infection and the severity of the disease in humans.

Keywords: Rotavirus; Saponins; Diarrhea; Quillaja; saponaria; Molina; Microbiocide; Antiviral

Resistance testing of clinical varicella-zoster virus strains by Andreas Sauerbrei; Johanna Taut; Roland Zell; Peter Wutzler (pp. 242-247).

Acyclovir resistance of varicella-zoster virus (VZV) has been reported in rare cases of immunocompromised patients. In this study, the natural polymorphism of the thymidine kinase (TK) and DNA polymerase (pol) genes was examined in 51 clinical VZV isolates sensitive to acyclovir (ACV). In addition, 16 VZV strains with clinical resistance to ACV were analyzed. None of the ACV-sensitive strains of the clades 1, 3 and 5 showed gene polymorphism of the TK. By contrast, the DNA pol gene exhibited polymorphism-related substitutions as a function of the VZV clade. The novel substitutions M286I, E824Q, R984H and H1089Y were detected in strains of clades 3 and 5. In the TK gene of 7 VZV strains with clinical ACV resistance, the novel substitutions L73I, A163stop, W225R, T256M, N334stop and the deletion of nucleotides 19–223 were found to be associated most likely with resistance. In one strain showing the substitution W225R, ACV resistance could be confirmed by the viral phenotype. In the DNA pol gene, the novel amino acid substitutions T237K and A955T could be detected, but their significance remains unclear. In conclusion, the characterization of resistance using genetic analysis of the TK and DNA pol genes has to be considered the method of choice for the determination of VZV resistance to antiviral drugs. In a considerable number of patients with clinical ACV-resistant VZV infections, resistance cannot be verified by virological methods.

Keywords: Varicella-zoster virus; Phenotypic resistance; Genotypic resistance; Thymidine kinase; DNA polymerase

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Antiviral Research (v.90, #3)

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Antiviral Research (v.90, #3)