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Antiviral Research (v.78, #3)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Applications of bioluminescence imaging to antiviral research and therapy: Multiple luciferase enzymes and quantitation by Kathryn E. Luker; Gary D. Luker (pp. 179-187).

Bioluminescence imaging (BLI) has emerged as a powerful new modality for studies of viral infection and therapy in small animal models. BLI technology captures the light emitted from different luciferase enzymes to detect sites of viral infection and quantify viral replication in the context of a living animal. In this review, we discuss the biochemical features of various luciferase enzymes and modifications to these enzymes that can greatly enhance their ability to image viral infection, host responses and the effects of therapy. We also describe BLI instrumentation and technical aspects of BLI needed to optimize imaging data. Examples of BLI for quantitative analysis of viral infection and in vivo monitoring of antiviral and antibacterial therapy are presented to highlight the potential for BLI to accelerate discovery of new antiviral agents and determine efficacy of antiviral compounds. Ongoing research to use multiple luciferase enzymes to image viral infection, host immune signaling pathways, and cell trafficking in the same animal will continue to advance BLI for longitudinal, real-time quantification and analyses of viral infection and pre-clinical testing of promising therapeutic agents.

Keywords: Bioluminescence imaging; Fluorescence; Reporter virus; Quantitation; Antiviral therapy; Pathogenesis; Vaccinia virus; Herpes simplex virus; Interferon

In vitro inhibition of classical swine fever virus replication by siRNAs targeting N^pro and NS5B genes by Xingran Xu; Huancheng Guo; Chang Xiao; Yunfeng Zha; Zixue Shi; Xianzhu Xia; Changchun Tu (pp. 188-193).

Classical swine fever (CSF) is a highly contagious disease of pigs, which causes important economic losses worldwide. In the present study, the specific effect of RNA interference on the replication of CSF virus (CSFV) was explored. Three species of small interfering RNA (siRNA), targeting different regions of CSFV N^pro and NS5B genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with CSFV, the viral proliferation within the cells was examined by indirect immunofluorescence microscopy. At 72h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, while most untreated virus-infected cells were positive. Treatment with the siRNAs caused a 4–12-fold reduction in viral genome copy number as assessed by real time RT-PCR. Transfection with the siRNAs also suppressed the production of infectious virus by up to 467-fold as assessed by TCID₅₀ assay. These results suggested that the three species of siRNAs can efficiently inhibit CSFV genome replication and infectious virus production, with the inhibition persisting for 72–84h.

Keywords: CSFV; Replication; siRNA

PA subunit of RNA polymerase as a promising target for anti-influenza virus agents by Misako Nakazawa; Shin-etsu Kadowaki; Izumi Watanabe; Youko Kadowaki; Masaya Takei; Hideyuki Fukuda (pp. 194-201).

RNA polymerase of influenza virus is a specific enzyme necessary for the viral replication. A siRNA against the RNA polymerase and the RNA polymerase inhibitor L-742,001 reduced accumulation of viral RNAs in the infected cells. L-742,001 strongly inhibited virus re-growth after removal of the agent from the culture, whereas the neuraminidase inhibitor zanamivir did not. L-742,001-resistant mutants showed a Thr-20 to Ala substitution in the PA subunit of RNA polymerase. The drug-resistant virus showed a slight reduction in the susceptibility to L-742,001 in both the plaque assay (threefold reduction) and enzyme assay (two- to three-fold reduction). The resistance levels were lower than those of zanamivir-resistant mutants in the plaque assay. Against zanamivir-resistant mutants, L-742,001 retained the same antiviral activity as against the wild-type strain. These results indicate that L-742,001 is most likely to act at the PA subunit, and possesses a unique profile. It is suggested that PA subunit of RNA polymerase is a promising target for anti-influenza virus agents.

Keywords: Influenza virus; RNA polymerase; PA subunit

Rectal immunization generates protective immunity in the female genital tract against herpes simplex virus type 2 infection: Relative importance of myeloid differentiation factor 88 by Sara Tengvall; Derek O’Hagan; Ali M. Harandi (pp. 202-214).

The present study was undertaken to examine the potential of rectal route of immunization for induction of protective immunity in the female genital tract against genital herpes infection in mice. A single rectal immunization of female C57Bl/6 mice with live attenuated herpes simplex virus type 2 lacking thymidine kinase (HSV-2 TK⁻) was shown to confer HSV-specific cellular and humoral immune responses as well as protection against an otherwise lethal vaginal challenge with a virulent HSV-2 strain. The immunity afforded by rectal immunization with HSV-2 TK⁻ was shown to be independent of sex hormonal influence and the usage of the adaptor protein myeloid differentiation factor 88 (MyD88). Next, the impact of rectal immunization with HSV-2 glycoprotein D (gD) in combination with CpG oligodeoxynucleotide (ODN) or cholera toxin (CT) on induction of immunity against HSV-2 was investigated. Rectal immunization of mice with gD+CpG failed to generate gD specific immune responses and protection against genital herpes infection. Conversely, rectal immunization with gD+CT elicited potent gD-specific cellular immune responses and protection against genital herpes infection through a MyD88-dependent manner. These results highlight the potential of rectal route for the development of novel immunization strategies to elicit immunity in the female genital tract against genital herpes and presumably other sexually transmitted diseases.

Keywords: Rectal immunization; Genital herpes; Sex hormones; MyD88; Mucosal vaccine; Adjuvant

A luciferase-based screening method for inhibitors of alphavirus replication applied to nucleoside analogues by Leena Pohjala; Vladimir Barai; Alex Azhayev; Seppo Lapinjoki; Tero Ahola (pp. 215-222).

Several members of the widespread alphavirus group are pathogenic, but no therapy is available to treat these RNA virus infections. We report here a quantitative assay to screen for inhibitors of Semliki Forest virus (SFV) replication, and demonstrate the effects of 29 nucleosides on SFV and Sindbis virus replication. The anti-SFV assay developed is based on a SFV strain containing Renilla luciferase inserted after the nsP3 coding region, yielding a marker virus in which the luciferase is cleaved out during polyprotein processing. The reporter-gene assay was miniaturized, automated and validated, resulting in a Z′ value of 0.52. [³H]uridine labeling for 1h at the maximal viral RNA synthesis time point was used as a comparative method. Anti-SFV screening and counter-screening for cell viability led to the discovery of several new SFV inhibitors. 3′-Amino-3′-deoxyadenosine was the most potent inhibitor in this set, with an IC₅₀ value of 18μM in the reporter-gene assay and 2μM in RNA synthesis rate detection. Besides the 3′-substituted analogues, certain N⁶-substituted nucleosides had similar IC₅₀ values for both SFV and Sindbis replication, suggesting the applicability of this methodology to alphaviruses in general.

Keywords: Semliki Forest virus; Sindbis virus; RNA replication; Luciferase; Antiviral screening

MegaRibavirin aerosol for the treatment of influenza A virus infections in mice by Brian E. Gilbert; Matthew T. McLeay (pp. 223-229).

While newer neuraminidase inhibitors have been used recently to treat influenza A and B virus infections, emergence of drug resistance poses potential problems. Previous ribavirin aerosol treatments of influenza were effective and drug resistance was not observed. To make ribavirin aerosol treatment a quicker process and limited to once or twice daily treatments, a MegaRibavirin formulation (100mg of ribavirin/mL) was developed that when used with the Aerotech II nebulizer was effective in preventing death in a lethal influenza A virus mouse model. Aerosol generated using the Aerotech II nebulizer flowing at 10L of air/min produced aerosol droplets that contained 2.3mg of ribavirin/L with a mass median aerodynamic diameter of 1.8μm. Using this system for treatment, a single daily 30-min exposure on days 1–4 produced a survival rate of greater than 90%. Delaying the start of aerosol treatment for 48 or 72h and treating once daily for 30min for two days (days 2–3 and 3–4, respectively) still significantly increased the number of survivors and mean time to death. For the treatment of influenza in general and for pandemic avian influenza, the MegaRibavirin-Aerotech II method of aerosol treatment allows for short treatment periods, minimizes environmental issues and costs less.

Keywords: Aerosol; Ribavirin; Influenza; Mice; Treatment

C3H/HeN mouse model for the evaluation of antiviral agents for the treatment of Venezuelan equine encephalitis virus infection by Justin G. Julander; Ramona Skirpstunas; Venkatraman Siddharthan; Kristiina Shafer; Justin D. Hoopes; Donald F. Smee; John D. Morrey (pp. 230-241).

The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4±0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1α, IL-1β, IL-6, IL-12, MCP-1, IFNγ, TNFα, MIP-1α, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-α B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.

Keywords: Venezuelan equine encephalitis; Virus; Antiviral; Therapy; Interferon; C3H/HeN; Mouse

Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro by Jun Xu; Jue Wang; Fei Deng; Zhihong Hu; Hualin Wang (pp. 242-249).

Hepatitis B virus (HBV) infection is endemic in Asia and causes major public health problems worldwide. Present treatment strategies for HBV infections are not satisfactory and the clinical limitation of current antiviral drugs for HBV, such as lamivudine, is causing rapid emergence of drug-resistant viral strains during the prolonged therapeutic treatment. In this research, the efficacy of a natural green tea extract (GTE) against HBV in a stably expressed HBV cell line HepG2-N10 is examined. The expression of viral antigens, HBsAg and HBeAg, were determined by using enzyme linked immuno-absorbent assay (ELISA). Quantitative real-time-PCR (Q-PCR) was used for the determination of extracellular HBV DNA and intracellular replicative intermediates and nuclear covalent closed circular DNA (cccDNA). HBV mRNAs were also analyzed by reverse transcription PCR (RT-PCR). Results showed that the 50% effective concentration (EC₅₀) of GTE on HBsAg, HBeAg, extracellular HBV DNA and intracellular HBV DNA were 5.02, 5.681, 19.81, and 10.76μg/ml, respectively. While the concentration of GTE with the inhibition percentage of 50% on proliferating cells (CC₅₀) was 171.8μg/ml. Similar analysis of the principal component of GTE, epigallocatechin gallate (EGCG), revealed it has relative weaker efficacy compared to GTE.

Keywords: Hepatitis B virus; Green tea extract; Epigallocatechin gallate; HBeAg; HBsAg HBV DNA; Repliction intermediates; cccDNA

Expression of the human cytomegalovirus UL97 gene in a chimeric guinea pig cytomegalovirus (GPCMV) results in viable virus with increased susceptibility to ganciclovir and maribavir by Alistair McGregor; K. Yeon Choi; Xiaohong Cui; Michael A. McVoy; Mark R. Schleiss (pp. 250-259).

In lieu of a licensed vaccine, antivirals are being considered as an intervention to prevent congenital human cytomegalovirus (HCMV) infection. Ideally, antiviral therapies should undergo pre-clinical evaluation in an animal model prior to human use. Guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV. However, GPCMV is not susceptible to the most commonly used HCMV antiviral, ganciclovir (GCV), rendering in vivo study of this agent problematic in the guinea pig model. Human cytomegalovirus (HCMV) susceptibility to GCV is linked to the UL97 gene. We hypothesized that GPCMV susceptibility to GCV could be improved by inserting the HCMV (Towne) UL97 gene into the GPCMV genome in place of the homolog, GP97. A chimeric GPCMV (GPCMV:: UL97) expressed UL97 protein, and replicated efficiently in cell culture, with kinetics similar to wild-type GPCMV. In contrast, deletion of GP97 resulted in a virus (GPCMVd GP97) that grew poorly in culture. GPCMV:: UL97 had substantially improved susceptibility to the inhibitory effects of GCV in comparison to wild-type GPCMV. Additionally, GPCMV:: UL97 exhibited improved susceptibility to another antiviral undergoing clinical trials, maribavir (MBV; benzimidazole riboside 1263W94), which also acts through UL97.

Keywords: Guinea pig cytomegalovirus; Ganciclovir; Maribavir; Congenital CMV infection; Antiviral therapy; Bacterial artificial chromosome

APOBEC and iNOS are not the main intracellular effectors of IFN-γ-mediated inactivation of Hepatitis B virus replication by Sandra Proto; John A. Taylor; Shilpa Chokshi; Naveenan Navaratnam; Nikolai V. Naoumov (pp. 260-267).

Interferon-gamma (IFN-γ) produced by activated T-cells is the principle mediator of non-cytolytic Hepatitis B virus (HBV) inactivation; however the intracellular pathways responsible are poorly defined. We investigated the role of IFN-γ–inducible nitric oxide synthase (iNOS) and APOBEC3 (A3) enzyme family in the inhibition of HBV replication by IFN-γ.Hepatoma-cell lines transfected with HBV DNA were treated with IFN-γ. Viral replication, iNOS and A3 mRNAs were quantitated by TaqMan^®PCR and the direct nitric oxide (NO) effect on HBV replication was investigated using an NO-donor. A3G antiviral activity was verified by co-transfection with its inhibitor, human immunodeficiency virus (HIV)-associated virion infectivity factor (Vif).IFN-γ caused a dose-dependent reduction (>50%) of HBV DNA in the absence of cytotoxicity. Although iNOS mRNA increased 45-fold in IFN-γ treated cells, NO₂⁻ was not detectable in supernatants and the use of an NO-donor did not inhibit HBV replication. A3 enzyme mRNAs varied between cells and were >10-fold higher in lymphocytes than in liver tissue. IFN-γ up-regulated A3G mRNA by three-fold, associated with significant HBV DNA decrease. However, A3G degradation by Vif did not abolish the antiviral effect of IFN-γ against HBV.IFN-γ inhibits HBV replication and up-regulates both iNOS and A3G. However, other pathways appear to have a greater role in IFN-γ-induced HBV inactivation in the liver.

Keywords: Interferon-γ; Hepatitis B virus; Inducible nitric oxide synthase; Apobec3 enzymes

Penciclovir is a potent inhibitor of feline herpesvirus-1 with susceptibility determined at the level of virus-encoded thymidine kinase by Islam T.M. Hussein; Rebecca V. Menashy; Hugh J. Field (pp. 268-274).

Feline herpesvirus-1 (FHV-1) is the causative agent of a severe ocular disease in cats for which a safe potent antiviral chemotherapeutic agent is highly demanded. The sensitivity of FHV-1 to inhibition by three anti-herpetic nucleoside analogues [acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV)] was tested by means of yield reduction assay. ACV showed very poor ability to inhibit FHV-1 replication. At low multiplicity of infection (MOI), both PCV and CDV were nearly equally effective with IC₅₀ values ranging between 6 and 8μg/ml. However, when the MOI was raised to 3PFU/cell, the activity of CDV was markedly reduced (IC₅₀ 25μg/ml), while that of PCV remained relatively low (IC₅₀ 10μg/ml). Although FHV-1 is normally insensitive to ACV, it exhibited >1000-fold increase in sensitivity when the thymidine kinase (TK) encoded by herpes simplex virus-1 (HSV-1) was supplied in trans. Furthermore, three PCV-resistant FHV-1 variants selected in vitro were shown to carry mutations in the TK gene. Taken together, these data provided direct evidence that PCV is a potent selective inhibitor of FHV-1 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues.

Keywords: Feline herpesvirus; Thymidine kinase; Penciclovir

Potent inhibition of drug-resistant HIV protease variants by monoclonal antibodies by Vanda Bartoňová; Vlastimil Král; Irena Sieglová; Jiří Brynda; Milan Fábry; Magdalena Hořejší; Milan Kožíšek; Klára Grantz Šašková; Jan Konvalinka; Juraj Sedláček; Pavlína Řezáčová (pp. 275-277).

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1–6) and flap residues 36–46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5–11nM and 13–169nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

Keywords: HIV protease; Drug resistance; Inhibiting antibody; Dissociative inhibition

R75761, a lead compound for the development of antiviral drugs in late stage poliomyelitis eradication strategies and beyond by Bert Thys; Armando M. De Palma; Johan Neyts; Koen Andries; Raf Vrijsen; Bart Rombaut (pp. 278-281).

In this study the antiviral activity of a panel of 18 out of 240 pyridazinamine analogues was evaluated against the Sabin strains of the three poliovirus types. We found one compound, R75761 which had a comparable 50% effective concentration (EC₅₀) value against all three poliovirus Sabin strains. Virus multiplication was reduced by 10^4.0-fold, 10^6.2-fold and 10^6.6-fold for poliovirus type 1, type 2 and type 3, respectively. R75761 could be considered as a lead compound for development of anti-poliovirus drugs to be used during the late stage of poliovirus eradication and beyond.

Keywords: Anti-poliovirus compound; Polio eradication; Pyridazinamine analogue; Picornavirus; Poliovirus

Contents of Volume (pp. 282-284).

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Antiviral Research (v.78, #3)