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Antiviral Research (v.91, #3)

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

Prelim II: Editorial Board (pp. ii).

Prospects of control and eradication of capripox from the Indian subcontinent: A perspective by V. Bhanuprakash; M. Hosamani; R.K. Singh (pp. 225-232).

Sheeppox and goatpox, two endemic capripox infections in India, pose a significant economic threat to small ruminant productivity in the subcontinent. Vaccination of all susceptible sheep and goats is the feasible and sustainable means of control. Availability of effective live attenuated vaccines that are inherently thermostable and development of improved diagnostics provide the opportunities to initiate effective control measures for capripox. All animals older than 4months can be vaccinated with the current homologous vaccines using a single vaccination by intradermal or subcutaneous routes. The success of the control program needs to be monitored by active surveillance particularly for the presence of virus, as sero-monitoring does not enable the differentiation of infection and vaccination. And also the sero-conversion following capripox vaccination is not detectable enough by the available tools. Sustained control efforts call for socio-economic and political stability, adequate infrastructure and logistic support to store and transport vaccines for reaching out vaccines to the remote end users. Availability of veterinary services, improved extension services for increased awareness among farmers, contribute significantly to the control campaigns. Poor vaccination coverage and in-adequate infrastructure in major parts of the country are some of the major elements that come in the way of effective implementation of building herd immunity through immunization.

Keywords: Sheeppox and goatpox; Vaccines and diagnostics; Veterinary infrastructure; Public private partnership; Control and eradication; Indian subcontinent

Mutations in HCV non-structural genes do not contribute to resistance to nitazoxanide in replicon-containing cells by Changsuek Yon; Prasanth Viswanathan; Jean-François Rossignol; Brent Korba (pp. 233-240).

Nitazoxanide (NTZ) exhibits potent antiviral activity against hepatitis C virus (HCV) in cell culture. Previously, HCV replicon-containing cell lines resistant to NTZ were selected, but transfer the HCV NTZ-resistance phenotype was not observed following transfection of whole cell RNA. To further explore the nature of the resistance of HCV to NTZ, full length HCV replicon sequences were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines. Numerous nucleotide changes were observed in individual HCV genomes relative to the RP7 HCV consensus sequence, but no common mutations in the HCV non-structural genes or 3′-UTR were detected. A cluster of single nucleotide mutations was found within a 5-base portion of the 5′-UTR in 20/21 HCV replicon sequences from both resistant cell lines. Three mutations (5′-UTR G17A, G18A, C20U) were individually inserted into CON1 (‘wild-type’) HCV replicons, showed reduced replication (5 to 50-fold), but none conferred resistance to NTZ. RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under interferon. Transfection of cured NTZ-11 and TIZ-9 with either whole cell RNAs from RP7, NTZ-11, or TIZ-9, ‘wild-type’ or the 5′-UTR mutation-containing replicon RNAs exhibited an NTZ-resistance phenotype. TIZ (the active metabolite of NTZ) was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. These data confirm previous speculations that HCV resistance to NTZ is not due to mutations in the virus, and demonstrate that HCV resistance and most likely the antiviral activity of TIZ are due to interactions with cellular target(s).

Keywords: Nitazoxanide; Hepatitis C virus; Drug resistance; Replicon; Cell culture

High-throughput screening identification of poliovirus RNA-dependent RNA polymerase inhibitors by Grace Campagnola; Peng Gong; Olve B. Peersen (pp. 241-251).

► Fluorescence based in vitro screen for elongation phase inhibitors of a viral RNA-dependent RNA polymerase. ► Subtle structural changes associated with viral RNA-dependent RNA polymerase catalysis may be difficult to inhibit. ► Crystallographic observation of inhibitors bound to the poliovirus polymerase elongation complex active site.Viral RNA-dependent RNA polymerase (RdRP) enzymes are essential for the replication of positive-strand RNA viruses and established targets for the development of selective antiviral therapeutics. In this work we have carried out a high-throughput screen of 154,267 compounds to identify poliovirus polymerase inhibitors using a fluorescence based RNA elongation assay. Screening and subsequent validation experiments using kinetic methods and RNA product analysis resulted in the identification of seven inhibitors that affect the RNA binding, initiation, or elongation activity of the polymerase. X-ray crystallography data show clear density for five of the compounds in the active site of the poliovirus polymerase elongation complex. The inhibitors occupy the NTP binding site by stacking on the priming nucleotide and interacting with the templating base, yet competition studies show fairly weak IC₅₀ values in the low μM range. A comparison with nucleotide bound structures suggests that weak binding is likely due to the lack of a triphosphate group on the inhibitors. Consequently, the inhibitors are primarily effective at blocking polymerase initiation and do not effectively compete with NTP binding during processive elongation. These findings are discussed in the context of the polymerase elongation complex structure and allosteric control of the viral RdRP catalytic cycle.

Keywords: RNA-dependent RNA polymerase; RdRP; Inhibitor; Poliovirus; HTS; RNA

The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus by Tatsuya Suda; Masaaki Kawano; Yasuhisa Nogi; Naohito Ohno; Toshitaka Akatsuka; Masanori Matsui (pp. 252-258).

► We examine if the immunization route influences the recruitment of pulmonary CTLs. ► Mice are immunized with adenovirus expressing influenza M1 via various routes. ► The immunization route impacts the recruitment of CTLs in the lung. ► Hock immunization is most effective. ► These results may improve our ability to design vaccines.Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ⁺ CD8⁺ T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8⁺ T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ⁺ CD8⁺ T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus.

Keywords: Cytotoxic T lymphocyte; Immunization route; Adenoviral vaccine; Hock immunization; Influenza A virus

CK2 inhibitors increase the sensitivity of HSV-1 to interferon-β by Miles C. Smith; Adam M. Bayless; Erica T. Goddard; David J. Davido (pp. 259-266).

Herpes simplex virus type 1 (HSV-1) requires the activities of cellular kinases for efficient replication. The host kinase, CK2, has been shown or is predicted to modify several HSV-1 proteins and has been proposed to affect one or more steps in the viral life cycle. Furthermore, potential cellular and viral substrates of CK2 are involved in antiviral pathways and viral counter-defenses, respectively, suggesting that CK2 regulates these processes. Consequently, we tested whether pharmacological inhibitors of CK2 impaired HSV-1 replication, either alone or in combination with the cellular antiviral factor, interferon-β (IFN-β). Our results indicate that the use of CK2 inhibitors results in a minor reduction in HSV-1 replication but enhanced the inhibitory effect of IFN-β on replication. This effect was dependent on the HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), which impairs several host antiviral responses, including that produced by IFN-β. Inhibitors of CK2 did not, however, impede the ability of ICP0 to induce the degradation of two cellular targets: the promyelocytic leukemia protein (PML) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Notably, this effect was only apparent for HSV-1, as the CK2 inhibitors did not enhance the antiviral effect of IFN-β on either vesicular stomatitis virus or adenovirus type 5. Thus, our data suggest that the activity of CK2 is required for an early function during viral infection that assists the growth of HSV-1 in IFN-β-treated cells.

Keywords: HSV-1; CK2; IFN; ICP0; Ad5; VSV

Inactivation of a non-enveloped RNA virus by artificial ribonucleases: Honey bees and Acute bee paralysis virus as a new experimental model for in vivo antiviral activity assessment by Antonina A. Fedorova; Klara Azzami; Elena I. Ryabchikova; Yulia E. Spitsyna; Vladimir N. Silnikov; Wolfgang Ritter; Hans J. Gross; Jürgen Tautz; Valentin V. Vlassov; Hildburg Beier; Marina A. Zenkova (pp. 267-277).

RNA-containing viruses represent a global threat to the health and wellbeing of humans and animals. Hence, the discovery of new approaches for the design of novel vaccines and antiviral compounds attains high attention. Here we describe the potential of artificial ribonucleases (aRNases), low molecular weight compounds capable to cleave phosphodiester bonds in RNA under mild conditions, to act as antiviral compounds via destroying the genome of non-enveloped RNA viruses, and the potential of utilizing honey bee larvae and adult bees ( Apis mellifera) as a novel experimental system for the screening of new antiviral compounds. Pre-incubation of an Acute bee paralysis virus (ABPV) suspension with aRNases D3–12, K-D-1 or Dp12F6 in a concentration-dependent manner increased the survival rate of bee larvae and adult bees subsequently infected with these preparations, whereas incubation of the virus with aRNases ABL3C3 or L2–3 had no effect at all. The results of RT-PCR analysis of viral RNA isolated from aRNase-treated virus particles confirmed that virus inactivation occurs via degradation of viral genomic RNA: dose-dependent inactivation of ABPV correlates well with the cleavage of viral RNA. Electron microscopy analysis revealed that the morphology of ABPV particles inactivated by aRNases remains unaffected as compared to control virus preparations. Altogether the obtained results clearly demonstrate the potential of aRNases as a new virus inactivation agents and bee larvae/ABPV as a new in vivo system for the screening of antiviral compounds.

Keywords: Artificial ribonucleases; Virus inactivation; Antiviral activity; Killed-virus vaccine; Acute bee paralysis virus; Bee larvae

Evaluation of resistance development and viability recovery by a non-enveloped virus after repeated cycles of aPDT by Liliana Costa; João P.C. Tomé; Maria G.P.M.S. Neves; Augusto C. Tomé; José A.S. Cavaleiro; Maria A.F. Faustino; Ângela Cunha; Newton C.M. Gomes; Adelaide Almeida (pp. 278-282).

► Tri-Py⁺-Me-PF effectively photoinactivate T4-like phages. ► Phages after aPDT do not recover their viability and do not develop resistance after 10 successive treatments.Nowadays, the emergence of drug resistant microorganisms is a public health concern. The antimicrobial photodynamic therapy (aPDT) has an efficient action against a wide range of microorganisms and can be viewed as an alternative approach for treating microbial infections. The aim of this study was to determine if a model target virus (T4-like bacteriophage), in the presence of the tricationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py⁺-Me-PF), can develop resistance to aPDT and recover its viability after photodynamic treatments. To assess the development of aPDT resistance after repeated treatments, a suspension of T4-like bacteriophage was irradiated with white light (40Wm⁻²) for 120min in the presence of 5.0μM of Tri-Py⁺-Me-PF (99.99% of inactivation) and new phage suspensions were produced from the surviving phages, after each cycle of light exposure. The procedure was repeated ten times. To evaluate the recovery of viral viability after photoinactivation, a suspension of T4-like bacteriophage was irradiated with white light for 120min in the presence of 5.0μM of Tri-Py⁺-Me-PF on five consecutive days. In each day, an aliquot of the irradiated suspension was plated and the number of lysis plaques was counted after 24, 48, 72, 96 and 120h of dark incubation at 37°C. The profile of bacteriophage photoinactivation did not change after ten consecutive cycles and no recovery of viability was detected after five accumulated cycles of photodynamic treatment. The results suggest that aPDT represents a valuable and promising alternative therapy to treat viral infections, overcoming the problem of microbial resistance.

Keywords: Antimicrobial photodynamic therapy (aPDT); Bacteriophages; Resistance; Viability recovery; Porphyrin

Characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus by Tatsuhiko Ozawa; Aishun Jin; Kazuto Tajiri; Masaya Takemoto; Tomoko Okuda; Kimiyasu Shiraki; Hiroyuki Kishi; Atsushi Muraguchi (pp. 283-287).

The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10⁻¹⁰M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.

Keywords: Influenza; M2e-antibody; Antibody therapeutics

Characterization of the binding affinities of peramivir and oseltamivir carboxylate to the neuraminidase enzyme by Shanta Bantia; Ramanda Upshaw; Y.S. Babu (pp. 288-291).

With the continued threat of morbidity and mortality from influenza and the development of resistance to influenza antiviral drugs, there is increasing interest in new treatments, such as the investigational intravenous drug peramivir, and in combination treatments. In this study, we determined the impact of oseltamivir carboxylate on the binding affinity of peramivir/neuraminidase (NA) enzyme complex and vice versa. Influenza NA was incubated with peramivir and oseltamivir carboxylate alone and in combination. Dissociation rates of the enzyme–inhibitor complex measured in the presence of NA substrate for peramivir alone and the combination were similar, suggesting that peramivir competitively inhibits the neuraminidase enzyme and that oseltamivir carboxylate when added to peramivir does not impact the binding affinity of peramivir to the NA enzyme.

Keywords: Peramivir; Oseltamivir carboxylate; Binding affinity; Enzyme–inhibitor complex; Neuraminidase (NA)

The new and less toxic protease inhibitor saquinavir–NO maintains anti-HIV-1 properties in vitro indistinguishable from those of the parental compound saquinavir by Filippo Canducci; Elisa Rita Ceresola; Diego Saita; Yousef Al-Abed; Gianni Garotta; Massimo Clementi; Ferdinando Nicoletti (pp. 292-295).

► The covalent addition of a NO group to the protease inhibitor Saq has not modified its antiviral activity. ► Since Saq–NO is less toxic than Saq both in vitro and in vivo, larger doses of Saq–NO could be used for a more effective treatment of HIV-1 patients. ► The reduced toxicity of Saq–NO may limit side effects at the current daily dosage or increase the genetic barrier of the PI based regimen if used at higher dosage.Although, the antiviral activity, tolerability and convenience of protease inhibitors have improved significantly in recent years, toxicity-associated adverse events including diarrhea, lipid alterations, disturbance of glucose homeostasis and liver enzyme elevations still remain a major concern during treatment of HIV-1 patients. We have recently shown that the covalent attachment of the NO moiety to the HIV-1 protease inhibitor saquinavir (Saq–NO) reduces its toxicity. In this study, we evaluated in vitro the anti-HIV activity of Saq–NO vs. its parental compound Saq. Site directed mutants with the most frequently identified Saq associated resistance mutations and their combinations were generated on proviral AD8-based backbones. Phenotypic assays were conducted using wild type clinical isolates and fully replicating recombinant viruses with Saq and Saq–NO in parallel on purified CD4+ T cells. The following recombinant viruses were generated and tested: L33F, M46I, G48V, I54V, I84V+L90M, M46I+L90M, G48V+L90M, M46I+I54V+L90M, L33F+M46I+L90M. The fold change resistance compared to the wild type viruses was between 1.3 and 7 for all single mutants, between 3.4 and 20 for double mutants and between 16.7 and 28.5 for viruses carrying three mutations for both compounds. The results clearly demonstrate that Saq–NO maintains an anti-HIV-1 profile very similar to that of Saq. The possibility to reduce Saq associated side effects and to increase the concentration of the drug in vivo may allow a higher and possibly more effective dosage of Saq–NO in HIV-1-infected patients and to increase the genetic barrier of this PI thus impairing the selection of resistant clones.

Keywords: Protease inhibitors; Genetic barrier; Toxicity; Drug resistance; Saquinavir

Inhibitory effects of tricin derivative from Sasa albo-marginata on replication of human cytomegalovirus by Kazuhiko Akuzawa; Rie Yamada; Zhuan Li; Ying Li; Hidetaka Sadanari; Keiko Matsubara; Kunitomo Watanabe; Mamoru Koketsu; Yuuzo Tuchida; Tsugiya Murayama (pp. 296-303).

► The anti-human cytomegalovirus (HCMV) activity of tricin was studied in the MRC-5 cells. ► Tricin significantly suppressed HCMV replication, but had no virucidal effect on cell-free HCMV. ► Tricin decreased the expression of IE antigen and COX-2 expression in HCMV-infected cells. ► Tricin is a novel compound with potential COX inhibitor-dependent anti-HCMV activity.The anti-human cytomegalovirus (HCMV) activity of tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), a derivative from Sasa albo-marginata, was studied in the human embryonic fibroblast cell line MRC-5. In a plaque assay, tricin and ganciclovir (GCV) showed concentration-dependent inhibitory properties from 0.05 to 3.6μM and 0.01 to 1.0μM, respectively. Tricin had no virucidal effects on cell-free HCMV. Treatment with tricin 1h before, or 1h or 3h after viral infection significantly suppressed HCMV replication. Moreover, tricin inhibited the expression of immediate early (IE) 2 mRNA and DNA polymerase (UL54) mRNA in HCMV-infected cells. Western blot analysis also demonstrated that tricin decreased the expression of IE antigen (especially IE2) and cyclooxygenase 2 (COX-2) expression in HCMV-infected cells. In the presence of tricin, prostaglandin E2 (PGE₂) accumulation by HCMV infection was completely inhibited. These results suggest that tricin is a novel compound with potential COX inhibitor-dependent anti-HCMV activity.

Keywords: Anti-cytomegalovirus activity; Tricin; HCMV; IE2; COX-2; Entry

Antiviral activity of the proteasome inhibitor VL-01 against influenza A viruses by Emanuel Haasbach; Eva-Katharina Pauli; Robert Spranger; David Mitzner; Ulrich Schubert; Ralf Kircheis; Oliver Planz (pp. 304-313).

► Inhibition of the proteasome with VL-01 leads to reduced virus titer in vitro and in vivo. ► Proteasome inhibition shows a reduction of cytokines/chemokines after LPS or H5N1 infection. ► Aerosol treatment with VL-01 shows no toxic side effects in mice.The appearance of highly pathogenic avian influenza A viruses of the H5N1 subtype being able to infect humans and the 2009 H1N1 pandemic reveals the urgent need for new and efficient countermeasures against these viruses. The long-term efficacy of current antivirals is often limited, because of the emergence of drug-resistant virus mutants. A growing understanding of the virus–host interaction raises the possibility to explore alternative targets involved in the viral replication. In the present study we show that the proteasome inhibitor VL-01 leads to reduction of influenza virus replication in human lung adenocarcinoma epithelial cells (A549) as demonstrated with three different influenza virus strains, A/Puerto Rico/8/34 (H1N1) (EC₅₀ value of 1.7μM), A/Regensburg/D6/09 (H1N1v) (EC₅₀ value of 2.4μM) and A/Mallard/Bavaria/1/2006 (H5N1) (EC₅₀ value of 0.8μM). In in vivo experiments we could demonstrate that VL-01-aerosol-treatment of BALB/c mice with 14.1mg/kg results in no toxic side effects, reduced progeny virus titers in the lung (1.1±0.3log₁₀pfu) and enhanced survival of mice after infection with a 5-fold MLD₅₀ of the human influenza A virus strain A/Puerto Rico/8/34 (H1N1) up to 50%. Furthermore, treatment of mice with VL-01 reduced the cytokine release of IL-α/β, IL-6, MIP-1β, RANTES and TNF-α induced by LPS or highly pathogen avian H5N1 influenza A virus. The present data demonstrates an antiviral effect of VL-01 in vitro and in vivo and the ability to reduce influenza virus induced cytokines and chemokines.

Keywords: Influenza A virus; Proteasome inhibitor; Drug-resistance

Synergistic activity of baicalein with ribavirin against influenza A (H1N1) virus infections in cell culture and in mice by Lili Chen; Jie Dou; Zhenzhen Su; Huimin Zhou; Hui Wang; Weidong Zhou; Qinglong Guo; Changlin Zhou (pp. 314-320).

► We investigated the synergistic activity of baicalein with ribavirin against influenza virus in cell culture and in mice. ► Study demonstrated that the compound inhibited the synthesis of viral matrix protein gene greater than ribavirin alone. ► Higher survival rate, lower body weight loss, fewer inflammatory responses in the lungs of infected were observed.Baicalein is a flavonoid derived from the root of Scutellaria baicalensis, a traditional Chinese medicine that has been used for hundreds of years; baicalein has also been demonstrated to have antiviral activity with low toxicity. The synergistic activity of baicalein with ribavirin against influenza virus infections in cell culture and in mice was investigated for the first time in our research. In vitro, maximal synergy at lower concentrations of baicalein (0.125μg/ml) and ribavirin (12.5μg/ml) was observed, and the reduced expression of the viral matrix protein (M) gene suggested that drug combinations caused greater inhibition than ribavirin alone, especially the combination of 0.5μg/ml baicalein and 5μg/ml ribavirin. In vivo, combinations of baicalein and ribavirin provided a higher survival rate and lower body weight loss. Moreover, fewer inflammatory responses in the lungs of mice infected with virus and treated with baicalein and ribavirin were observed; the mean scores were 1.0, 0.8, and 1.2 with the doses of ribavirin at 50mg/kg/d combined with baicalein at 100mg/kg/d, 200mg/kg/d, and 400mg/kg/d respectively, while the placebo group had a mean pathology score of 3.2. Thus, the data demonstrates that combinations of baicalein and ribavirin provide better protection against influenza infection than each compound used alone and could potentially be clinically useful.

Keywords: Synergistic activity; Baicalein; Ribavirin; Antiviral activity

High-throughput assay to identify inhibitors of Vpu-mediated down-regulation of cell surface BST-2 by Quan Zhang; Zhenlong Liu; Zeyun Mi; Xiaoyu Li; Pingping Jia; Jinming Zhou; Xiao Yin; Xuefu You; Liyan Yu; Fei Guo; Jing Ma; Chen Liang; Shan Cen (pp. 321-329).

► Blocking Vpu-mediated down-regulation of cell surface BST-2 is viewed as a new opportunity for developing anti-HIV drugs. ► Establishment of the first high-throughput assay to screen novel anti-HIV drug targeting BST-2 down-regulation by Vpu. ► The simple and economic assay shows an excellent correlation with with results obtained by flow cytometry (FACS). ► Provide a proof-of-concept of implementing the same strategy to develop new drugs against other viruses sensitive to BST-2.Bone marrow stromal cell antigen 2 (BST-2, also known as Tetherin) inhibits HIV-1 release and thereby severely impairs viral replication. HIV-1 accessory protein Vpu induces the down-regulation of cell surface BST-2, and counteracts the antiviral function of BST-2. Blocking Vpu-mediated down-regulation of cell surface BST-2 is viewed as a new opportunity for developing anti-HIV drugs. In this study, we have developed a high-throughput cell-based ELISA to identify small molecules that antagonize HIV-1 Vpu function and consequently inhibit HIV-1 replication through rescuing the antiviral activity of host BST-2. This cell-ELISA shows an excellent correlation with results obtained by flow cytometry (FACS). Under optimal conditions, a Z′ factor of 0.605 was achieved in a 96-well format. Together, these results demonstrate that this assay can be used to quantify the cell surface level of BST-2 and be adapted to a high-throughput screening for novel anti-HIV compounds.

Keywords: HIV-1; Vpu; BST-2; High-throughput assay

Antiviral effect of a selective COX-2 inhibitor on H5N1 infection in vitro by Suki M.Y. Lee; W.W. Gai; Timothy K.W. Cheung; J.S.M. Peiris (pp. 330-334).

A selective cyclooxygenase-2 (COX-2) inhibitor has been previously shown to suppress the hyper-induced pro-inflammatory responses in H5N1 infected primary human cells. Here, we demonstrate that COX-2 inhibitors suppress H5N1 virus replication in human macrophages suggesting that H5N1 virus replication (more so than seasonal H1N1 virus) is dependent on activation of COX-2 dependent signaling pathways in host cells. COX-2 and its downstream signaling pathways deserve detailed investigation as a novel therapeutic target for treatment of H5N1 disease.

Keywords: Influenza; H5N1; Antiviral; Selective COX-2 inhibitor

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Antiviral Research (v.91, #3)