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Antiviral Research (v.79, #1)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Rapid sequence-based diagnosis of viral infection by Phenix-Lan Quan; Thomas Briese; Gustavo Palacios; W. Ian Lipkin (pp. 1-5).

With globalization of microbial threats and an increasing appreciation for the role of infection in chronic as well as acute diseases, there is burgeoning interest in the development of specific antiviral drugs. Less attention has been focused on the establishment and implementation of rapid viral diagnostic methods, without which it will not be possible to obtain the full benefit of new therapies. Here we review the current status of viral diagnostics and the utility of various sequence-based diagnostic platforms for applications in clinical microbiology, surveillance and pathogen discovery.

Keywords: Viral infection; Molecular diagnosis; Pathogen discovery; Polymerase chain reaction (PCR); DNA microarray; High-throughput sequencing

Inhibitory effects of some derivatives of glycyrrhizic acid against Epstein-Barr virus infection: Structure–activity relationships by Jung-Chung Lin; Jaw-Ming Cherng; Man-Shan Hung; Lidia A. Baltina; Lia Baltina; Rimma Kondratenko (pp. 6-11).

Glycyrrhizic acid (18β-GL or GL) is a herbal drug with a broad spectrum of antiviral activities and pharmacological effects and multiple sites of action. Previously we showed that GL inhibits Epstein-Barr virus (EBV) infection in vitro by interfering with an early step of the EBV replication cycle (possibly attachment/penetration). Here we tested the effects of 15 GL derivatives against EBV infection by scoring the numbers of cell expressing viral antigens and quantifying EBV DNA copy numbers in superinfected Raji cells. The derivatives were made either by transformation of GL on carboxyl and hydroxyl groups or by conjugation of amino acid residues into the carbohydrate part. We identified seven compounds active against EBV and all showed dose-dependent inhibition as determined by both assays. Among these active compounds, the introduction of amino acid residues into the GL carbohydrate part enhanced the antiviral activity in three of the seven active compounds. However, when Glu(OH)-OMe was substituted by Glu(OMe)-OMe, its antiviral activity was completely abolished. Introduction of potassium or ammonium salt to GL reduced the antiviral activity with no significant effect on cytotoxicity. The α-isomer (18α-GL) of 18β-GL was as potent as the β-form, but its sodium salt lost antiviral activity. The metabolic product of GL, 18β-glycyrrhetinic acid (18β-GA or GA), was 7.5-fold more active against EBV than its parental compound GL but, concomitantly, exhibited increased cytotoxicity resulting in a decreased therapeutic index.

Keywords: Glycyrrhizic acid derivatives; Epstein-Barr virus; Antiviral activity

Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules by Zhujun Ao; Ami Patel; Kaylie Tran; Xinying He; Keith Fowke; Kevin Coombs; Darwyn Kobasa; Gary Kobinger; Xiaojian Yao (pp. 12-18).

In this study, we have generated and characterized an avian influenza H5N1 hemagglutinin (HA), neuraminidase (NA) and M2 ion channel pseudotyped HIV-based vector system (HaNaM-pseudotyped HIV vector). The cleavage site of the HA protein was modified to necessitate trypsin-dependent maturation of the glycoprotein. HA, NA and M2 were efficiently incorporated in HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Results also showed that the presence of avian influenza M2 and NA proteins maximized both vector production and transduction and that transduction was highly sensitive to the specific NA inhibitor oseltamivir (Tamiflu). H5N1 HaNaM-pseudotyped HIV vector system was also adapted for cell-based high throughput screening of drug candidates against influenza virus infection, and its high sensitivity to the specific oseltamivir validates its potential utility in the identification of new influenza inhibitors. Overall, the trypsin-dependent H5N1-pseudotyped HIV vector can mimic avian influenza virus infection processes with sufficient precision to allow for the identification of new antivirals and to study avian influenza virus biology in a lower biosafety level laboratory environment.

Keywords: Avian influenza virus; Hemagglutinin; Neuraminidase; M2 protein; H5N1-pseudotyped HIV vector system

Synthesis of 1-β-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus by Dong-Hoon Chung; Sidath C. Kumarapperuma; Yanjie Sun; Qianjun Li; Yong-Kyu Chu; Jeffrey B. Arterburn; William B. Parker; Jeffrey Smith; Kristin Spik; Harish N. Ramanathan; Connie S. Schmaljohn; Colleen B. Jonsson (pp. 19-27).

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-β-ribosides and identified one with antiviral activity, 1-β-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC₅₀ value of 10 and 4.4μM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a ∼25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1±0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5′-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.

Keywords: Hemorrhagic fever with renal syndrome; HFRS; Hantaan virus HTNV; Andes virus; Ribavirin; Crimean Congo hemorrhagic fever virus; Rift Valley fever virus

Genotypic detection of acyclovir-resistant HSV-1: Characterization of 67 ACV-sensitive and 14 ACV-resistant viruses by Emilie Frobert; Jean-Claude Cortay; Tadamasa Ooka; Fatiha Najioullah; Danielle Thouvenot; Bruno Lina; Florence Morfin (pp. 28-36).

Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24h.

Keywords: Herpes simplex virus; ACV resistance; Thymidine kinase gene; Direct genotyping

Structure and sequence motifs of siRNA linked with in vitro down-regulation of morbillivirus gene expression by Renata Servan de Almeida; Djénéba Keita; Geneviève Libeau; Emmanuel Albina (pp. 37-48).

The most challenging task in RNA interference is the design of active small interfering RNA (siRNA) sequences. Numerous strategies have been published to select siRNA. They have proved effective in some applications but have failed in many others. Nonetheless, all existing guidelines have been devised to select effective siRNAs targeting human or murine genes. They may not be appropriate to select functional sequences that target genes from other organisms like viruses. In this study, we have analyzed 62 siRNA duplexes of 19 bases targeting three genes of three morbilliviruses. In those duplexes, we have checked which features are associated with siRNA functionality. Our results suggest that the intramolecular secondary structure of the targeted mRNA contributes to siRNA efficiency. We also confirm that the presence of at least the sequence motifs U13, A or U19, as well as the absence of G13, cooperate to increase siRNA knockdown rates. Additionally, we observe that G11 is linked with siRNA efficacy. We believe that an algorithm based on these findings may help in the selection of functional siRNA sequences directed against viral genes.

Keywords: siRNA; RNAi; Morbillivirus; Sequence motifs; Secondary structure; Target sequence selection

Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo by Mark Schleiss; Jan Eickhoff; Sabrina Auerochs; Martina Leis; Silke Abele; Sabine Rechter; Yeon Choi; Jodi Anderson; Gillian Scott; William Rawlinson; Detlef Michel; Stephan Ensminger; Bert Klebl; Thomas Stamminger; Manfred Marschall (pp. 49-61).

Cytomegalovirus infection is associated with severe disease in immunocompromised individuals. Current antiviral therapy faces several limitations. In a search of novel drug candidates, we describe here the anti-cytomegaloviral properties of two compounds of the chemical class of quinazolines, gefitinib (Iressa^®) and Ax7396 (RGB-315389). Both compounds showed strong inhibitory effects in vitro against human and animal cytomegaloviruses with IC₅₀s in a low micromolar range. Cytotoxicity did not occur at these effective concentrations. The antiviral mode of action was based on the inhibition of protein kinase activity, mainly directed to a viral target kinase (UL97/M97) in addition to cellular target candidates. This was demonstrated by a high sensitivity of the respective protein kinases in vitro and by infection experiments with viral mutants carrying genomic alterations in the ORF UL97/M97 modulating viral drug sensitivity. In a guinea pig model, gefitinib showed inhibition of cytomegaloviral loads in blood and lung tissue. Importantly, the rate of mortality of infected animals was reduced by gefitinib treatment. In contrast to the in vitro data, Ax7396 showed no significant antiviral activity in a mouse model. Further in vivo analyses have to assess the potential use of gefitinib in the treatment of cytomegalovirus disease.

Keywords: Human cytomegalovirus; Protein kinase inhibitors; Antiviral activity; Mode of action; Animal models; Drug development

Anti-HSV activity of digitoxin and its possible mechanisms by Chun-Ting Su; John T.-A. Hsu; Hsing-Pang Hsieh; Pi-Han Lin; Ting-Chi Chen; Chuan-Liang Kao; Chun-Nan Lee; Sui-Yuan Chang (pp. 62-70).

Herpes simplex virus type 1 (HSV-1) can establish latent infection in the nervous system and usually leads to life-threatening diseases in immunocompromised individuals upon reactivation. Treatment with conventional nucleoside analogue such as acyclovir is effective in most cases, but drug-resistance may arise due to prolonged treatment in immunocompromised individuals. In this study, we identified an in-use medication, digitoxin, which actively inhibited HSV-1 replication with a 50% effective concentration (EC₅₀) of 0.05μM. The 50% cytotoxicity concentration (CC₅₀) of digitoxin is 10.66μM and the derived selective index is 213. Several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also showed anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication and the virus release stage. The observation that digitoxin can inhibit acyclovir-resistant viruses further implicates that digitoxin represents a novel drug class with distinct antiviral mechanisms from traditional drugs.

Keywords: Digitoxin; HSV; Cardiac glycoside

Efficacy of cationic lipid–DNA complexes (CLDC) on hepatitis B virus in transgenic mice by John D. Morrey; Neil E. Motter; Brandon Taro; Marla Lay; Jeffery Fairman (pp. 71-79).

Cationic lipid–DNA (non-coding) complexes (CLDC) are activators of the innate immune response that increase survival of rodents with some acute viral infections and cancers. CLDC were evaluated for their ability to impact viral DNA levels in transgenic mice carrying an infectious clone of hepatitis B virus (HBV). Mice used in the studies were diet-restricted as nursing pups from solid food, because the expression of HBV DNA in the liver was increased above background levels in some mice with this restriction. Survival surgery was performed on these mice to obtain liver biopsies from which to select animals with suitable levels of liver HBV DNA for entry into the experimental protocols. Intravenous administration of 5μg/mouse of CLDC on days 1, 7 and 13 reduced liver HBV DNA to similar low levels achieved with the positive control, adefovir dipivoxil. In a subsequent experiment, the same treatment schedule was used to determine that the minimal effective CLDC dose was between 0.5 and 0.05μg/mouse. Selective cytokines were increased in the livers of CLDC-treated compared to placebo-treated mice in a dose-responsive manner. CLDC were effective in reducing liver HBV DNA and could be considered for further evaluation in other hepatitis models.

Keywords: Cationic lipid–DNA complex; CLDC; Hepatitis B virus; Transgenic mice

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Antiviral Research (v.79, #1)