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Antiviral Research (v.90, #1)

Notes to Authors (pp. co3).

Editorial Board (pp. ii).

A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus by Sarah A. Goodchild; Lyn M. O’Brien; John Steven; Mischa R. Muller; Oliver J. Lanning; Christopher H. Logue; Riccardo V. D’Elia; Robert J. Phillpotts; Stuart D. Perkins (pp. 1-8).

In murine models of Venezuelan equine encephalitis virus (VEEV) infection, the neutralising monoclonal antibody 1A3B-7 has been shown to be effective in passive protection from challenge by the aerosol route with serogroups I, II and Mucambo virus (formally VEE complex subtype IIIA). This antibody is able to bind to all serogroups of the VEEV complex when used in ELISA and therefore is an excellent candidate for protein engineering in order to derive a humanised molecule suitable for therapeutic use in humans. A Complementarity Determining Region (CDR) grafting approach using human germline IgG frameworks was used to produce a panel of humanised variants of 1A3B-7, from which a single candidate molecule with retained binding specificity was identified. Evaluation of humanised 1A3B-7 (Hu1A3B-7) in in vitro studies indicated that Hu1A3B-7 retained both broad specificity and neutralising activity. Furthermore, in vivo experiments showed that Hu1A3B-7 successfully protected mice against lethal subcutaneous and aerosol challenges with VEEV strain TrD (serogroup I). Hu1A3B-7 is therefore a promising candidate for the future development of a broad-spectrum antiviral therapy to treat VEEV disease in humans.

Keywords: Venezuelan equine encephalitis virus; Monoclonal antibody; Immunotherapy; Humanisation

Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells by Tomoichiro Oka; Hirotaka Takagi; Yukinobu Tohya; Kosuke Murakami; Naokazu Takeda; Takaji Wakita; Kazuhiko Katayama (pp. 9-16).

Feline calicivirus (FCV) is an important veterinary pathogen and causes respiratory disease in cats. Because it grows well in cell culture, FCV is often used as a model virus of non-culturable caliciviruses. In this study, a cell-free and two cell culture-based biosensor assay systems were established to detect FCV protease activity. The assays utilize luciferase sensor technology or second-generation bioluminescence resonance energy transfer (BRET²). A luciferase sensor was designed to contain an FCV protease cleavage motif within the permutated luciferase (GloSensor). The BRET²-based probe contained the same cleavage motif flanked by a renilla luciferase and a variant of green fluorescent protein. To confirm the specificity of these assay systems, GloSensor or a BRET²-based probe containing a mutation in the cleavage motif was also constructed. In a cell-free assay, GloSensor showed increased luminescence in proportion to the amount of FCV protease, while no signal change was observed when the construct harboring the mutant cleavage motif was used. A feline cell line stably expressing GloSensor or the BRET²-based probe was established. Increased levels of GloSensor luminescence, and decreased levels of BRET² signals were observed according to input FCV titers. In contrast, no significant signal change was observed in the cells stably expressing the mutant cleavage motif. GloSensor and the BRET²-based probe were capable of detecting the inhibitory activity of ribavirin in FCV-infected cells. Our results demonstrate that these biosensors are useful to detect FCV protease activity induced in infected cells, and well worth consideration for screening of anti-FCV protease compounds in cell-free system as well as anti-FCV compounds in cultured cells.

Keywords: Biosensor; Luciferase; Bioluminescence resonance energy transfer; Feline calicivirus; Protease; Ribavirin

A single intramuscular injection of neuraminidase inhibitor peramivir demonstrates antiviral activity against novel pandemic A/California/04/2009 (H1N1) influenza virus infection in mice by Shanta Bantia; Debra Kellogg; Cynthia Parker; Ramanda Upshaw; Natalia A. Ilyushina; Y.S. Babu (pp. 17-21).

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss ( p<0.001) and mortality ( p<0.05) in mice infected with LD₉₀ dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8g for the 1, 3, 10 and 30mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10mg/kg/d twice a day, orally for 5days) was 90, 30 and 20% at 24, 48 and 72h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models.

Keywords: Peramivir; Influenza; Mouse model; Intramuscular; Pandemic

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin by Yohichi Kumaki; Miles K. Wandersee; Aaron J. Smith; Yanchen Zhou; Graham Simmons; Nathan M. Nelson; Kevin W. Bailey; Zachary G. Vest; Joseph K.-K. Li; Paul Kay-Sheung Chan; Donald F. Smee; Dale L. Barnard (pp. 22-32).

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1±0.4μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD₅₀ (575PFU) of virus for 4h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0mg/kg/day for 4 days. Treatment with UDA at 5mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV ( p<0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss ( p<0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells.

Keywords: BALB/c mouse; SARS-CoV; Urtica dioica; agglutinin (UDA)

Within-host co-evolution of Gag P453L and protease D30N/N88D demonstrates virological advantage in a highly protease inhibitor-exposed HIV-1 case by Junko Shibata; Wataru Sugiura; Hirotaka Ode; Yasumasa Iwatani; Hironori Sato; Hsinyi Tsang; Masakazu Matsuda; Naoki Hasegawa; Fengrong Ren; Hiroshi Tanaka (pp. 33-41).

To better understand the mechanism of HIV group-specific antigen (Gag) and protease (PR) co-evolution in drug-resistance acquisition, we analyzed a drug-resistance case by both bioinformatics and virological methods. We especially considered the quality of sequence data and analytical accuracy by introducing single-genome sequencing (SGS) and Spidermonkey/Bayesian graphical models (BGM) analysis, respectively. We analyzed 129 HIV-1 Gag–PR linkage sequences obtained from 8 time points, and the resulting sequences were applied to the Spidermonkey co-evolution analysis program, which identified ten mutation pairs as significantly co-evolving. Among these, we focused on associations between Gag-P453L, the P5′ position of the p1/p6 cleavage-site mutation, and PR-D30N/N88D nelfinavir-resistant mutations, and attempted to clarify their virological significance in vitro by constructing recombinant clones. The results showed that P453L^Gag has the potential to improve replication capacity and the Gag processing efficiency of viruses with D30N^PR/N88D^PR but has little effect on nelfinavir susceptibility. Homology modeling analysis suggested that hydrogen bonds between the 30th PR residue and the R452^Gag are disturbed by the D30N^PR mutation, but the impaired interaction is compensated by P453L^Gag generating new hydrophobic interactions. Furthermore, database analysis indicated that the P453L^Gag/D30N^PR/N88D^PR association was not specific only to our clinical case, but was common among AIDS patients.

Keywords: HIV; Protease; Gag; Drug resistance; Co-evolution

HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro by Valentina Svicher; Emanuela Balestra; Valeria Cento; Loredana Sarmati; Luca Dori; Ina Vandenbroucke; Roberta D’Arrigo; Anna Rita Buonomini; Herwig Van Marck; Matteo Surdo; Patrizia Saccomandi; Wendy Mostmans; Jeroen Aerssens; Stefano Aquaro; Lieven J. Stuyver; Massimo Andreoni; Francesca Ceccherini-Silberstein; Carlo Federico Perno (pp. 42-53).

Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4⁺-CCR5⁺-/CXCR4⁺-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed.Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5⁺/X4), and 2 in CXCR4-expressing cells (R5/X4⁺). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability.Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5⁺/X4-tropic isolates. In both pure-X4 and R5/X4⁺-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2months) showed no-emergence of X4-tropic variants for all R5- and R5⁺/X4-isolates tested (while X4-virus remained fully-resistant).In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5⁺/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5⁺/X4-tropic viruses may be a therapeutic-option that deserves further investigations.

Keywords: HIV-1 tropism; Maraviroc; Pyrosequencing; Phenotyping; Env mutations

A mammalian two-hybrid system-based assay for small-molecular HIV fusion inhibitors targeting gp41 by Xiaoxi Shui; Xinhua Lu; Yan Gao; Chen Liu; Fengzhi Ren; Qin Jiang; Hua Zhang; Baohua Zhao; Zhihui Zheng (pp. 54-63).

gp41 is a major component of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) responsible for fusion of the viral envelope with the target cellular membrane. The formation of the trimer-of-hairpins core structure of gp41, via the interaction between its N-terminal heptad repeat (NHR) and its C-terminal heptad repeat (CHR) plays a key role in the membrane fusion process. Hence, inhibitors of trimer-of-hairpins formation have become a promising new class of HIV therapeutics. In the present study, based on the mammalian two-hybrid system, we developed a cell-based assay for detecting small-molecular HIV-1 fusion inhibitors targeting gp41. The optimized assay can be adapted to high-throughput screening in 96- and 384-well microplates with high signal-to-background ratios and acceptable Z′ factors. The known small-molecular gp41 inhibitors, ADS-J1, XTT formazan and tannin acid, tested positive in this assay, with half-maximal inhibitory concentration (IC₅₀) values of 4.9μM, 5.6μM and 0.8μM, respectively. These data suggested that this novel assay is robust, sensitive and specific for identifying small-molecular HIV-1 gp41 inhibitors.

Keywords: HIV fusion inhibitor; gp41; Mammalian two-hybrid system; Small molecular compound

Emodin inhibits current through SARS-associated coronavirus 3a protein by Silvia Schwarz; Kai Wang; Wenjing Yu; Bing Sun; Wolfgang Schwarz (pp. 64-69).

The open-reading-frame 3a of SARS coronavirus (SARS-CoV) had been demonstrated previously to form a cation-selective channel that may become expressed in the infected cell and is then involved in virus release. Drugs that inhibit the ion channel formed by the 3a protein can be expected to inhibit virus release, and would be a source for the development of novel therapeutic agents. Here we demonstrate that emodin can inhibit the 3a ion channel of coronavirus SARS-CoV and HCoV-OC43 as well as virus release from HCoV-OC43 with a K_1/2 value of about 20μM. We suggest that viral ion channels, in general, may be a good target for the development of antiviral agents.

Keywords: 3a protein; Corona virus; Emodin; Voltage clamp; Ion channel; Virus release

Evaluation of Lassa antiviral compound ST-193 in a guinea pig model by Kathleen A. Cashman; Mark A. Smith; Nancy A. Twenhafel; Ryan A. Larson; Kevin F. Jones; Robert D. Allen III; Dongcheng Dai; Jarasvech Chinsangaram; Tove’ C. Bolken; Dennis E. Hruby; Sean M. Amberg; Lisa E. Hensley; Mary C. Guttieri (pp. 70-79).

Lassa virus (LASV), a member of the Arenaviridae family, causes a viral hemorrhagic fever endemic to West Africa, where as many as 300,000 infections occur per year. Presently, there are no FDA-approved LASV-specific vaccines or antiviral agents, although the antiviral drug ribavirin has shown some efficacy. A recently identified small-molecule inhibitor of arenavirus entry, ST-193, exhibits submicromolar antiviral activity in vitro. To determine the antiviral utility of ST-193 in vivo, we tested the efficacy of this compound in the LASV guinea pig model. Four groups of strain 13 guinea pigs were administered 25 or 80mg/kg ST-193, 25mg/kg of ribavirin, or the vehicle by the intraperitoneal (i.p.) route before infection with a lethal dose of LASV, strain Josiah, and continuing once daily for 14 days. Control animals exhibited severe disease, becoming moribund between days 10 and 15 postinfection. ST-193-treated animals exhibited fewer signs of disease and enhanced survival when compared to the ribavirin or vehicle groups. Body temperatures in all groups were elevated by day 9, but returned to normal by day 19 postinfection in the majority of ST-193-treated animals. ST-193 treatment mediated a 2–3-log reduction in viremia relative to vehicle-treated controls. The overall survival rate for the ST-193-treated guinea pigs was 62.5% (10/16) compared with 0% in the ribavirin (0/8) and vehicle (0/7) groups. These data suggest that ST-193 may serve as an improved candidate for the treatment of Lassa fever.

Keywords: Lassa virus; Arenavirus; Small molecule inhibitor; Fusion inhibitor; Glycoprotein; Guinea pig

Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs by Emily M. Kimmel; Maria Jerome; Jeff Holderness; Deann Snyder; Sharon Kemoli; Mark A. Jutila; Jodi F. Hedges (pp. 80-86).

Oligomeric procyanidins (OPCs) have been shown to have antiviral and immunostimulatory effects. OPCs isolated from non-ripe apple peel were tested for capacity to reduce dengue virus (DENV) titers. Similar to published accounts, OPCs exhibited direct antiviral activity. The possibility of enhanced innate immune protection was also tested by measuring and characterizing gene and protein expression induced by OPCs during DENV infection. Treatment of DENV-infected human PBMCs with OPCs decreased viral titers and affected the expression of critical innate antiviral immune products. OPCs enhanced expression of MXI and IFNB transcripts in high MOI DENV infected PBMC cultures, and phosphorylation of STAT2 in response to recombinant type I IFN (IFN I). During low MOI infection, addition of OPCs increased expression of STAT1 transcripts, MHC I and TNFα protein production. Thus, OPCs exhibited innate immune stimulation of cells in DENV-infected cultures and uninfected cells treated with IFN I. While OPCs from a number of sources are known to exhibit antiviral effects, their mechanisms are not precisely defined. The capacity of OPCs to increase sensitivity to IFN I could be broadly applicable to many viral infections and two separate antiviral mechanisms suggest that OPCs may represent a novel, robust antiviral therapy.

Keywords: Procyanidins; Dengue; PBMC; Interferon; Innate

A comparison of pyrosequencing and neuraminidase inhibition assays for the detection of oseltamivir-resistant pandemic influenza A(H1N1) 2009 viruses by Yi-Mo Deng; Natalie Caldwell; Aeron Hurt; Tim Shaw; Anne Kelso; Glenys Chidlow; Simon Williams; David Smith; Ian Barr (pp. 87-91).

Currently most pandemic influenza A(H1N1) 2009 (H1N1pdm) viruses are sensitive to oseltamivir, but a single point mutation (H275Y) in the neuraminidase (NA) gene of H1N1pdm can lead to resistance and such viruses have been reported from several countries. In this study we compare the performance of a pyrosequencing-based method for the detection of the H275Y mutation in H1N1pdm viruses with a conventional NA inhibition assay. Pyrosequencing could detect as little as 5% H275Y mutants in a mixed viral population, while mixtures with 25% or greater mutant virus were required before a significant increase in IC₅₀ value could be detected. However, the sensitivity of the NA inhibition assay could be enhanced by using a more sophisticated curve-fitting analysis to generate similar results to the pyrosequencing assay. Of 181 H1N1pdm clinical samples examined by pyrosequencing, nine samples from five patients were found to contain H275Y mutant viruses, four of whom were under oseltamivir treatment. Changes in the ratio of H275Y mutant to wild-type viruses were observed in serial clinical specimens from two patients over the duration of their treatment. This study highlights the need for close monitoring of the H275Y mutation in clinical samples, in particular from severely ill patients infected with H1N1pdm. The use of pyrosequencing and the NA inhibition assay provide powerful tools for the rapid detection and quantitation of resistant influenza viruses in mixed populations.

Keywords: Abbreviations; RT-PCR; reverse transcriptase polymerase chain reaction; WHO; World Health Organisation; RNA; ribonucleic acid; DNA; deoxyribonucleic acid; HA; hemagglutinin; NA; neuraminidase; H275Y; histidine to tyrosine substitution at position 275; NAI; neuraminidase inhibitor; H1N1pdm; pandemic influenza A(H1N1) 2009; ICU; intensive care unit; MDCK; Madin-Darby canine kidney; SD; standard deviation; LDR; log dose–responseOseltamivir resistance; H275Y mutation; Pandemic influenza A(H1N1) 2009 virus; Pyrosequencing; Neuraminidase inhibition assay

HCV RNA decline in the first 24h exhibits high negative predictive value of sustained virologic response in HIV/HCV genotype 1 co-infected patients treated with peginterferon and ribavirin by N. Laufer; F. Bolcic; M.J. Rolón; A. Martinez; R. Reynoso; H. Pérez; H. Salomón; P. Cahn; J. Quarleri (pp. 92-97).

Treatment with Peg-interferon and ribavirin (PEG-IFN/RBV) for HIV patients co-infected with hepatitis C virus (HCV) genotype 1 has suboptimal rates of response. Viral kinetics has emerged as one of the best prognostic factors of treatment outcome.Twenty HIV/HCV genotype 1 co-infected patients in treatment with PEG-IFN/RBV, had blood drawn at baseline, 24h, 4, 12, 24, 48, and 72 weeks. HCV-RNA levels were evaluated at each time point. ROC curves were used to evaluate the log₁₀HCV-RNA decay at 24h that exhibits the best predictive value of achieving response. Genomic characterization of HCV NS5A at both interferon sensitivity-determining region (ISDR) and protein-kinase binding (PKRBD) domains were performed in order to evaluate its heterogeneity and association with 24h HCV-RNA decay and SVR.Non-responder patients exhibited a mean of 0.7log₁₀ (SD 0.74log₁₀) HCV-RNA decay at 24h, whereas responder-patients presented 1.6log₁₀ (SD 0.28log₁₀), p=0.04. A reduction in HCV viral load from baseline to 24h of <1.4 had a negative predictive value for achieving SVR of 100% and a positive predictive value of 50%. HCV genotype 1 isolates from patients with a decrease of HCV-RNA at 24h >1.4log₁₀, exhibited 3.1(SD 1.5) amino acids substitutions in ISDR and 4.8(SD 2.3) in PKRBD regions and 1.6(SD 0.7) and 2.4(SD1.3), respectively, in those patients presenting lower reduction in HCV-RNA.HIV/HCV genotype 1 co-infected patients with a decrease in HCV-VL at 24h >1.4log₁₀ are more likely to achieve SVR when treated with PEG-IFN/RBV than those with lower levels of HCV-RNA decay. Along with other host-related and viral-related prognostic factors in HIV/HCV co-infected patients, this very early time point of evaluation could be of relevance in the management of HCV-specific treatment.

Keywords: 24; h; Viral kinetics; SVR; HIV/HCV co-infection; HCV treatment; NS5A

Potential of adenovirus and baculovirus vectors for the delivery of shRNA against morbilliviruses by Zaheer Ahmed Nizamani; Gunther M. Keil; Emmanuel Albina; Carine Holz; Cécile Minet; Olivier Kwiatek; Geneviève Libeau; Renata Servan de Almeida (pp. 98-101).

Morbilliviruses are important pathogens of humans, ruminants, carnivores and marine mammals. Although good vaccines inducing long-term immunity are available, recurrent outbreaks of measles, canine distemper and peste des petits ruminants (PPR) are observed. In control strategies, antivirals thus could be useful to confine virus spread and application of interfering RNAs is a promising approach, provided they can be delivered efficiently into the host cells. We have constructed recombinant adenovirus and baculovirus vectors expressing short hairpin RNAs (shRNAs) against the PPR virus (PPRV) and compared them in vitro. It was found that both recombinant viruses inhibited PPRV replication with the baculovirus vector, which inhibited generation of infectious progeny by more than 2log₁₀ and the nucleoprotein expression of PPRV by 73%, being the more efficient. The results show that baculoviral shRNA-expressing vectors have the potential for therapeutic use against morbillivirus infections.

Keywords: RNAi; Morbillivirus; Adenovirus; Baculovirus; PPRV

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Antiviral Research (v.90, #1)