Check out our New Publishers' Select for Free Articles

Antiviral Research (v.87, #2)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

The VIZIER project: Overview; expectations; and achievements by Bruno Coutard; Bruno Canard (pp. 85-94).

VIZIER is an acronym for a research project entitled “Comparative Structural Genomics of Viral Enzymes Involved in Replication” funded by the European Commission between November 1st, 2004 and April 30th, 2009. It involved 25 partners from 12 countries. In this paper, we describe the organization of the project and the culture created by its multidisciplinary essence. We discuss the main thematic sections of the project and the strategy adopted to optimize the integration of various scientific fields into a common objective: to obtain crystal structures of the widest variety of RNA virus replication enzymes documented and validated as potential drug targets. We discuss the thematic sections and their overall organization, their successes and bottlenecks around the protein production pipeline, the “low hanging fruit” strategy, and measures directed to problem solving. We discuss possible future options for such large-scale projects in the area of antiviral drug design. In a series of accompanying papers in Antiviral Research, the project and its achievements are presented for each virus family.

Keywords: Viral structural genomics; Crystal structure; Emerging virus; RNA virus; Antiviral; Replication

Practical application of bioinformatics by the multidisciplinary VIZIER consortium by Alexander E. Gorbalenya; Philippe Lieutaud; Mark R. Harris; Bruno Coutard; Bruno Canard; Gerard J. Kleywegt; Alexander A. Kravchenko; Dmitry V. Samborskiy; Igor A. Sidorov; Andrey M. Leontovich; T. Alwyn Jones (pp. 95-110).

This review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960, active from 1 November 2004 to 30 April 2009), to achieve its goals. From the management of the information flow of the project, to bioinformatics-mediated selection of RNA viruses and prediction of protein targets, to the analysis of 3D protein structures and antiviral compounds, these technologies provided a communication framework and integrated solutions for steady and timely advancement of the project. RNA viruses form a large class of major pathogens that affect humans and domestic animals. Such RNA viruses as HIV, Influenza virus and Hepatitis C virus are of prime medical concern today, but the identities of viruses that will threaten human population tomorrow are far from certain. To contain outbreaks of common or newly emerging infections, prototype drugs against viruses representing the Virus Universe must be developed. This concept was championed by the VIZIER project which brought together experts in diverse fields to produce a concerted and sustained effort for identifying and validating targets for antivirus therapy in dozens of RNA virus lineages.

Keywords: VIZIER; VaZyMolO; VirAliS; Xtrack; EDBase

Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control by E.A. Gould; B. Coutard; H. Malet; B. Morin; S. Jamal; S. Weaver; A. Gorbalenya; G. Moureau; C. Baronti; I. Delogu; N. Forrester; M. Khasnatinov; T. Gritsun; X. de Lamballerie; B. Canard (pp. 111-124).

The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project—VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37–46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.

Keywords: Alphavirus; Genomics; Structure/function studies; Replicative enzymes; Antivirals; Evolution; Classification; Biogeography; VIZIER

Structure and functionality in flavivirus NS-proteins: Perspectives for drug design by Michela Bollati; Karin Alvarez; René Assenberg; Cécile Baronti; Bruno Canard; Shelley Cook; Bruno Coutard; Etienne Decroly; Xavier de Lamballerie; Ernest A. Gould; Gilda Grard; Jonathan M. Grimes; Rolf Hilgenfeld; Anna M. Jansson; Hélène Malet; Erika J. Mancini; Eloise Mastrangelo; Andrea Mattevi; Mario Milani; Grégory Moureau; Johan Neyts; Raymond J. Owens; Jingshan Ren; Barbara Selisko; Silvia Speroni; Holger Steuber; David I. Stuart; Torsten Unge; Martino Bolognesi (pp. 125-148).

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Keywords: Abbreviations; BVDV; bovine viral diarrhea virus; C; capsid protein; CSFV; classical swine fever virus; CCHFV; Crimean-Congo hemorrhagic fever virus; CPE; cyto-pathogenic effect; dsRNA; double-stranded RNA; ER; endoplasmic reticulum; E; envelope protein; GMP; guanosine monophosphate; GTP; guanosine triphosphate; GTase; guanylyltransferase; NS3Hel; helicase; HIV; Human Immunodeficiency Virus I; HCV; hepatitis C virus; HBS; high affinity binding site; IMP; Inosine 5′-monophosphate; LBS; low-affinity binding site; M; membrane protein; NS5MTase; methyltransferase; N7MTase; (guanine-N7)-methyltransferase; 2′OMTase; (nucleoside-2′-O-)-methyltransferase; NS; non-structural; NLS; nuclear localization sequences; NS3Pro; protease; RC; replication-competent complex; RSV; respiratory syncytial virus; NS5RdRp; RNA-dependent RNA polymerase; NS3RTPase; RNA triphosphatase; AdoMet; S-adenosyl-L-methionine; ssRNA; single-stranded RNA; T-705 RMP; T-705-ribofuranosyl-5′-monophosphate; VIZIER; Viral Enzymes Involved in ReplicationFlavivirus; Flaviviral NS3 protein; Flaviviral NS5 protein; Protease; Helicase; Polymerase; Methyltransferase; Flavivirus protein structure; Antivirals; VIZIER Consortium

Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription by R. Assenberg; O. Delmas; B. Morin; S.C. Graham; X. De Lamballerie; C. Laubert; B. Coutard; J.M. Grimes; J. Neyts; R.J. Owens; B.W. Brandt; A. Gorbalenya; P. Tucker; D.I. Stuart; B. Canard; H. Bourhy (pp. 149-161).

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

Keywords: Rhabdovirus; Viral replication; Viral evolution; RNA viruses; Mononegavirales; Antiviral therapy

Antiviral strategies to control calicivirus infections by Jacques Rohayem; Mirko Bergmann; Julia Gebhardt; Ernest Gould; Paul Tucker; Andrea Mattevi; Torsten Unge; Rolf Hilgenfeld; Johan Neyts (pp. 162-178).

Caliciviridae are human or non-human pathogenic viruses with a high diversity. Some members of the Caliciviridae, i.e. human pathogenic norovirus or rabbit hemorrhagic disease virus (RHDV), are worldwide emerging pathogens. The norovirus is the major cause of viral gastroenteritis worldwide, accounting for about 85% of the outbreaks in Europe between 1995 and 2000. In the United States, 25 million cases of infection are reported each year. Since its emergence in 1984 as an agent of fatal hemorrhagic diseases in rabbits, RHDV has killed millions of rabbits and has been dispersed to all of the inhabitable continents.In view of their successful and apparently increasing emergence, the development of antiviral strategies to control infections due to these viral pathogens has now become an important issue in medicine and veterinary medicine. Antiviral strategies have to be based on an understanding of the epidemiology, transmission, clinical symptoms, viral replication and immunity to infection resulting from infection by these viruses. Here, we provide an overview of the mechanisms underlying calicivirus infection, focusing on the molecular aspects of replication in the host cell. Recent experimental data generated through an international collaboration on structural biology, virology and drug design within the European consortium VIZIER is also presented. Based on this analysis, we propose antiviral strategies that may significantly impact on the epidemiological characteristics of these highly successful viral pathogens.

Keywords: Calicivirus; Antiviral therapy; Viral enzymes; VIZIER

Characterization of HIV-1 resistance to a fusion inhibitor, N36, derived from the gp41 amino-terminal heptad repeat by Kazuki Izumi; Shota Nakamura; Hiroaki Nakano; Kazuya Shimura; Yasuko Sakagami; Shinya Oishi; Susumu Uchiyama; Tadayasu Ohkubo; Yuji Kobayashi; Nobutaka Fujii; Masao Matsuoka; Eiichi N. Kodama (pp. 179-186).

A transmembrane glycoprotein of HIV-1, gp41, plays a central role in membrane fusion of HIV-1 and host cells. Peptides derived from the amino- and carboxyl-terminal heptad repeat (N-HR and C-HR, respectively) of gp41 inhibit this fusion. The mechanism of resistance to enfuvirtide, a C-HR-derived peptide, is well defined; however the mechanism of resistance to N-HR-derived peptides remains unclear. We characterized an HIV-1 isolate resistant to the N-HR-derived peptide, N36. This HIV-1 acquired a total of four amino acid substitutions, D36G, N126K and E137Q in gp41, and P183Q in gp120. Among these substitutions, N126K and/or E137Q conferred resistance to not only N36, but also C34, which is the corresponding C-HR-derived peptide fusion inhibitor. We performed crystallographic and biochemical analysis of the 6-helix bundle formed by synthetic gp41-derived peptides containing the N126K/E137Q substitutions. The structure of the 6-helix bundle with N126K/E137Q was identical to that in wild-type HIV-1 except for the presence of a new hydrogen bond. Denaturing experiments revealed that the stability of the 6-helix bundle of N126K/E137Q is greater than in the wild-type. These results suggest that the stabilizing effect of N126K/E137Q provides resistance to N36 and C34.

Keywords: HIV-1; Fusion; Amino-terminus; gp41; Resistance

Inhibition of heat-shock protein 90 reduces Ebola virus replication by Darci R. Smith; Sarah McCarthy; Andrew Chrovian; Gene Olinger; Andrea Stossel; Thomas W. Geisbert; Lisa E. Hensley; John H. Connor (pp. 187-194).

Ebola virus (EBOV), a negative-sense RNA virus in the family Filoviridae, is known to cause severe hemorrhagic fever in humans and other primates. Infection with EBOV causes a high mortality rate and currently there is no FDA-licensed vaccine or therapeutic treatment available. Recently, heat-shock protein 90 (Hsp90), a molecular chaperone, was shown to be an important host factor for the replication of several negative-strand viruses. We tested the effect of several different Hsp90 inhibitors including geldanamycin, radicicol, and 17-allylamino-17-demethoxygeldanamycin (17-AAG; a geldanamycin analog) on the replication of Zaire EBOV. Our results showed that inhibition of Hsp90 significantly reduced the replication of EBOV. Classic Hsp90 inhibitors reduced viral replication with an effective concentration at 50% (EC₅₀) in the high nanomolar to low micromolar range, while drugs from a new class of Hsp90 inhibitors showed markedly more potent inhibition. These compounds blocked EBOV replication with an EC₅₀ in the low nanomolar range and showed significant potency in blocking replication in primary human monocytes. These results validated that Hsp90 is an important host factor for the replication of filoviruses and suggest that Hsp90 inhibitors may be therapeutically effective in treating EBOV infection.

Keywords: Ebola virus; Hsp90; Therapeutic

Treatment with cationic liposome–DNA complexes (CLDCs) protects mice from lethal Western equine encephalitis virus (WEEV) challenge by Christopher H. Logue; Aaron T. Phillips; Eric C. Mossel; Jeremy P. Ledermann; Thomas Welte; Steve W. Dow; Ken E. Olson; Ann M. Powers (pp. 195-203).

Having recently characterized a CD-1 outbred mouse model of pathogenesis for Western equine encephalitis virus, we examined the possible protective effects of cationic liposome–DNA complexes (CLDCs) against encephalitic arboviral infection. In this investigation, mice were pre-treated, co-treated, or post-treated with CLDC then challenged with a subcutaneous or aerosol dose of the highly virulent WEEV-McMillan strain, lethal in mice 4–5 days after inoculation. Pre-treatment with CLDCs provided a significant protective effect in mice, which was reflected in significantly increased survival rates. Further, in some instances a therapeutic effect of CLDC administration up to 12h after WEEV challenge was observed. Mice treated with CLDC had significantly increased serum IFN-γ, TNF-α, and IL-12, suggesting a strong Th1-biased antiviral activation of the innate immune system. In virus-infected animals, large increases in production of IFN-γ, TNF-α, MCP-1, IL-12, and IL-10 in the brain were observed by 72h after infection, consistent with neuroinvasion and viral replication in the CNS. These results indicate that strong non-specific activation of innate immunity with an immune therapeutic such as CLDC is capable of eliciting significant protective immunity against a rapidly lethal strain of WEEV and suggest a possible prophylactic option for exposed individuals.

Keywords: Alphavirus; WEEV; CLDC; Immunotherapy

Potent inhibition of influenza virus replication with novel siRNA-chimeric-ribozyme constructs by Prashant Kumar; Vikas Sood; Rajesh Vyas; Nidhi Gupta; Akhil C. Banerjea; Madhu Khanna (pp. 204-212).

A multitarget approach is needed for effective gene silencing for RNA viruses that combines, more than one antiviral approach. Towards this end, we designed a wild-type (wt) chimeric construct, that consisted of small hairpin siRNA joined by a short intracellular cleavable linker to a known, hammerhead ribozyme (Rz), both targeted against M1 genome segment of influenza A virus. When this, wt chimeric RNA construct was introduced into a mammalian cell line, along with the M1 substrate, encoding DNA, very significant (67%) intracellular down regulation in the levels of target RNA was, observed. When the siRNA portion of this chimeric construct was mutated keeping the Rz region, unchanged, it caused only 33% intracellular reduction. On the contrary, when only the Rz was made, catalytically inactive, keeping the siRNA component unchanged, about 20% reduction in the target M1, specific RNA was observed. This wt chimeric construct showed impressive (>80%) protection against, virus challenge, on the other hand, the selectively disabled mutant constructs were less effective. Thus, in this proof of concept study we show that varying levels of protection against virus challenge was, observed with novel mutant versions of the chimeric constructs.

Keywords: Hammerhead Rz; siRNA; M1; Influenza A; Chimeric RNA; Mutants

Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-d-2′-ethynyl-7-deaza-adenosine triphosphate by Derek R. Latour; Andreas Jekle; Hassan Javanbakht; Robert Henningsen; Peter Gee; Ina Lee; Patricia Tran; Suping Ren; Alan K. Kutach; Seth F. Harris; Sandra M. Wang; Stephen J. Lok; David Shaw; Jim Li; Gabrielle Heilek; Klaus Klumpp; David C. Swinney; Jerome Deval (pp. 213-222).

Dengue virus (DENV), an emerging pathogen from the Flaviviridae family with neither vaccine nor antiviral treatment available, causes a serious worldwide public health threat. In theory, there are several ways by which small molecules could inhibit the replication cycle of DENV. Here, we show that the nucleoside analogue beta-d-2′-ethynyl-7-deaza-adenosine inhibits representative strains of all four serotypes of DENV with an EC₅₀ around or below 1μM. Using membrane-associated native replicase complex as well as recombinant RNA polymerase from each DENV serotype in enzymatic assays, we provide evidence that beta-d-2′-ethynyl-7-deaza-adenosine triphosphate (2′E-7D-ATP) targets viral replication at the polymerase active site by competing with the natural nucleotide substrate with an apparent K_i of 0.060±0.016μM. In single-nucleotide incorporation experiments, the catalytic efficiency of 2′E-7D-ATP is 10-fold lower than for natural ATP, and the incorporated nucleotide analogue causes immediate chain termination. A combination of bioinformatics and site-directed mutagenesis demonstrates that 2′E-7D-ATP is equipotent across all serotypes because the nucleotide binding site residues are conserved in dengue virus. Overall, beta-d-2′-ethynyl-7-deaza-adenosine provides a promising scaffold for the development of inhibitors of dengue virus polymerase.

Keywords: Abbreviations; AMP; adenosine monophosphate; ATP; adenosine triphosphate; BVDV; Bovine viral diarrhea virus; CPE; cytopathic effect; DENV; dengue virus; HCV; hepatitis C virus; IU; international unit; MTase; methyltransferase; NS5; non-structural protein 5; NS5pol; polymerase domain of NS5; FL; full length; NGC; new Guinea C; ssRNA; single-stranded RNA; YFV; yellow fever virus; WNV; West Nile virus; 2′E-7D-ATP; beta-; d; -2′-ethynyl-7-deaza-adenosine triphosphateDengue virus; NS5 polymerase; Nucleotide; Chain terminator; Single-nucleotide incorporation; Steady-state kinetics

A single amino acid in the C-terminus of VP3 protein influences the replication of attenuated infectious bursal disease virus in vitro and in vivo by Yongqiang Wang; Xiaole Qi; Zhonghui Kang; Fei Yu; Liting Qin; Honglei Gao; Yulong Gao; Xiaomei Wang (pp. 223-229).

The very virulent infectious bursal disease virus (vvIBDV) Gx strain causes over 60% mortality in chickens but cannot replicate in CEF cultures. The attenuated Gt strain, however, is not virulent in chickens and replicates well in CEF cultures. The two strains display differences in 6 amino acids in VP4 and 4 amino acids in VP3. To determine whether VP4 and VP3 are involved in the virulence and replication of IBDV, three chimeric viruses, in which the VP4/VP3/3′UTR, VP3/3′UTR or VP4 region of Gt were replaced by the corresponding region of Gx, were constructed and characterized in vitro and in vivo. The substituted regions in VP4 or VP3 did not affect virulence of Gt. While the substituted region in VP4 had no effect on viral replication of Gt in CEF cultures, substitution of the VP3/3′UTR region did reduce the replicative capacity of the virus. Through site-directed mutagenesis, three rescued recombinant viruses with a single amino acid substitution in the C-terminus of VP3 of the Gt strain (L981P, A990V and T1005A) were characterized in a similar manner. Amino acid substitution at position 990 reduced viral replication of Gt and reduced its efficacy of protection against vvIBDV Gx challenge in vivo. This study provides important information for the design and development of more effective IBDV vaccines using reverse genetics.

Keywords: Infectious bursal disease virus; Reverse genetic system; VP3; Viral replication

Use of preconditioned human phagocytes for extracorporeal adsorption of viruses by Felix Oberender; Heiderose Oberender; Jens Altrichter; Emil C. Reisinger; Steffen R. Mitzner (pp. 230-234).

Conventional treatment of severe viral disease is limited by the narrow choice as well as the often-significant side effects or lack of clear efficacy of antiviral chemotherapy. At the same time, however, it is known that a reduction in viral load leads to significant clinical improvement in a number of important viral diseases. In this paper we discuss the possibility of using preconditioned human phagocytes in an extracorporeal biohybrid system for adsorption of viral pathogens. We present data from in vitro experiments testing adsorption of an enterovirus and of hepatitis B virus (HBV) by a preconditioned human promyelocytic cell line. While no clearance of HBV could be detected, the results revealed a near elimination of enterovirus with the cell line displaying robust viability. Enterovirus titers of 1000 (reciprocal) were reduced to a mean titer of 10^0.6 CCID₅₀ with no virus detectable after adsorption in two out of five samples. Titers of 10000 (reciprocal) were in turn reduced to a mean of 10^1.4 CCID₅₀. The kinetics of the process was remarkable with this near elimination of the pathogen occurring within only 15min. Extracorporeal viral adsorption by a cellular biohybrid system appears feasible. Pairing target pathogens with suitable cell lines may offer a versatile antiviral technology.

Keywords: Extracorporeal; Biohybrid; Bioreactor; Leucocytes; HL-60; Viremia; Enterovirus; HBV

Enhancement of the immunogenicity of an infectious laryngotracheitis virus DNA vaccine by a bicistronic plasmid encoding glycoprotein B and interleukin-18 by Hong-Ying Chen; Li Zhao; Zhan-Yong Wei; Bao-An Cui; Zhen-Ya Wang; Xin-Sheng Li; Ping-An Xia; Jin-Peng Liu (pp. 235-241).

A DNA vaccine against infectious laryngotracheitis virus (ILTV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To determine if co-expression of a cytokine can result in a more potent ILTV DNA vaccine, immunogenicity and protective efficacy of a monocistronic vector encoding the glycoprotein B (gB) of ILTV was compared to that of a bicistronic vector separately encoding the gB and chicken interleukin-18.Humoral and cellular responses induced by the DNA vaccines administered to the quadriceps muscle of chickens were evaluated. There were significant differences in antibody levels elicited by either monocistronic or bicistronic DNA vaccines as determined by ELISA. The percentages of CD3⁺, CD3⁺CD8⁺ and CD3⁺CD4⁺ subgroups of peripheral blood T-lymphocytes in chickens immunized with the bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent CG strain of ILTV, the protective efficacy was enhanced significantly after immunization with the bicistronic DNA vaccine. These results demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing ILTV DNA vaccine immunogenicity.

Keywords: Chicken interleukin-18; DNA vaccine; Eukaryotic expression; Infectious laryngotracheitis virus

Oseltamivir-resistant influenza A(H1N1) viruses in south of France, 2007/2009 by Jean-Sébastien Casalegno; Maude Bouscambert-Duchamp; Valérie Caro; Isabelle Schuffenecker; Murielle Sabatier; Aurélien Traversier; Martine Valette; Bruno Lina; Olivier Ferraris; Vanessa Escuret (pp. 242-248).

Influenza A(H1N1) viruses resistant to oseltamivir carboxylate (OC) emerged in 2007/2008 in the absence of antiviral pressure. These OC-resistant A(H1N1) viruses had a better fitness than the sensitive ones as they were 100% prevalent in 2008/2009.To better understand the role of the neuraminidase (NA) affinity in the emergence of these OC-resistant A(H1N1) viruses we compared the NA properties among A(H1N1) clinical isolates in south of France between 2005 and 2009 and reference strains from 1977 to 2007, using NA inhibition assays, kinetic analyses of NA activities, and sequence analysis of viral NA and hemagglutinin (HA).In 2007/2008, among 374 A(H1N1) isolates tested, 38% were resistant to OC with a mean IC₅₀ of 564±357nM. The mean Km of OC-sensitive isolates (H275) was significantly lower (22.6±4.7μM) than the Km of previous reference strains (44.9±5μM) and the mean Km of the OC-resistant isolates (Y275) (37.2±7.7μM). The combination of different amino acid mutations in N1 particularly the D344N could explain the higher NA affinity of A/Brisbane/59/2007 related variants compared to the previous A(H1N1) strains and the H275Y mutation allowed to retrieve Km values near 40μM.

Keywords: A(H1N1) influenza viruses; Resistance to oseltamivir; H275Y mutation; Kinetic neuraminidase parameters

Modulation of miR-122 on persistently Borna disease virus infected human oligodendroglial cells by Jun Qian; Aixia Zhai; Wenping Kao; Yujun Li; Wuqi Song; Yingmei Fu; Xiaobei Chen; Qingmeng Zhang; Jing Wu; Hui Li; Zhaohua Zhong; Hong Ling; Fengmin Zhang (pp. 249-256).

Using RNAhybrid software we found the predicted binding of complementary sequences between miR-122 and viral mRNAs, may be important for the antiviral effect of miR-122 on Borna disease virus (BDV). A moderate expression of miR-122 was identified in human oligodendroglial cells (OL), but with a much lower level of miR-122 in BDV persistent infection (OL/BDV) and cells transfected with BDV gene expression vectors. Over-expression of miR-122 and specific blocking experiments demonstrated that miR-122 was able to specifically inhibit BDV protein synthesis, viral gene replication and transcription, and induce the secretion/synthesis of interferon (IFN) in OL and OL/BDV cells. The abolishment of miR-122 by AMO-122 inhibited endogenous IFN induction by IFN-beta. These results indicate that miR-122 can exert direct antiviral function by inhibiting BDV translation and replication on one hand, while acting indirectly through IFN to increase the host innate immunity to modulate the virus–host interactions on the other hand.

Keywords: microRNA-122; Borna disease virus; Human oligodendroglial cells; Interferon; Persistent infection

In vitro antiviral activity of single domain antibody fragments against poliovirus by Bert Thys; Lise Schotte; Serge Muyldermans; Ulrich Wernery; Gholamreza Hassanzadeh-Ghassabeh; Bart Rombaut (pp. 257-264).

VHHs or Nanobodies^® are single-domain antigen-binding fragments derived from heavy chain antibodies found in camelids. It has already been shown that complex protein mixtures and even whole organisms elicit good immune responses in camelids; therefore we hypothesized that VHHs selected from a dromedary immunized with poliovirus type 1 might inhibit the in vitro replication of poliovirus through binding to essential biological sites on the viral capsid. In this study, we aimed to determine whether VHHs inhibit wild-type and vaccine strains of poliovirus type 1. Interestingly, VHHs showed a potent antipolio activity with EC₅₀ values in the low nanomolar range. Moreover, these antibody fragments completely blocked viral multiplication at higher concentrations. Remarkably, no (immune) escape variants against some of these VHHs could be generated. In conclusion, VHHs fulfil several in vitro requirements to be assigned as potential antiviral compounds for further development of an anti-poliovirus drugs.

Keywords: Abbreviations; VHH; variable part of the heavy chain of a heavy chain antibody; mAb; monoclonal antibody; CDR; complementarity determining regionAnti-poliovirus compound; Poliovirus; Antiviral; VHH; Nanobody

Transgenic mouse model integrating siRNA targeting the foot and mouth disease virus by Wang Pengyan; Jiang Jianjun; Li Ning; Sheng Jinliang; Ren Yan; Chen Chuangfu; Guo Zhiru (pp. 265-268).

We have constructed 2 small interfering RNAs (siRNAs) specifically targeting homogenous 3D and 2B1 regions of 7 serotypes of the foot and mouth disease virus (FMDV) and tested the ability of siRNAs to inhibit virus replication in baby hamster kidney (BHK-21) cells and suckling mice. In this study, we generated transgenic mouse models integrating short hairpin RNA (shRNA) targeting microinfected FMDV. When examined at the 7th passage in transgenic mice, the target gene was still found by PCR to be integrated in the genome. Compared to the control mice, the transgenic mice showed only slightly abnormal pathology when they were infected with the FMDV serotype Asia 1. The number of viruses in the tissues of the transgenic mouse was very low and in some tissues no virus could be detected by immunohistochemistry.

Keywords: Foot and mouth disease virus (FMDV); Short hairpin RNA (shRNA); Transgenic mouse models; Immunohistochemistry

Macrophage HIV-1 infection in duodenal tissue of patients on long term HAART by Alberto Zalar; Maria Ines Figueroa; Beatriz Ruibal-Ares; Patricia Baré; Pedro Cahn; Maria Marta de E. de Bracco; Liliana Belmonte (pp. 269-271).

Mucosal surfaces play a major role in human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis. Since the role of intestinal macrophages as viral reservoirs during chronic HIV-1 infection has not been elucidated, we investigated the effects of successful therapy on intestinal HIV-1 persistence. Intestinal macrophage infection was demonstrated by the expression of p24 antigen by flow cytometry and by the presence of proviral DNA, assessed by PCR. Proviral DNA was detected in duodenal mucosa of HIV-infected patients under treatment with undetectable plasma viral load. These findings confirm that intestinal macrophages can act as viral reservoirs and permit HIV-1 production even after viral suppression following antiretroviral therapy.

Keywords: Intestinal macrophages; HIV-1; HAART; Tissue reservoirs

The resistance mutation R155K in the NS3/4A protease of hepatitis C virus also leads the virus to escape from HLA-A*68-restricted CD8 T cells by Shadi Salloum; Silvia F. Kluge; Arthur Y. Kim; Michael Roggendorf; Joerg Timm (pp. 272-275).

The NS3/4A serine protease of the hepatitis C virus (HCV) is one of the most attractive targets for specific antiviral agents. However, mutations conferring resistance may decrease the efficacy of these drugs. Although the level of resistance associated with specific mutations differs between different compounds, substitutions R155K and A156T reduce susceptibility to all protease inhibitors published so far. Interestingly, variants harboring the resistant mutation R155K were also detected as the predominant quasispecies in some treatment-naïve patients. Of note, key positions for resistance overlap with the HLA-A*68-restricted epitope HAVGIFRAAV_1175–1184. The aim of our study was to analyze the impact of protease inhibitor resistance mutations on the replication level and the antiviral CD8 T cell response against this HCV epitope. Our findings suggest that the R155K variant is associated with a relatively high replication level and with a substantial loss of cross-recognition by specific CD8 T cells targeting the epitope HAVGIFRAAV_1175–1184, providing a possible explanation for its existence in the absence of drug selection pressure.

Keywords: CD8 T cell; Immune escape; Drug resistance; STAT-C; Protease inhibitor; BILN 2061

Baculovirus treatment fully protects mice against a lethal challenge of FMDV by Paula Molinari; Soledad García-Nuñez; M. José Gravisaco; Elisa Carrillo; Analía Berinstein; Oscar Taboga (pp. 276-279).

Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease that affects cattle, swine, goat and sheep among others. FMDV is able to overcome the initial host innate immune response by inhibiting the induction of antiviral molecules at both the transcriptional and the translational levels. It has been demonstrated that FMDV A/Arg/2001 causes the death of adult C57Bl/6 mice within 72h. We evaluated the capacity of Autographa californica nuclear polyhedrosis virus (AcNPV), an insect virus with potent innate immunostimulating effects, to promote early protection against FMDV A/Arg/2001 challenge in C57Bl/6 mice. Groups of 8–9 weeks old female mice were injected intravenously with AcNPV and challenged with a lethal dose of FMDV at different times post-administration. Our results showed that pretreatment of mice with a single injection of AcNPV 3h or 3 days before FMDV challenge resulted in complete abrogation of mortality and complete or partial suppression of viremia, respectively. Furthermore, no signs of disease were observed. AcNPV could be a valuable tool to improve the design of a novel vaccine that protects as an adjuvant at early times post-vaccination.

Keywords: Baculovirus; FMDV; Mice

Sections

Personal tools

Antiviral Research (v.87, #2)

Sections

Personal tools

Local resources

Antiviral Research (v.87, #2)