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Antiviral Research (v.97, #2)

Prelim II: Editorial Board (pp. ii).

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

Prelim II: Editorial Board (pp. ii).

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

Targeting virulence mechanisms for the prevention and therapy of arenaviral hemorrhagic fever by Lisa McLay; Aftab Ansari; Yuying Liang; Hinh Ly (pp. 81-92).

► Lassa virus and the South American arenaviruses cause severe hemorrhagic fever (HF). ► Arenaviral HF is characterized by high viremia and immunosuppression. ► Arenaviruses encode four gene products: GPC, L polymerase, NP and the Z matrix protein.A number of arenaviruses are pathogenic for humans, but they differ significantly in virulence. Lassa virus, found in West Africa, causes severe hemorrhagic fever (HF), while the other principal Old World arenavirus, lymphocytic choriomeningitis virus, causes mild illness in persons with normal immune function, and poses a threat only to immunocompromised individuals. The New World agents, including Junin, Machupo and Sabia virus, are highly pathogenic for humans. Arenaviral HF is characterized by high viremia and general immune suppression, the mechanism of which is unknown. Studies using viral reverse genetics, cell-based assays, animal models and human genome-wide association analysis have revealed potential mechanisms by which arenaviruses cause severe disease in humans. Each of the four viral gene products (GPC, L polymerase, NP, and Z matrix protein) and several host-cell factors (e.g., α-dystroglycan) are responsible for mediating viral entry, genome replication, and the inhibition of apoptosis, translation and interferon-beta (IFNβ) production. This review summarizes current knowledge of the role of each viral protein and host factor in the pathogenesis of arenaviral HF. Insights from recent studies are being exploited for the development of novel therapies.

Keywords: Arenavirus; Lassa virus; Viral hemorrhagic fever; Virulence mechanisms; Antiviral therapy

Targeting virulence mechanisms for the prevention and therapy of arenaviral hemorrhagic fever by Lisa McLay; Aftab Ansari; Yuying Liang; Hinh Ly (pp. 81-92).

Keywords: Arenavirus; Lassa virus; Viral hemorrhagic fever; Virulence mechanisms; Antiviral therapy

The amino acid substitutions rtP177G and rtF249A in the reverse transcriptase domain of hepatitis B virus polymerase reduce the susceptibility to tenofovir by Bo Qin; Bettina Budeus; Liang Cao; Chunchen Wu; Yun Wang; Xiaoyong Zhang; Simon Rayner; Daniel Hoffmann; Mengji Lu; Xinwen Chen (pp. 93-100).

► Potential HBV TDF-resistant sites were selected based on bioinformatics analysis. ► Replication capacity and resistance phenotype were analyzed in vitro and in vivo. ► Two HBV mutants (rtP177G and rtF249A) showed enhanced resistance to TDF. ► The resistant mechanisms are discussed based on the RTs structural comparison.Long term antiviral therapy with nucleoside/nucleotide analogs have been routinely used to treat chronic hepatitis B virus (HBV) infection but may lead to the emergence of drug-resistant viral mutants. However, the HBV resistance mutations for tenofovir (TDF) remain controversial. It is speculated that the genetic barrier for TDF resistance may be high for HBV. We asked whether selected amino acid substitutions in HBV polymerase may reduce susceptibility to TDF. A series of amino acids in HBV polymerase were selected based on bioinformatics analysis for mutagenesis. The replication competence and susceptibility to TDF of the mutated HBV clones were determined both in vitro and in vivo. nineteen mutations in HBV polymerase were included and impaired the replication competence of HBV genome in different degrees. The mutations at rtL77F (sS69C), rtF88L (sF80Y), and rtP177G (sR169G) also significantly affected HBsAg expression. The HBV mutants with rtP177G and rtF249A were found to have reduced susceptibility to TDF in vitro with a resistance index of 2.53 and 12.16, respectively. The testing in in vivo model based on the hydrodynamic injection revealed the antiviral effect of TDF against wild type and mutated HBV genomes and confirmed the reduced the susceptibility of mutant HBV to TDF.

Keywords: Hepatitis B virus; Drug resistance; Tenofovir; Nucleoside/nucleotide analogs; Replication competence; Hydrodynamic injection mouse model

SOCS1 abrogates IFN’s antiviral effect on hepatitis C virus replication by Run-Xuan Shao; Leiliang Zhang; Zhi Hong; Kaku Goto; Du Cheng; Wen-Chi Chen; Nikolaus Jilg; Kattareeya Kumthip; Dahlene N. Fusco; Lee F. Peng; Raymond T. Chung (pp. 101-107).

► Silencing of SOCS1 by shRNA increased the anti-HCV effect of IFN. ► Overexpression of SOCS1 prior to IFN treatment inhibited JAK-STAT signaling pathway. ► SOCS1 negatively regulates IFN signaling during HCV treatment.Suppressor of cytokine signaling 1 (SOCS1) and suppressor of cytokine signaling 3 (SOCS3) have been thought to block type I interferon (IFN) signaling. We have previously reported that SOCS3 suppresses HCV replication in an mTOR-dependent manner. However, the relationship between SOCS1 and HCV replication remains unclear. Here, we found that overexpression of SOCS1 alone did not have an effect on HCV RNA replication. However, suppression of HCV replication by IFN-α was rescued by SOCS1 overexpression. The upregulation of HCV replication by SOCS1 overexpression in the presence of IFN is likely a result of the impairment of IFN signaling by SOCS1 and subsequent induction of ISGs. Knockdown of SOCS1 alone with specific shRNA enhanced the antiviral effect of IFN compared with negative control. Thus, SOCS1 acts as a suppressor of type I IFN function against HCV.

Keywords: Hepatitis C virus; SOCS1; Interferon; ISG; JFH1

Catheterized guinea pigs infected with Ebola Zaire virus allows safer sequential sampling to determine the pharmacokinetic profile of a phosphatidylserine-targeting monoclonal antibody by Stuart Dowall; Irene Taylor; Paul Yeates; Leonie Smith; Antony Rule; Linda Easterbrook; Christine Bruce; Nicola Cook; Kara Corbin-Lickfett; Cyril Empig; Kyle Schlunegger; Victoria Graham; Mike Dennis; Roger Hewson (pp. 108-111).

► Catheterized guinea pigs can be used successfully at high containment. ► Catheterization reduces the requirement for sharps. ► Simultaneous blood samples can easily be collected from guinea pigs. ► Ebola virus infection had no effect on decay profile of a PS-targeting antibody.Sequential sampling from animals challenged with highly pathogenic organisms, such as haemorrhagic fever viruses, is required for many pharmaceutical studies. Using the guinea pig model of Ebola virus infection, a catheterized system was used which had the benefits of allowing repeated sampling of the same cohort of animals, and also a reduction in the use of sharps at high biological containment. Levels of a PS-targeting antibody (Bavituximab) were measured in Ebola-infected animals and uninfected controls. Data showed that the pharmacokinetics were similar in both groups, therefore Ebola virus infection did not have an observable effect on the half-life of the antibody.

Keywords: Ebola; Catheterized; Model; Pharmacokinetic

Novel polymeric inhibitors of HCoV-NL63 by Aleksandra Milewska; Justyna Ciejka; Kamil Kaminski; Anna Karewicz; Dorota Bielska; Slawomir Zeglen; Wojciech Karolak; Maria Nowakowska; Jan Potempa; Berend Jan Bosch; Krzysztof Pyrc; Krzysztof Szczubialka (pp. 112-121).

► Polymer HTCC and its hydrophobic derivative HM-HTCC can inhibit human coronavirus NL63 and murine hepatitis virus. ► HTCC binds the S protein and most likely hampers viral entry to susceptible cells.The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.

Keywords: HCoV-NL63; Coronavirus; Polymers; Human coronavirus NL63; MHV; Murine hepatitis virus

The large tumor antigen: A “Swiss Army knife” protein possessing the functions required for the polyomavirus life cycle by D. Topalis; G. Andrei; R. Snoeck (pp. 122-136).

► A comparison between the L-Tag and E1 shows high similarity in the binding to the ORI. ► Polyoma- and papillomavirus helicases adopt a hexameric form to unwind the viral DNA. ► E6 and E7 share conserved motifs with the L-Tag for the inactivation of p53 and Rb. ► Compounds targeting the helicases of polyoma- and papillomavirus are presented.The SV40 large tumor antigen (L-Tag) is involved in the replication and cell transformation processes that take place during the polyomavirus life cycle. The ability of the L-Tag to interact with and to inactivate the tumor suppressor proteins p53 and pRb, makes this polyfunctional protein an interesting target in the search for compounds with antiviral and/or antiproliferative activities designed for the management of polyomavirus-associated diseases. The severe diseases caused by polyomaviruses, mainly in immunocompromised hosts, and the absence of licensed treatments, make the discovery of new antipolyomavirus drugs urgent.Parallels can be made between the SV40 L-Tag and the human papillomavirus (HPV) oncoproteins (E6 and E7) as they are also able to deregulate the cell cycle in order to promote cell transformation and its maintenance. In this review, a presentation of the SV40 L-Tag characteristics, regarding viral replication and cellular transformation, will show how similar these two processes are between the polyoma- and papillomavirus families. Insights at the molecular level will highlight similarities in the binding of polyoma- and papillomavirus replicative helicases to the viral DNA and in their disruptions of the p53 and pRb tumor suppressor proteins.

Keywords: Polyomavirus; Papillomavirus; Large T antigen; Replicative helicase; Oncoproteins; Merkel cell polyomavirus

The large tumor antigen: A “Swiss Army knife” protein possessing the functions required for the polyomavirus life cycle by D. Topalis; G. Andrei; R. Snoeck (pp. 122-136).

Keywords: Polyomavirus; Papillomavirus; Large T antigen; Replicative helicase; Oncoproteins; Merkel cell polyomavirus

Exploring the binding of peptidic West Nile virus NS2B–NS3 protease inhibitors by NMR by CongBao Kang; Shovanlal Gayen; Weiling Wang; Rene Severin; Angela Shuyi Chen; Huichang Annie Lim; Cheng San Brian Chia; Andreas Schüller; Danny Ngoc Phouc Doan; Anders Poulsen; Jeffrey Hill; Subhash G. Vasudevan; Thomas H. Keller (pp. 137-144).

► P3 residues are not necessary for potent inhibition of WNV protease. ► Acetyl-KR-aldehyde has a IC₅₀ of 170nM against WNV protease. ► Acetyl-K-agmatine (without an aldehyde warhead) does not inhibit WNV protease. ► Small modifications of inhibitor structure can lead to major changes in the conformation of the WNV protease.West Nile virus (WNV) NS2B–NS3 protease is an important drug target since it is an essential protein for the replication of the virus. In order to determine the minimum pharmacophore for protease inhibition, a series of dipeptide aldehydes were synthesized. The 50% inhibitory concentration (IC₅₀) measurements revealed that a simple acetyl-KR-aldehyde was only threefold less active than 4-phenyl-phenylacetyl-KKR-aldehyde (1) (Stoermer et al., 2008) that was used as the reference compound. The ligand efficiency of 0.40kcal/mol/HA (HA=heavy atom) for acetyl-KR-aldehyde is much improved compared to the reference compound1 (0.23kcal/mol/HA). The binding of the inhibitors was examined using¹H-¹⁵N-HSQC experiments and differential chemical shifts were used to map the ligand binding sites. The biophysical studies show that the conformational mobility of WNV protease has a major impact on the design of novel inhibitors, since the protein conformation changes profoundly depending on the structure of the bound ligand.

Keywords: WNV protease; Peptide inhibitors; NMR study; Induced fit

Poly(I:C) combined with multi-epitope protein vaccine completely protects against virulent foot-and-mouth disease virus challenge in pigs by Yimei Cao; Zengjun Lu; Yanli Li; Pu Sun; Dong Li; Pinghua Li; Xingwen Bai; Yuanfang Fu; Huifang Bao; Chunxue Zhou; Baoxia Xie; Yingli Chen; Zaixin Liu (pp. 145-153).

► Developing a multi-epitope vaccine for FMD with optimal immunogenicity. ► Combination poly(I:C) with this vaccine conferred 100% protection in pigs. ► Poly(I:C) up-regulated T-cell immunity in pigs. ► Poly(I:C) is an effective adjuvant for this epitope-based vaccine of FMDV.We designed a series of epitope proteins containing the G–H loops of three topotypes of foot-and-mouth disease virus (FMDV) serotype O and promiscuous artificial Th sites and selected one epitope protein (designated as B4) with optimal immunogenicity and cross-reactivity. Three out of five pigs immunized intramuscularly with this B4 were protected against virulent FMDV challenge after a single inoculation, while all pigs co-immunized with B4 and polyinosinic–cytidylic acid [poly(I:C)] conferred complete protection following FMDV challenge. Additionally, we demonstrated that all pigs co-immunized with B4 and poly(I:C) elicited FMDV-specific neutralizing antibodies, total IgG antibodies, type I interferon (IFN-α/β) and cytokines IFN-γ. In contrast, some pigs immunized with B4 alone produced parameters mentioned above, while some not, suggesting that poly(I:C) reduced animal-to-animal variations in both cellular and humoral responses often observed in association with epitope-based vaccines and up-regulated T-cell immunity often poorly observed in protein-based vaccines. We propose that poly(I:C) is an effective adjuvant for this epitope-based vaccine of FMDV. This combination could yield an effective and safe candidate vaccine for the control and eradication of FMD in pigs.

Keywords: FMDV; Multi-epitope protein vaccine; Poly(I:C); Pig

Synergistic activity of amenamevir (ASP2151) with nucleoside analogs against herpes simplex virus types 1 and 2 and varicella-zoster virus by Koji Chono; Kiyomitsu Katsumata; Hiroshi Suzuki; Kimiyasu Shiraki (pp. 154-160).

► ASP2151 is a helicase–primase inhibitor against Alphaherpesvirinae viruses. ► Combination effect of ASP2151 with existing antiherpes agents was studied. ► ASP2151 combined with ACV showed synergistic effect against HSV-1, HSV-2, and VZV. ► ASP2151 combined with VACV was more potent than each monotherapy in mouse model.ASP2151 (amenamevir) is a helicase–primase complex inhibitor with antiviral activity against herpes simplex virus HSV-1, HSV-2, and varicella-zoster virus (VZV). To assess combination therapy of ASP2151 with existing antiherpes agents against HSV-1, HSV-2, and VZV, we conducted in vitro and in vivo studies of two-drug combinations . The combination activity effect of ASP2151 with nucleoside analogs acyclovir (ACV), penciclovir (PCV), or vidarabine (VDB) was tested via plaque-reduction assay and MTS assay, and the data were analyzed using isobolograms and response surface modeling. In vivo combination therapy of ASP2151 with valaciclovir (VACV) was studied in an HSV-1-infected zosteriform spread mouse model. The antiviral activity of ASP2151 combined with ACV and PCV against ACV-susceptible HSV-1, HSV-2, and VZV showed a statistically significant synergistic effect ( P<0.05). ASP2151 with VDB was observed to have additive effects against ACV-susceptible HSV-2 and synergistic effects against VZV. In the mouse model of zosteriform spread, the inhibition of disease progression via combination therapy was more potent than that of either drugs as monotherapy ( P<0.05). These results indicate that the combination therapies of ASP2151 with ACV and PCV have synergistic antiherpes effects against HSV and VZV infections and may be feasible in case of severe disease, such as herpes encephalitis or in patients with immunosuppression.

Keywords: Helicase–primase inhibitor; HSV-1; HSV-2; VZV; Amenamevir; Combination therapy

Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease by Yunjeong Kim; Sivakoteswara Rao Mandadapu; William C. Groutas; Kyeong-Ok Chang (pp. 161-168).

► Feline infectious peritonitis virus (FIPV) is a causative agent of a fatal disease among cats. ► Protease inhibitors targeting virus 3C/3CL protease potently inhibited feline infectious peritonitis virus in cells. ► The antiviral effects of inhibitors were compared with a cathepsin B inhibitor, an entry blocker. ► The inhibitors exerted strong synergistic effects with a cathepsin B inhibitor against a FIPV in cells.Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC₅₀ in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

Keywords: Feline coronaviruses; Feline infectious peritonitis virus; Protease inhibitor; Cathepsin B; Synergy; 3CL protease

Experimental therapies for yellow fever by Justin G. Julander (pp. 169-179).

► Proposes methods for the development of countermeasures for the treatment of yellow fever. ► Includes discussion on the classification and replication of yellow fever virus. ► Discusses in vitro and in vivo model systems used for the discovery of antiviral agents. ► Identifies recently discovered viral pathways and their potential as targets for drug development. ► Discusses potential avenues of host response pathway manipulation in order to ameliorate yellow fever disease.A number of viruses in the family Flaviviridae are the focus of efforts to develop effective antiviral therapies. Success has been achieved with inhibitors for the treatment of hepatitis C, and there is interest in clinical trials of drugs against dengue fever. Antiviral therapies have also been evaluated in patients with Japanese encephalitis and West Nile encephalitis. However, no treatment has been developed against the prototype flavivirus, yellow fever virus (YFV). Despite the availability of the live, attenuated 17D vaccine, thousands of cases of YF continue to occur each year in Africa and South America, with a significant mortality rate. In addition, a small number of vaccinees develop severe systemic infections with the 17D virus. This paper reviews current efforts to develop antiviral therapies, either directly targeting the virus or blocking detrimental host responses to infection.

Keywords: Flavivirus; Yellow fever virus; Antiviral therapy

Experimental therapies for yellow fever by Justin G. Julander (pp. 169-179).

Keywords: Flavivirus; Yellow fever virus; Antiviral therapy

Nucleotide embargo by SAMHD1: A strategy to block retroviral infection by Eduardo Pauls; Ester Ballana; José A. Esté (pp. 180-182).

► SAMHD1 is a new HIV-1 restriction factor in myeloid cells and resting CD4+ T lymphocytes. ► SAMHD1 controls the size of the intracellular dNTP pool available for reverse transcription. ► Small molecules affecting SAMHD1 function may represent a novel antiviral strategy.SAMHD1 (sterile alpha motif and histidine/aspartic acid (HD) domain-containing protein 1) has been identified as a novel HIV-1 restriction factor in myeloid cells and resting CD4+ T lymphocytes. SAMHD1 restriction is antagonized by the lentiviral protein Vpx. Here, we comment on the latest knowledge of SAMHD1 biology, focusing on how it regulates the pool of intracellular nucleotides to control HIV replication. We discuss how HIV restriction by SAMHD1 and viral counter-restriction mechanisms may suggest new strategies for therapeutic intervention.

Keywords: HIV-1; Restriction factor; Vpx; SAMHD1; Immune response; Antiviral target

Nucleotide embargo by SAMHD1: A strategy to block retroviral infection by Eduardo Pauls; Ester Ballana; José A. Esté (pp. 180-182).

Keywords: HIV-1; Restriction factor; Vpx; SAMHD1; Immune response; Antiviral target

Antiviral drug discovery for the treatment of enterovirus 71 infections by Luqing Shang; Mengying Xu; Zheng Yin (pp. 183-194).

► Enterovirus 71 (EV71) causes hand, foot and mouth disease in children. ► A small percentage of patients develop severe central nervous system infection. ► Although vaccines have been in use for poliovirus, none has been developed for EV71. ► No antiviral drugs are approved for the treatment of EV71 infection. ► A range of compounds have been identified that inhibit steps in EV71 replication.Enterovirus 71 (EV71) is a small, positive-sense, single-stranded RNA virus in the genus Enterovirus, family Picornavirus. It causes hand, foot and mouth disease in infants and children, which in a small percentage of cases progresses to central nervous system infection, ranging from aseptic meningitis to fatal encephalitis. Sporadic cases of EV71 infection occur throughout the world, but large epidemics have occurred recently in Southeast Asia and China. There are currently no approved vaccines or antiviral therapies for the prevention or treatment of EV71 infection. This paper reviews efforts to develop antiviral therapies against EV71.

Keywords: Picornavirus; Enterovirus; Enterovirus 71; Antiviral therapy

Antiviral drug discovery for the treatment of enterovirus 71 infections by Luqing Shang; Mengying Xu; Zheng Yin (pp. 183-194).

Keywords: Picornavirus; Enterovirus; Enterovirus 71; Antiviral therapy

Ezetimibe blocks hepatitis B virus infection after virus uptake into hepatocytes by Julie Lucifora; Knud Esser; Ulrike Protzer (pp. 195-197).

► Ezetimibe is a drug known to block lipids transport. ► We analyzed the effect of Ezetimibe on HBV infection using an in vitro model. ► Ezetimibe inhibits HBV infection at a post-entry step before formation of cccDNA. ► Ezetimibe rather targets a host factor than the virus itself. ► Our data suggest that HBV hijacks lipid transport pathways for its establishment.Current treatment of chronic hepatitis B virus (HBV) infection mainly targets viral replication in hepatocytes and leads to curing only in exceptional cases. Despite their potential to improve therapeutic success, no drugs interfering with early infection steps of the hepatotropic pathogen HBV are available to date. Recently, entry of the hepatitis C virus (HCV) has been shown to occur along hepatic cholesterol uptake pathways and ezetimibe, a drug which blocks this lipid transport, has been shown to inhibit HCV infection. We here investigated the effect of ezetimibe on HBV infection using differentiated HepaRG cells as a cell-culture infection model. Treatment with ezetimibe inhibited establishment of intrahepatic cccDNA and expression of viral replication markers when cells were infected with HBV virions, while we observed no effect when the HBV viral genome was transduced via an adenoviral vector. Our data suggest that modulating hepatic cholesterol uptake by ezetimibe inhibits early HBV infection and that ezetimibe sensitive lipid transport pathways represent new targets for antiviral therapy in HBV infection.

Keywords: Ezetimibe; Hepatitis B virus; Infection; Hepatocytes; Antiviral

Ezetimibe blocks hepatitis B virus infection after virus uptake into hepatocytes by Julie Lucifora; Knud Esser; Ulrike Protzer (pp. 195-197).

Keywords: Ezetimibe; Hepatitis B virus; Infection; Hepatocytes; Antiviral

Development and characterization of a stable eGFP enterovirus 71 for antiviral screening by Baodi Shang; Chenglin Deng; Hanqing Ye; Wenbo Xu; Zhiming Yuan; Pei-Yong Shi; Bo Zhang (pp. 198-205).

► An eGFP reporter EV71 cDNA clone of epidemic strain was developed. ► The reporter virus was infectious to Vero cells and stable. ► The reporter virus was demonstrated the utility in antiviral screening.Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5′ untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10⁶PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.

Keywords: Abbreviations; EV71; enterovirus 71; HTS; high-throughput screen; hpi; hour post-infection; pt; post-transfection; pi; post-infection; eGFP; enhanced green fluorescent protein; GuHCl; guanidine hydrochlorideEnterovirus; Reporter virus; High-throughput assay; Antiviral drug discovery

Development and characterization of a stable eGFP enterovirus 71 for antiviral screening by Baodi Shang; Chenglin Deng; Hanqing Ye; Wenbo Xu; Zhiming Yuan; Pei-Yong Shi; Bo Zhang (pp. 198-205).

Influenza antiviral resistance in the Asia-Pacific region during 2011 by Sook-Kwan Leang; Yi-Mo Deng; Robert Shaw; Natalie Caldwell; Pina Iannello; Naomi Komadina; Philippe Buchy; Malinee Chittaganpitch; Dominic E. Dwyer; Peter Fagan; Ann-Claire Gourinat; Frances Hammill; Paul F. Horwood; Q.S. Huang; Peng Kei Ip; Lance Jennings; Alison Kesson; Tuckweng Kok; Jacob L. Kool; Avram Levy; Cui Lin; Katie Lindsay; Osmali Osman; Gina Papadakis; Fahimeh Rahnamal; William Rawlinson; Craig Redden; Jennifer Ridgway; I-Ching Sam; Suzanne Svobodova; Amado Tandoc; Geethani Wickramasinghe; Jan Williamson; Noelene Wilson; Mohd Apandi Yusof; Anne Kelso; Ian G. Barr; Aeron C. Hurt (pp. 206-210).

► Adamantane resistance remains extremely high in the Asia-Pacific. ► Oseltamivir-resistance in untreated community patients increased in 2011. ► Novel B variants with reduced susceptibility were detected. ► Surveillance of community specimens for resistant strains remains important.Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Keywords: Influenza; Antiviral resistance; Surveillance; Asia-Pacific; WHO

Engineered DNA modifying enzymes: Components of a future strategy to cure HIV/AIDS by Frank Buchholz; Joachim Hauber (pp. 211-217).

► Curing HIV infection must address the pool of cells that harbor latent virus. ► Gene therapy strategies may be required to eradicate infected cells. ► Specific endonucleases can directly target integrated HIV proviral DNA. ► Tre-recombinase can remove integrated HIV-1 from infected cells. ► A Tre-recombinase recognizing most HIV-1 subtypes should be developed.Despite phenomenal advances in AIDS therapy transforming the disease into a chronic illness for most patients, a routine cure for HIV infections remains a distant goal. However, a recent example of HIV eradication in a patient who had received CCR5-negative bone marrow cells after full-body irradiation has fuelled new hopes for a cure for AIDS. Here, we review new HIV treatment strategies that use sophisticated genome engineering to target HIV infections. These approaches offer new ways to tackle the infection, and alone or in conjunction with already established treatments, promise to transform HIV into a curable disease.

Keywords: HIV; Cure; Eradication; DNA modifying enzymes; Nucleases; Recombinases

Engineered DNA modifying enzymes: Components of a future strategy to cure HIV/AIDS by Frank Buchholz; Joachim Hauber (pp. 211-217).

Keywords: HIV; Cure; Eradication; DNA modifying enzymes; Nucleases; Recombinases

Melittin-loaded immunoliposomes against viral surface proteins, a new approach to antiviral therapy by Alberto Falco; Enrique Barrajón-Catalán; María P. Menéndez-Gutiérrez; Julio Coll; Vicente Micol; Amparo Estepa (pp. 218-221).

► First time description of an AMP loaded-immunoliposome system targeted to virus. ► Direct inactivation of VHSV particles by melittin loaded-immunoliposomes in vitro. ► Melittin loaded-immunoliposomes also reduce VHSV spread in vitro.In this study, melittin, a well-characterized pore-forming lytic amphiphilic peptide susceptible to be vehiculized in lipid membranes, has been utilized to study their antiviral properties. For this purpose, an assay based on melittin loaded-immunoliposomes previously described by our group was adapted to antiviral purposes by means of monoclonal antibodies targeting the surface G glycoprotein of the fish viral haemorrhagic septicemia rhabdovirus (VHSV). We also studied the antiviral action of these immunoliposomes in vitro and the results showed that they are capable of inhibiting the VHSV infectivity by 95.2% via direct inactivation of the virus. Furthermore, the inhibition of the infectivity when treatments were added at different times post-infection and the analysis of the infection foci sizes suggested altogether that they also act by reducing the VHSV spread in cell culture and by killing the infected cells which express the G glycoprotein in their plasmatic membranes.

Keywords: Immunoliposomes; Melittin; Antiviral therapy; VHSV; G glycoprotein

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Antiviral Research (v.97, #2)