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Antiviral Research (v.96, #3)

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).

The protective immune response against Pseudorabies virus induced by DNA vaccination is impaired if the plasmid harbors a functional Porcine circovirus type 2 rep and origin of replication by Florence Faurez; Béatrice Grasland; Véronique Béven; Roland Cariolet; André Keranflec’h; Aurélie Henry; André Jestin; Daniel Dory (pp. 271-279).

► A replicative plasmid harboring PCV2 Ori- rep was evaluated as a DNA vaccine vector. ► This plasmid was compared to a non-replicative PCV2-derived plasmid. ► These plasmids were used to vaccinate pigs against Pseudorabies virus infection. ► Pigs were more affected by PrV-infection with the replicative plasmid. ► This study shows an in vivo deleterious effect of PCV2 Ori- rep on PrV protection.A plasmid rendered replicative in mammalian cells by inserting the Porcine circovirus 2 (PCV2) origin of replication and replicase gene (Ori- rep) has been previously constructed. The aim of the present study was to evaluate if the replication capacity of this plasmid could be advantageously used to improve the protective immunity induced by DNA vaccination. In this case we used the porcine Pseudorabies virus (PrV) DNA vaccination model. The replicative capacity of the DNA vaccine did not improve the protective immunity against PrV in pigs, but on the contrary the presence of the PCV2 Ori- rep sequence was harmful in the induction of this immunity compared to an equivalent but non-replicative DNA vaccine. In addition, the distribution and the persistence of the replicative and non-replicative plasmids inside the body were the same. This is the first study showing an in vivo deleterious effect of the replicative active PCV2 Ori- rep on the natural and specific protection against PrV infection.

Keywords: DNA vaccination; Pseudorabies virus; porcine Circovirus type 2; replicative plasmid

Crucial role of the N-glycans on the viral E-envelope glycoprotein in DC-SIGN-mediated dengue virus infection by Marijke M.F. Alen; Kai Dallmeier; Jan Balzarini; Johan Neyts; Dominique Schols (pp. 280-287).

► First time generation of a dengue virus resistant to a carbohydrate-binding agent. ► This mutant dengue virus deleted both N-glycans on the viral E-glycoprotein. ► The mutant virus cannot infect its main target cell, the DC-SIGN⁺ dendritic cell. ► The mutant virus could replicate in (DC-SIGN⁻) mammalian and insect cell lines. ► The viral N-glycans are crucial for DC-SIGN receptor interaction and infection.We generated in the mosquito cell line C6/36 a dengue virus (DENV) resistant to Hippeastrum hybrid agglutinin (HHA), a carbohydrate-binding agent (CBA). The genotype and phenotype were characterized of the HHA resistant (HHA^res) DENV compared to the wild-type (WT) DENV. Sequencing the structural proteins of HHA^res resulted in two mutations, N67D and T155I, indicating a deletion of both N-glycosylation sites on the viral envelope E-glycoprotein. The HHA^res DENV could replicate in mammalian and mosquito cells that are lacking dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expression. In contrast, DC-SIGN expressing human cells namely monocyte-derived dendritic cells as well as DC-SIGN-transfected cells were no longer susceptible to HHA^res DENV. This demonstrates a crucial role of the N-glycans in the E-glycoprotein in the infection of dendritic cells, which constitute primary target cells of DENV during viral pathogenesis in the human body.

Keywords: Dengue virus; DC-SIGN; Dendritic cells; Carbohydrate binding agent; N-glycan; Viral entry

Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency by B. Mohana Subramanian; M. Madhanmohan; Rajan Sriraman; R.V. Chandrasekhar Reddy; S. Yuvaraj; Kankipati Manikumar; S. Rajalakshmi; S.B. Nagendrakumar; Samir Kumar Rana; V.A. Srinivasan (pp. 288-295).

► Development of FMDV type O virus like particles (VLP) in insect cell line. ► Evaluation of the efficacy and immunogenicity of the VLP vaccine in cattle. ► The 50% protective dose of the VLP vaccine formulation was 5.01.Foot-and-mouth disease (FMD) is an economically significant viral disease that rampage dairy and other livestock industries in many countries. The disease is being controlled by the use of an inactivated vaccine. However, a recombinant marker vaccine, which avoids the use of live virus, may be an option for the unambiguous differentiation of infected animals from vaccinated animals. A recombinant baculovirus clone containing P1-2A-3C coding sequences of foot-and-mouth disease virus (FMDV) serotype O₁ Manisa was generated. The FMDV structural proteins along with the 3C protease were expressed in Sf9 cells and the generation of virus like particles (VLP) was studied. The recombinant protein was formulated as vaccine using an oil adjuvant, ISA 206 and potency of the vaccine was tested in cattle. The vaccine had a potency value (PD₅₀) of 5.01 and most of the vaccinated animals exhibited neutralizing antibody titers after two immunizations.

Keywords: FMDV; VLP vaccine; Cattle challenge

Protein kinase inhibitors that inhibit induction of lytic program and replication of Epstein–Barr virus by R. Goswami; S. Gershburg; A. Satorius; E. Gershburg (pp. 296-304).

► Protein kinase inhibitors of PI3K, MAPK, and NF-κB pathways inhibit EBV replication. ► Out of 80 tested protein kinase inhibitors only K252A inhibited activity of EBV-PK. ► PI3K inhibitors seem to act during the reactivation; MAPK inhibitor U0126 acts later.Signaling pathways mediating Epstein–Barr virus (EBV) reactivation by Ag-bound B-cell receptor (BCR) were analyzed using a panel of 80 protein kinase inhibitors. Broad range protein kinase inhibitors Staurosporine, K252A, and PKC-412 significantly reduced the EBV genome copy numbers measured 48h after reactivation perhaps due to their higher toxicity. In addition, selected inhibitors of the phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways, glycogen synthase kinase 3β (GSK-3β), platelet-derived growth factor receptor-associated tyrosine kinase (PDGFRK), and epidermal growth factor receptor-associated tyrosine kinase (EGFRK) significantly reduced the EBV genome copy numbers. Of those, only U0126 and Erbstatin analog, which inhibit MAPK pathway and EGFRK, respectively, did not inhibit viral reactivation assessed by expression of the EBV early protein, EA-D. None of the tested compounds, except for K252A, affected the activity of the EBV-encoded protein kinase in vitro. These results show that EBV reactivation induced by BCR signaling is mainly mediated through PI3K and PKC, whereas MAPK might be involved in later stages of viral replication.

Keywords: Abbreviations; BCR; B-cell receptor; EBV; Epstein–Barr virus; CDK; cyclin-dependent kinase; GSK-3β; glycogen synthase kinase 3β; PI3K; phosphatidylinositol-3-kinase; PCR; polymerase chain reaction; PDGFRK; platelet-derived growth factor receptor-associated tyrosine kinase; EGFRK; epidermal growth factor receptor-associated tyrosine kinase; NF-κB; nuclear factor κB; PLCγ2; phospholipase Cγ2; MAPK; mitogen-activated protein kinase; Btk; Bruton’s tyrosine kinase; PIP2; phosphatidylinositol diphosphate; DAG; diacylglycerol; IP3; inositol triphosphate; PKC; protein kinase C; ERK; extracellular signal-regulated kinase; JNK; c-Jun NH2 terminal kinase; CHPK; conserved herpesvirus protein kinase; HCMV; human cytomegalovirus; NGIC-I; non-glucosidic indolocarbazole I; BL; Burkitt’s lymphoma; CDK; cyclin-dependent kinase; DMSO; dimethylsulfoxide; ACV; acyclovir; qPCR; quantitative real-time PCR; PKI; protein kinase inhibitor; GST; glutathione-S-transferase; MBP; myelin basic protein; DTT; dithiothreitol; MEK1/2; mitogen-activated protein kinase kinases 1 and 2; CKI; casein kinase I; CKII; casein kinase II; DRB; 5,6-dichloro-1β-; d; -ribofuranosilbenzimidazoleEpstein–Barr virus (EBV); Protein kinase inhibitors (PKI); Signal transduction; EBV BGLF4 protein kinase; Viral reactivation

Protein kinase inhibitors that inhibit induction of lytic program and replication of Epstein–Barr virus by R. Goswami; S. Gershburg; A. Satorius; E. Gershburg (pp. 296-304).

Establishment of a robust dengue virus NS3–NS5 binding assay for identification of protein–protein interaction inhibitors by Hirotaka Takahashi; Chikako Takahashi; Nicole J. Moreland; Young-Tae Chang; Tatsuya Sawasaki; Akihide Ryo; Subhash G. Vasudevan; Youichi Suzuki; Naoki Yamamoto (pp. 305-314).

► Wheat-germ cell-free protein system simplified the production of DENV NS3 and NS5. ► A robust assay for NS3–NS5 interaction was established by AlphaScreen technology. ► Protease and helicase domains of NS3 were required for the binding to NS5. ► Both domains of NS3 exhibited competitive actions to the NS3–NS5 interaction.Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3–NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z′ factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins.

Keywords: Dengue virus; NS3; NS5; Wheat-germ cell-free protein synthesis; AlphaScreen technology; Protein–protein interaction inhibitors

Poly(A)-binding protein interacts with the nucleocapsid protein of porcine reproductive and respiratory syndrome virus and participates in viral replication by Xiaoye Wang; Juan Bai; Lili Zhang; Xianwei Wang; Yufeng Li; Ping Jiang (pp. 315-323).

► The interaction of poly(A)-binding protein with the N protein of PRRSV was found in porcine alveolar macrophages (PAMs). ► The interactive domains between the poly(A)-binding protein and N protein were mapped. ► The levels of poly(A)-binding protein expression could affect PRRSV replication in vitro.Interactions between host factors and the viral protein play important roles in host adaptation and regulation of virus replication. Poly(A)-binding protein (PABP), a host cellular protein that enhances translational efficiency by circularizing mRNAs, was identified by yeast two-hybrid screening as a cellular partner for PRRSV nucleocapsid (N) protein in porcine alveolar macrophages. The specific interaction of PRRSV N protein with PABP was confirmed in infected cells by co-immunoprecipitation and in vitro by GST pull-down assay. We showed by confocal microscopy that the PABP co-localized with the PRRSV N protein. Using a series of deletion mutants, the interactive domain of N protein with PABP was mapped to a region of amino acids 52–69. For PABP, C-terminal half, which interestingly interacts other translation regulators, was determined to be the domain interactive with N protein. Short hairpin RNA (shRNA)-mediated silencing of PABP in cells resulted in significantly reduced PRRSV RNA synthesis, viral encoded protein expression and viral titer. Overall, the results presented here point toward an important role for PABP in regulating PRRSV replication.

Keywords: PRRSV; N protein; PABP; Yeast two-hybrid

Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain by Yuhang Wang; Shifu Kan; Shouwen Du; Yanxin Qi; Jinhui Wang; Liming Liu; Huifan Ji; Dongyun He; Na Wu; Chang Li; Baorong Chi; Xiao Li; Ningyi Jin (pp. 324-332).

► We construct an attenuated TE3L deficient vaccinia virus Tian Tan strain. ► Cell toxicity of the attenuated virus is significantly decreased. ► The in vivo virulence of the attenuated virus is reduced. ► The attenuated virus is a candidate vector with increased safety.An attenuated vaccinia virus (VACV), TE3L⁻VTT, was evaluated for virulence and safety to determine its potential use as a vaccine or as a recombinant virus vector to express foreign genes. The virulence of TE3L⁻VTT was compared with that of the wild-type VTT both in vivo and in vitro. The humoral and cellular immune responses were detected in a mouse model to test the vaccine efficacy of the TE3L mutant. The results suggested that deletion of the TE3L gene decreased the virulence and neurovirulence significantly in mice and rabbit models, yet retained the immunogenicity. Thus, the deletion of TE3L improved the safety of the VTT vector; this approach may yield a valuable resource for studies of recombinant VACV-vectored vaccines.

Keywords: Attenuated vaccinia virus vector; Tian Tan strain; TE3L; Virulence; Immunogenicity

Anti-hepatitis B virus activities of α-DDB–FNC, a novel nucleoside–biphenyldicarboxylate compound in cells and ducks, and its anti-immunological liver injury effect in mice by Qinghua Yang; Xuejie Zhao; Limin Zang; Xianzhen Fang; Jing Zhao; Xiaorui Yang; Qingduan Wang; Liyun Zheng; Junbiao Chang (pp. 333-339).

► The nucleoside–biphenyldicarboxylate compound α-DDB–FNC has a novel chemical structure. ► The compound α-DDB–FNC has anti-HBV activity both in vitro and in vivo. ► We analyse the hepatoprotective effect of α-DDB–FNC to ConA-induced liver injury in mice.Infection with hepatitis B virus (HBV) continues to be a major global cause of acute and chronic liver disease with high mortality. Herein, we examined both the anti-HBV and hepatoprotective activity of α-DDB–FNC. In human HBV-transfected liver cell line HepG2.2.15, α-DDB–FNC effectively suppressed the secretion of HBV antigens in a time and dose-dependent manner with 25.11% inhibition on HBeAg and 43.68% on HBsAg at 2.5μM on day 9. Consistent with the HBV antigen reduction, α-DDB–FNC (2.5μM) also reduced HBV DNA level by 77.74% extracellularly and 78.94% intracellularly on day 9. In the duck hepatitis B virus (DHBV) infected ducks, after α-DDB–FNC was given once daily for 10days, the serum and liver DHBV DNA levels were reduced markedly with 96.81% and 97.21% at 10mgkg⁻¹ on day 10, respectively. In Con A-induced immunological liver-injury mice, α-DDB–FNC significantly inhibited the elevation of serum ALT, AST, TBiL and liver MDA, NO levels. Furthermore, significant improvement of the liver was observed after α-DDB–FNC treatment both in ducks and mice, as evaluated by the histopathological analysis. In conclusion, our results demonstrated that α-DDB–FNC possesses both antiviral activity against HBV and hepatoprotective effect to Con A-induced liver-injury mice.

Keywords: α-DDB–FNC; HepG2.2.15; Duck hepatitis B virus (DHBV); Liver injury

Neutralization of dengue virus in the presence of Fc receptor-mediated phagocytosis distinguishes serotype-specific from cross-neutralizing antibodies by Ryan S.L. Wu; Kuan Rong Chan; Hwee Cheng Tan; Angelia Chow; John C. Allen Jr.; Eng Eong Ooi (pp. 340-343).

► Distinguishing serotype-specific from cross-neutralizing antibodies is important. ► Convalescent sera neutralize homologous but not heterologous serotype intracellularly. ► This approach can clarify the serotype of the infection and hence immunity serologically. ► Our findings could serve vaccine and therapeutic antibody selection for development.Although several vaccine candidates are presently in various phases of clinical trials, the field still lacks an effective tool to determine protective immunity. The presence of cross-neutralizing antibodies limits a serological approach to identify the etiology and distinguish lifelong from short-lived humoral protection. A recent study indicated that cross-reactive but not serotype-specific antibodies require high antibody concentration to co-ligate FcγRIIB and inhibit infection. Here, we tested if these differences could allow us to distinguish serotype-specific from cross-neutralizing antibodies. Using 30 blinded early convalescent serum samples from patients with virologically confirmed dengue, we demonstrate that neutralization in the presence of FcγR-mediated phagocytosis in THP-1 correctly identifies the DENV serotype of the infection in 93.3% of the cases compared to 76.7% with plaque reduction neutralization test. Our findings could provide a new approach for evaluating DENV neutralization and suggest that in addition to blocking specific ligand–receptor interactions for viral entry, antibodies must prevent viral uncoating during FcγR-mediated phagocytosis for complete humoral protection.

Keywords: Dengue virus; Neutralization; FcγR-mediated phagocytosis; Immunity

Influenza virus neuraminidase contributes to the dextran sulfate-dependent suppressive replication of some influenza A virus strains by Hiroshi Yamada; Eiko Moriishi; Ahmad M. Haredy; Nobuyuki Takenaka; Yasuko Mori; Koichi Yamanishi; Shigefumi Okamoto (pp. 344-352).

► Dextran sulfate (DS) inhibited the replication of some influenza A virus strains. ► The suppression by DS was observed at the late stage of viral replication. ► The influenza NA contributes to the DS-suppressible viral replication.Dextran sulfate (DS), a negatively charged, sulfated polysaccharide, suppresses the replication of an influenza A virus strain, and this suppression is associated with inhibition of the hemagglutinin (HA)-dependent fusion activity. However, it remains unknown whether the replication of all or just some influenza A virus strains is suppressed by DS, or whether HA is the only target for the replication suppression. In the present study, we found that DS inhibited the replication of some, but not all influenza A virus strains. The suppression in the DS-sensitive strains was dose-dependent and neutralized by diethylaminoethyl-dextran (DD), which has a positive charge. The suppression by DS was observed not only at the initial stage of viral infection, which includes viral attachment and entry, but also at the late stage, which includes virus assembly and release from infected cells. Electron microscopy revealed that the DS induced viral aggregation at the cell surface. The neuraminidase (NA) activity of the strains whose viral replication was inhibited at the late stage was also more suppressed by DS than that of the strains whose replication was not inhibited, and this inhibition of NA activity was also neutralized by adding positively charged DD. Furthermore, we found that replacing the NA gene of a strain in which viral replication was inhibited by DS at the late stage with the NA gene from a strain in which viral replication was not inhibited, eliminated the DS-dependent suppression. These results suggest that the influenza virus NA contributes to the DS-suppressible virus release from infected cells at the late stage, and the suppression may involve the inhibition of NA activity by DS’s negative charge.

Keywords: Influenza A virus; Dextran sulfate; Suppressive replication; Neuraminidase; Negative electric charge

Kyasanur forest disease by Michael R. Holbrook (pp. 353-362).

► Kyasanur forest disease (KFD), caused by a tick-borne flavivirus, is seen in a limited area of India. ► The disease affects both wild primates and humans living near forested areas. ► KFD is characterized by hemorrhagic fever, with a case fatality rate in the range of 1–3%. ► Viruses related to KFDV have been identified in China and Saudi Arabia. ► A formalin-inactivated vaccine is in use, but no effective therapies have been identified.In the spring of 1957, an outbreak of severe disease was documented in people living near the Kyasanur forest in Karnataka state, India, which also affected wild nonhuman primates. Collection of samples from dead animals and the use of classical virological techniques led to the isolation of a previously unrecognized virus, named Kyasanur forest disease virus (KFDV), which was found to be related to the Russian spring-summer encephalitis (RSSE) complex of tick-borne viruses. Further evaluation found that KFD, which frequently took the form of a hemorrhagic syndrome, differed from most other RSSE virus infections, which were characterized by neurologic disease. Its association with illness in wild primates was also unique. Hemaphysalis spinigera was identified as the probable tick vector. Despite an estimated annual incidence in India of 400–500 cases, KFD is historically understudied. Most of what is known about the disease comes from studies in the late 1950s and early 1960s by the Virus Research Center in Pune, India and their collaborators at the Rockefeller Foundation. A report in ProMED in early 2012 indicated that the number of cases of KFD this year is possibly the largest since 2005, reminding us that there are significant gaps in our knowledge of the disease, including many aspects of its pathogenesis, the host response to infection and potential therapeutic options. A vaccine is currently in use in India, but efforts could be made to improve its long-term efficacy.

Keywords: Kyasanur forest disease; Alkhurma virus; Tick-borne encephalitis; Flavivirus

Kyasanur forest disease by Michael R. Holbrook (pp. 353-362).

Keywords: Kyasanur forest disease; Alkhurma virus; Tick-borne encephalitis; Flavivirus

Prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection by Thessicar E. Antoine; Yogendra K. Mishra; James Trigilio; Vaibhav Tiwari; Rainer Adelung; Deepak Shukla (pp. 363-375).

► Zinc oxide tetrapods as anti-HSV-2 agents. ► Zinc oxide tetrapods as virus trapping agents. ► Antiviral neutralizing effects of zinc oxide tetrapods. ► Therapeutic effects of viral entry blocking. ► Zinc oxide tetrapods block viral spread.The attachment of Herpes simplex virus type-2 (HSV-2) to a target cell requires ionic interactions between negatively charged cell surface co-receptor heparan sulfate (HS) and positively charged residues on viral envelop glycoproteins, gB and gC. Effective blocking of this first step of HSV-2 pathogenesis demonstrates significant prophylactic effects against the viral disease; any in vitro therapeutic effects of blocking this interaction, however, are not clear. Here, we provide new evidence that zinc oxide tetrapod micro-nanostructures synthesized by flame transport approach significantly block HSV-2 entry into target cells and, in addition, demonstrate the potential to stop the spread of the virus among already infected cells. The zinc oxide tetrapods (ZnOTs) also exhibit the ability to neutralize HSV-2 virions. Natural target cells such as human vaginal epithelial and HeLa cells showed highly reduced infectivity when infected with HSV-2 virions that were pre-incubated with the ZnOTs. The mechanism behind the ability of ZnOTs to prevent, neutralize or reduce HSV-2 infection relies on their ability to bind the HSV-2 virions. We used fluorescently labeled ZnOTs and GFP-expressing HSV-2 virions to demonstrate the binding of the ZnOTs with HSV-2. We also show that the binding and hence, the antiviral effects of ZnOTs can be enhanced by illuminating the ZnOTs with UV light. Our results provide new insights into the anti-HSV-2 effects of ZnOT and rationalize their development as a HSV-2 trapping agent for the prevention and/or treatment of infection. The observed results also demonstrate that blocking HSV-2 attachment can have prophylactic as well as therapeutic applications.

Keywords: Herpes; Heparan sulfate; Zinc oxide; Herpes simplex virus; Entry; Glycoproteins

Comparison of the protection of ferrets against pandemic 2009 influenza A virus (H1N1) by 244 DI influenza virus and oseltamivir by Nigel J. Dimmock; Brian K. Dove; Bo Meng; Paul D. Scott; Irene Taylor; Linda Cheung; Bassam Hallis; Anthony C. Marriott; Miles W. Carroll; Andrew J. Easton (pp. 376-385).

► We compared the ability of DI RNA and Tamiflu to protect ferrets from influenza. ► We treated ferrets with one 2μg intranasal dose of DI RNA delivered as DI virus. ► Or we treated ferrets with 10 oral doses of Tamiflu totalling 25mg over 5days. ► Pandemic A/California/04/09 amplified the DI RNA by >25,000-fold. ► DI virus was more effective than Tamiflu in combatting pandemic A/California/04/09.The main antivirals employed to combat seasonal and pandemic influenza are oseltamivir and zanamivir which act by inhibiting the virus-encoded neuraminidase. These have to be deployed close to the time of infection and antiviral resistance to the more widely used oseltamivir has arisen relatively rapidly. Defective interfering (DI) influenza virus is a natural antiviral that works in a different way to oseltamivir and zanamivir, and a cloned version (segment 1 244 DI RNA in a cloned A/PR/8/34 virus; 244/PR8) has proved effective in preclinical studies in mice. The active principle is the DI RNA, and this is thought to interact with all influenza A viruses by inhibiting RNA virus synthesis and packaging of the cognate virion RNA into nascent DI virus particles. We have compared the ability of DI virus and oseltamivir to protect ferrets from intranasal 2009 pandemic influenza virus A/California/04/09 (A/Cal, H1N1). Ferrets were treated with a single 2μg intranasal dose of 244 DI RNA delivered as 244/PR8 virus, or a total of 25mg/kg body weight of oseltamivir given as 10 oral doses over 5days. Both DI virus and oseltamivir reduced day 2 infectivity and the influx of cells into nasal fluids, and permitted the development of adaptive immunity. However DI virus, but not oseltamivir, significantly reduced weight loss, facilitated better weight gain, reduced respiratory disease, and reduced infectivity on days 4 and 6. 244 DI RNA was amplified by A/Cal by >25,000-fold, consistent with the amelioration of clinical disease. Treatment with DI virus did not delay clearance or cause persistence of infectious virus or DI RNA. Thus in this system DI virus was overall more effective than oseltamivir in combatting pandemic A/California/04/09.

Keywords: Influenza virus; Defective interfering virus; Ferret; Protection; Oseltamivir

Impact of novel mutations of herpes simplex virus 1 and 2 thymidine kinases on acyclovir phosphorylation activity by Sonia Burrel; Pascale Bonnafous; Petr Hubacek; Henri Agut; David Boutolleau (pp. 386-390).

The acyclic analogue of guanosine acyclovir (ACV) constitutes the first-line drug for the treatment of herpes simplex virus (HSV) infections. ACV activation requires primophosphorylation by virus-encoded HSV thymidine kinase (TK). In 95% of cases, HSV resistance to ACV is associated with mutations located in TK. The aim of this work was to address the question of the potential involvement of novel HSV-1 and HSV-2 TK mutations in reduced susceptibility to ACV using a novel nonradioactive method, based on luminescent quantitation of ADP, for the evaluation of in vitro phosphorylation activity of TK. All recombinant TKs tested exhibited significantly lower ACV phosphorylation activities in comparison with those of reference KOS or gHSV-2 TKs ( p<0.015), therefore indicating that amino acid changes Y53D, L170P, R176W, A207P (HSV-1) and S66P, A72S, I101S, M183I (HSV-2) were likely to be involved in HSV resistance to ACV.

Keywords: Herpes simplex virus; Thymidine kinase; Acyclovir phosphorylation activity; Nonradioactive method

Targeting the host or the virus: Current and novel concepts for antiviral approaches against influenza virus infection by Suki Man-Yan Lee; Hui-Ling Yen (pp. 391-404).

► Novel antiviral compounds targeting influenza replication mechanism are under development. ► Sialidase and protease inhibitors may block viral entry at the host level. ► Strategies targeting virus–host interactions and host cellular mechanisms as treatment options.Influenza epidemics and pandemics are constant threats to human health. The application of antiviral drugs provides an immediate and direct control of influenza virus infection. At present, the major strategy for managing patients with influenza is through targeting conserved viral proteins critical for viral replication. Two classes of conventional antiviral drugs, the M2 ion channel blockers and the neuraminidase inhibitors, are frequently used. In recent years, increasing levels of resistance to both drug classes has become a major public health concern, highlighting the urgent need for the development of alternative treatments. Novel classes of antiviral compounds or biomolecules targeting viral replication mechanism are under development, using approaches including high-throughput small-molecule screening platforms and structure-based designs. In response to influenza virus infection, host cellular mechanisms are triggered to defend against the invaders. At the same time, viruses as obligate intracellular pathogens have evolved to exploit cellular responses in support of their efficient replication, including antagonizing the host type I interferon response as well as activation of specific cellular pathways at different stages of the replication cycle. Numerous studies have highlighted the possibility of targeting virus–host interactions and host cellular mechanisms to develop new treatment regimens. This review aims to give an overview of current and novel concepts targeting the virus and the host for managing influenza.

Keywords: Influenza; Antivirals; Immunomodulators; Neuraminidase inhibitor resistance; Cyclooxygenase-2 inhibitors

Targeting the host or the virus: Current and novel concepts for antiviral approaches against influenza virus infection by Suki Man-Yan Lee; Hui-Ling Yen (pp. 391-404).

Keywords: Influenza; Antivirals; Immunomodulators; Neuraminidase inhibitor resistance; Cyclooxygenase-2 inhibitors

The innate immune response to hepatitis B virus infection: Implications for pathogenesis and therapy by Jinhong Chang; Timothy M. Block; Ju-Tao Guo (pp. 405-413).

► Hepatitis B virus (HBV) does not efficiently activate pattern recognition receptor (PRR)-mediated innate immune responses. ► HBV engages with multiple components of PRR signal transduction pathways. ► Activation of toll-like receptors (TLRs) and RIG-I-like receptors potently inhibits HBV replication. ► Interferons target multiple steps of the HBV life cycle. ► TLR7 agonists have demonstrated encouraging therapeutic efficacy in preclinical studies.Pattern recognition receptor (PRR)-mediated innate immune responses play an essential role in defending the host from viral infections. Intriguingly, hepatitis B virus (HBV) has been shown to induce negligible innate immune responses during the early phase of infection. Whether this is due to the failure of the virus to activate PRRs or suppression of PRR signaling pathways by the virus remains controversial. However, a plethora of evidence suggests that HBV is sensitive to PRR ligand-induced antiviral responses. This review summarizes current understanding of the interaction between HBV and PRR-mediated host innate immunity, antiviral mechanisms of PRR responses against HBV and strategies to combat chronic HBV infection via induction of host innate antiviral responses.

Keywords: Hepatitis B virus; Pattern recognition receptors; Toll-like receptors; Interferons; TLR7 agonists

The innate immune response to hepatitis B virus infection: Implications for pathogenesis and therapy by Jinhong Chang; Timothy M. Block; Ju-Tao Guo (pp. 405-413).

Keywords: Hepatitis B virus; Pattern recognition receptors; Toll-like receptors; Interferons; TLR7 agonists

Modulation of TLR9 response in a mouse model of herpes simplex virus encephalitis by Nicolas Boivin; Rafik Menasria; Jocelyne Piret; Guy Boivin (pp. 414-421).

► TLR9 agonists improved outcome in mice when administered before the infection. ► TLR9 antagonist improved outcome in mice when administered after the infection. ► There is a balance in the TLR9 response that needs to be controlled. ► Preventing overproduction of pro-inflammatory cytokine is a key element. ► An appropriate modulation of the immune response is also necessary during HSE.We evaluated the effects of agonists and antagonist of toll-like receptor (TLR) 9 in comparison with a TLR3 agonist in a mouse model of herpes simplex virus type 1 (HSV-1) encephalitis (HSE). BALB/c mice received a single intranasal dose of either a TLR3 agonist (polyinosinic:polycytidylic acid; PIC), TLR9 agonists (oligodeoxynucleotides (ODNs) 1585, 1826 or 2395) or a TLR9 antagonist (ODN 2088), 1day before and, for selected groups, 3days after infection with HSV-1. Mice that received the pre-treatment with vehicle, PIC, ODNs 1585, 1826, 2395 and 2088 before infection had survival rates of 25%, 65%, 55%, 40%, 55% and 30%, respectively ( P<0.05 for PIC and ODNs 1585 and 2395 versus vehicle). Infected mice subsequently treated with vehicle, ODNs 2395 and 2088 had survival rates of 9%, 0% and 30%, respectively ( P<0.05, ODN 2088 versus other groups). The pre-treatment of mice with ODN 2395 reduced both the viral load ( P<0.05 at day 5) and the production of CCL2, IL-6 and CCL5 at days 3, 4 and 5 ( P<0.05 for IL-6 at day 3 and P<0.05 for CCL2 and CCL5 at day 4). Treatment of infected mice with ODN 2088 reduced the production of the same cytokines ( P=0.07 for CCL2 and P=0.09 for IL-6 at day 5). Pre-treatment of mice with TLR9 agonists before infection reduces brain viral load and cytokine levels resulting in increased HSE survival rates. On the other hand, TLR9 antagonists can be helpful to control the inflammatory response that could be detrimental after infection.

Keywords: Herpes; Encephalitis; Mouse; Toll-like receptors; Agonist and antagonist

Modulation of TLR9 response in a mouse model of herpes simplex virus encephalitis by Nicolas Boivin; Rafik Menasria; Jocelyne Piret; Guy Boivin (pp. 414-421).

Keywords: Herpes; Encephalitis; Mouse; Toll-like receptors; Agonist and antagonist

Genotype-specific mutations in the polymerase gene of hepatitis B virus potentially associated with resistance to oral antiviral therapy by Silvia Mirandola; Giada Sebastiani; Cristina Rossi; Emanuela Velo; Elke Maria Erne; Alessandro Vario; Diego Tempesta; Chiara Romualdi; Davide Campagnolo; Alfredo Alberti (pp. 422-429).

► Characterization of AA changes at 42 RT-positions according to HBV-genotype. ► HBV genotype D seems to have the highest genetic variability. ► Genotype-dependent mutation pattern in some LAM resistance positions. ► New genotype-specific AA substitutions potentially related to NAs resistance.The evolution of hepatitis B virus (HBV) and the role of different variants during antiviral therapy may be influenced by HBV genotype. We have therefore analysed substitutions potentially related to nucleos(t)ide analogues (NAs) resistance at 42 positions within RT-region in a cohort of patients with chronic hepatitis B in relation to HBV-genotype. RT mutations analysis was performed by direct sequencing in 200 NAs-naïve patients and in 64 LAM or LAM+ADV experienced patients with NAs resistance, infected mainly by HBV-genotypes D and A. 27 polymorphic-sites were identified among the 42 positions analysed and 64 novel mutations were detected in 23 positions. Genotype-D displayed the highest mutation frequency (6.4%) among all HBV-genotypes analysed. Single or multiple mutations were detected in 80% of naïve patients. Overall, the most frequent single mutations were at residues rt54, rt53 and rt91 which may associate with significantly lower HBV-DNA levels ( p=0.001). Comparison with sequencing data of patients failing LMV or LAM+ADV therapy revealed an higher frequency of novel genotype-specific mutations if compared with naïve patients: 3 mutations under LAM monotherapy in HBV-D (rtS85F; rtL91I; rtC256G) and 3 mutations under ADV therapy in HBV-A (rtI53V; rtW153R; rtF221Y). In HBV-D treated patients the dominant resistance mutation was rtL80V (31.4%) and rtM204I (60%) in LAM+ADV group while LAM-treated patients showed a preference of rtM204V (51.9%). Interestingly, none of HBV-A patients had mutation rtM204I under ADV add-on treatment but all of them had the “V” AA substitution. These results suggested that in patients with CHB, HBV-genotype might be relevant in the evolution and development of drug resistance showing also different mutation patterns in the YMDD motif between HBV genotype D and A.

Keywords: Abbreviations; AA; amino acid; ADV; adefovir-dipivoxil; CHB; chronic hepatitis B; ETV; entecavir; HBeAg; hepatitis B e Antigen; HBV; hepatitis B virus; HBV-A-D; hepatitis B genotype A-D; HBV-non D; hepatitis B genotype non D; LAM; lamivudine; LdT; telbivudine; NAs; nucleos(t)ide analogues; NAr; nucleos(t)ide analogues resistance; RT; reverse transcriptase; TDF; TenofovirHBV-genotype; Chronic hepatitis B; Nucleot(s)ide analogues therapy; YMDD mutations

Development and evaluation of a thermosensitive vaginal gel containing raltegravir+efavirenz loaded nanoparticles for HIV prophylaxis by Abhijit A. Date; Annemarie Shibata; Michael Goede; Bridget Sanford; Krista La Bruzzo; Michel Belshan; Christopher J. Destache (pp. 430-436).

► The PLGA nanoparticles (NPs) are <100nm with good encapsulation of raltegravir and efavirenz. ► The gel was made allowing the gel to be liquid at room temperature and solid at body temperature. ► The blank gel and raltegravir+efavirenz nanoparticles do not cause cytotoxicity over 14days to HeLa cells. ► Efavirenz had intracellular levels for 14days and raltegravir had levels for 6days by HPLC. ► Fluorescent-loaded NPs transfer from the gel within 30min and phagocytosized by HeLa cells.The objective of this investigation was to develop a thermosensitive vaginal gel containing raltegravir+efavirenz loaded PLGA nanoparticles (RAL+EFV–NPs) for pre-exposure prophylaxis of HIV. RAL+EFV–NPs were fabricated using a modified emulsion–solvent evaporation method and characterized for size and zeta potential. The average size and surface charge of RAL+EFV–NP were 81.8±6.4nm and −23.18±7.18mV respectively. The average encapsulation efficiency of raltegravir and efavirenz was 55.5% and 98.2% respectively. Thermosensitive vaginal gel containing RAL+EFV–NPs was successfully prepared using a combination of Pluronic F127 (20% w/v) and Pluronic F68 (1% w/v). Incorporation RAL+EFV–NPs in the gel did not result in nanoparticle aggregation and RAL+EFV–NPs containing gel showed thermogelation at 32.5°C. The RAL+EFV–NPs were evaluated for inhibition of HIV-1_NL4-3 using TZM-bl indicator cells. The EC₉₀ of RAL+EFV–NPs was lower than raltegravir+efavirenz (RAL+EFV) solution but did not reach significance. Compared to control HeLa cells without any treatment, RAL+EFV–NPs or blank gel were not cytotoxic for 14days in vitro. The intracellular levels of efavirenz in RAL+EFV–NPs treated HeLa cells were above the EC₉₀ for 14days whereas raltegravir intracellular concentrations were eliminated within 6days. Transwell experiments of NPs-in-gel demonstrated rapid transfer of fluorescent nanoparticles from the gel and uptake in HeLa cells within 30min. These data demonstrate the potential of antiretroviral NP-embedded vagina gels for long-term vaginal pre-exposure prophylaxis of heterosexual HIV-1 transmission.

Keywords: Raltegravir; Efavirenz; Pre-exposure prophylaxis; Nanoparticles; HIV; Thermosensitive gel

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Antiviral Research (v.96, #3)