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Antiviral Research (v.97, #3)

Prelim II: Editorial Board (pp. ii).

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

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus by Asit Kumar Manna; Anuj Kumar; Upasana Ray; Saumitra Das; Gautam Basu; Siddhartha Roy (pp. 223-226).

► Human La protein stimulates IRES dependent translation of HCV. ► The binding of La with HCV RNA occurs through a β-turn. ► A cyclic peptide mimicking the turn inhibits IRES mediated translation of HCV. ► The cyclic peptide also inhibits the growth of HCV replicon in a cell culture model. ► At 48h, the inhibition is much more potent than the corresponding linear peptide.Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the β-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

Keywords: Cyclic peptide; HCV translation; Replicon; Beta-turn

Protective immune responses in rabbits induced by a suicidal DNA vaccine of the VP60 gene of rabbit hemorrhagic disease virus by Yingjie Cheng; Zongyan Chen; Chuanfeng Li; Chun Meng; Run Wu; Guangqing Liu (pp. 227-231).

► A suicidal DNA vaccine was evaluated for the development of a vaccine against RHDV. ► RHDV-specific antibodies and cell immune response were well induced in rabbits. ► All the rabbits inoculated with the DNA vaccine were protected against RHDV. ► The DNA vaccine is a promising vaccine candidate for prevention of RHD.A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against rabbit hemorrhagic disease virus (RHDV). The VP60 gene of RHDV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/VP60, was transfected into BHK-21 cells, and the antigenicity of the expressed protein was confirmed using indirect immunofluorescence and a western blot assay. In addition, immunogenicity was studied in rabbits. Fifteen rabbits were injected intramuscularly twice with pSCA/VP60 at 2-week intervals. They were challenged with an RHDV isolate 2weeks after the second immunization. In all cases, anti-RHDV antibodies were detected by ELISA. Additionally, the lymphocyte proliferation response was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method, and neutralizing antibodies were measured by microneutralization tests. Our results showed that RHDV-specific antibodies and an RHDV-specific cell-mediated immune response were strongly induced in rabbits. Furthermore, all of the rabbits were protected against challenge with wild type RHDV. In conclusion, we demonstrated that the suicidal DNA vaccine is a promising vaccine candidate that facilitates the prevention of rabbit hemorrhagic disease caused by RHDV.

Keywords: Suicidal DNA vaccine; Rabbit hemorrhagic disease virus; VP60 gene; Rabbits

Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs by Hui Chen; Lihui Liu; Susan A. Jones; Nilesh Banavali; Jorden Kass; Zhong Li; Jing Zhang; Laura D. Kramer; Arun K. Ghosh; Hongmin Li (pp. 232-239).

► We synthesized five unnatural nucleoside analogs. ► Two analogs can efficiently inhibit WNV methyltransferase (MTase) activity. ► The anti-WNV MTase compounds did not inhibit human mRNA MTase. ► The mechanism of selectivity is different from what was reported previously.The flavivirus methyltransferase (MTase) sequentially methylates the N-7 and 2′-O positions of the viral RNA cap (GpppA-RNA→m⁷GpppA-RNA→m⁷GpppAm-RNA), using S-adenosyl-l-methionine (SAM) as a methyl donor. We report here the synthesis and biological evaluation of a series of novel nucleoside analogs. Two of these compounds can effectively and competitively inhibit the WNV MTase with IC₅₀ values in micromolar range and, more importantly, do not inhibit human MTase. The compounds can also suppress the WNV replication in cell culture.

Keywords: Flavivirus NS5; RNA cap methylation; West Nile virus; Methyltransferase; Antiviral development

Neuraminidase inhibitor susceptibility testing of influenza type B viruses in China during 2010 and 2011 identifies viruses with reduced susceptibility to oseltamivir and zanamivir by Dayan Wang; Katrina Sleeman; Weijuan Huang; Ha T. Nguyen; Marnie Levine; Yanhui Cheng; Xiyan Li; Minju Tan; Xiaohui Xing; Xiyan Xu; Alexander I. Klimov; Larisa V. Gubareva; Yuelong Shu (pp. 240-244).

► We report the drug resistance in country with low or non-existing NAI use. ► Influenza B viruses with reduced susceptibility to NAIs were identified. ► I222T and D197N mutation in NA gene was identified in China for the first time.Influenza type B viruses are responsible for substantial morbidity and mortality in humans. Antiviral drugs are an important supplement to vaccination for reducing the public health impact of influenza virus infections. Influenza B viruses are not sensitive to M2 inhibitors which limit the current therapeutic options to two neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, which are licensed in many countries. Drug resistance is a public health concern which has necessitated monitoring of influenza virus drug susceptibilities through active global surveillance. Here, we report the results of drug susceptibility surveillance of influenza type B viruses ( n=680) collected in mainland China during two calendar years, 2010 and 2011, assessed using functional neuraminidase (NA) inhibition (NI) assays. Four influenza B viruses exhibited reduced susceptibilities to oseltamivir, but not zanamivir, and shared the amino acid substitution I221T (ATC→ACC), at this conserved residue in the NA active site (I222T in N2 numbering). Additionally, a single virus with reduced susceptibility to both oseltamivir and zanamivir was identified and contained an amino acid substitution D197N (GAC→AAC) at another conserved residue in the NA active site (D198N in N2 numbering). This report underlies the importance of continued influenza antiviral susceptibility surveillance globally, even in countries where the use of NAIs has been low or non-existing.

Keywords: Neuraminidase inhibitor; Zanamivir; Oseltamivir; Influenza B

Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2) by Sergei V. Tillib; Tatiana I. Ivanova; Lev A. Vasilev; Marina V. Rutovskaya; Seda A. Saakyan; Irina Y. Gribova; Irina L. Tutykhina; Elena S. Sedova; Andrei A. Lysenko; Maxim M. Shmarov; Denis Y. Logunov; Boris S. Naroditsky; Alexander L. Gintsburg (pp. 245-254).

► We describe an efficient approach to generate antiviral single-domain antibodies. ► The activity of initially selected antibodies can be greatly increased by formatting. ► Described formatting procedure adds a trimerization domain to the antibody sequence. ► Formatted single-domain antibody can protect mice from influenza virus infection.This work continues a series of recently published studies that employ recombinant single-domain antibody (sdAb, or nanobody®) generation technologies to battle viruses by a passive immunization approach. As a proof of principle, we describe a modified technique to efficiently generate protective molecules against a particular strain of influenza virus within a reasonably short period of time. This approach starts with the immunization of a camel ( Camelus bactrianus) with the specified antigen-enriched material presented in as natural a form as possible. An avian influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) adapted for mice was used as a model source of antigens for both the immunization and phage display-based selection procedures. To significantly increase activities of initially selected monovalent single-domain antibodies, we propose a new type of sdAb formatting that involves the addition of a special type of coiled-coil sequence, the isoleucine zipper domain (ILZ). Presumably, the ILZ-containing peptides adopt trimeric parallel conformations. After the formatting, the biological activities (virus neutralization) of the initially selected anti-influenza virus (H5N2) sdAbs were significantly increased. Intraperitoneal or intranasal administration of the formatted sdAb at 2h before or 24h after viral challenge specifically protects mice from lethal infection with influenza virus. We hope that the described approach combined with the selection focused on particular conservative epitopes will lead to the generation of sdAb-based molecules protective against a broad spectrum of influenza virus subtypes.

Keywords: Abbreviations; Ab; antibody; sdAb; single-domain antibody; fsdAb; formatted single-domain antibody; HA; influenza virus hemagglutinin; HA1; membrane-distal, globular domain of hemagglutinin; aHAsdAb; anti-hemagglutinin sdAb; aHAfsdAb; anti-hemagglutinin formatted sdAb; hcAbs; Camelidae; -specific heavy chain-only antibodies (they lack the classical light-chain and are composed of a homodimer of heavy-chains); VHH; the; v; ariable domain of the; h; eavy chain of; h; cAbs consisting of one single-domain, antigen binding fragment (also called as single-domain antibody, sdAb, “nanobody” or “nanoantibody”); ILZ; isoleucine zipper; VN; virus neutralization; HI; hemagglutination inhibition; a-ck; single-domain antibody against cytokeratin 8; HRP; horseradish peroxidase; HA-tag; antigenic determinant, a fragment of 9 amino acids (YPYDVPDYA); PBS; phosphate buffered saline (137; mM NaCl, 2.7; mM KCl, 10; mM Na; ₂; HPO; ₄; , 2; mM KH; ₂; PO; ₄; , pH 7.4); HMR-analysis; a fingerprinting-like method of parallel restriction analysis of sdAb sequences (using three restriction enzymes – HinfI, MspI, and RsaI)Single-domain antibody; Recombinant antibody formatting; Influenza virus; Passive immunization

Comparison of factors that may affect the inhibitory efficacy of transgenic RNAi targeting of baculoviral genes in silkworm, Bombyx mori by Liang Jiang; Ping Zhao; Genhong Wang; Tingcai Cheng; Qiong Yang; Shengkai Jin; Ping Lin; Yang Xiao; Qiang Sun; Qingyou Xia (pp. 255-263).

► Resistance to BmNPV by RNAi of viral genes in transgenic silkworm. ► Comprehensive comparison of factors that may affect transgenic RNAi in individual. ► “Head to head” is a better connection pattern of gene fragments and spacer in RNAi. ► The efficiency of IE1 promoter combined with hr3 enhancer is better. ► The immediate early gene was the best target for suppressing viral replication. Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen affecting B. mori. This virus could be combated via RNAi of BmNPV genes in transgenic silkworm. However, several factors may affect the resistance of transgenic RNAi silkworm, such as the connection pattern of gene fragments and spacers (“head to head” or “tail to tail”), and the selection of promoters and target genes. In this study, we constructed several transgenic RNAi vectors using different phase genes ( ie-1, helicase, gp64, and vp39) and promoters (BmNPV IE1 promoter (IE1P), IE1P combined with hr3 enhancer of BmNPV, and B. mori A4 promoter (A4P)). Transgenic lines were generated via embryo microinjection using a practical silkworm strain. We analyzed the anti-BmNPV ability, virus gene mRNA level, and BmNPV content of these transgenic larvae. The results showed that “head to head” was better than “tail to tail,” IE1P combined with hr3 was better than IE1P and A4P, and an immediate early gene was the best target for RNAi.

Keywords: Antivirus; BmNPV; Resistance; RNAi; Silkworm; Transgenic

Rupintrivir is a promising candidate for treating severe cases of enterovirus-71 infection: Evaluation of antiviral efficacy in a murine infection model by Xiaonan Zhang; Zhigang Song; Boyin Qin; Xiaoling Zhang; Lixiang Chen; Yunwen Hu; Zhenghong Yuan (pp. 264-269).

► First proved low dose rupintrivir protects from lethal EV71 infection in vivo. ► Rupintrivir effectively alleviates EV71 induced pathologies in vivo. ► Previous safety records justify its immediate clinical trial In HFMD.Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.

Keywords: Enterovirus 71; Hand-foot-and-mouse disease; Rupintrivir

Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection by Katia E. Garcia-Crespo; Calvin C. Chan; Stanislaw J. Gabryszewski; Caroline M. Percopo; Peter Rigaux; Kimberly D. Dyer; Joseph B. Domachowske; Helene F. Rosenberg (pp. 270-279).

► Wild-type mice inoculated with Lactobacillus were fully protected against the lethal sequelae of subsequent PVM infection. ► Inflammation associated with Lactobacillus exposure is robust but transient. ► Lactobacillus clearance is rapid, but gDNA and PGN persist. ► Administration of Lactobacillus gDNA did not elicit inflammation or protect mice against PVM infection.We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities.

Keywords: Cytokines; Neutrophils; Peptidoglycan; Genomic DNA; Heterologous immunity

Shape of vaginal suppositories affects willingness-to-try and preference by Bangde Li; Toral Zaveri; Gregory R. Ziegler; John E. Hayes (pp. 280-284).

► 99 Sexually active women evaluated 5 microbicide prototypes in the laboratory. ► Willingness to use and ranked preference both differed as a function of shape. ► Even when volume was constant, perceived size varied as a function of length. ► Despite constant formulation perceived firmness varied as a function of width. ► Two promising candidates were identified for further optimization.HIV and other sexually transmitted infections (STIs) are a global threat to public health that may be countered, in part, by microbicides. A successful microbicide must be both biologically efficacious and highly acceptable to users. Sensory attributes have a direct influence on product acceptability. We created a series of vaginal suppositories appropriate for use as microbicides to investigate the influence of shape on women’s willingness-to-try. The influence of perceived size and firmness on acceptability was also assessed.Sexually-active women ( n=99) were invited to participate in an evaluation of vaginal suppositories in 5 different shapes including: Bullet, Long Oval, Round Oval, Teardrop and Tampon. The volume (3mL) and formulation for these five prototypes were identical. After manipulating prototypes ex vivo (in their hands), participants rated their willingness-to-try on a 100-point visual analog scale. The appropriateness of size and firmness were evaluated using 5-point just-about-right (JAR) scales. Each participant evaluated all five prototypes individually. Samples were presented in a counterbalanced monadic sequence using a Williams design.Mean willingness-to-try varied by shape, with Bullet and Long Oval receiving significantly higher scores. This was consistent with JAR data for size, as 70% and 65% of women indicated these shapes were ‘just-about-right’, respectively. In contrast, a minority of women endorsed the other 3 shapes as having a size that was ‘just-about-right’. The proportion of women who felt the firmness was ‘just-about-right’ was uniformly high, irrespective of shape, suggesting prior attempts to optimize the formula were successful. Perceptions of size and firmness were influenced by the physical length and width of the prototypes, in spite of having constant volume. Women showed high willingness-to-try when asked to assume they were at risk. These results are relevant for behavioral and formulation scientists working on microbicides, to better understand the influence of sensory attributes on acceptability, as acceptability and compliance ultimately impact effectiveness.

Keywords: Microbicide; Acceptability; Consumer behavior; Product optimization; Sensory perception; Just-about-right (JAR) scale

IL-17A but not IL-22 suppresses the replication of hepatitis B virus mediated by over-expression of MxA and OAS mRNA in the HepG2.2.15 cell line by Bing Wang; Xin-Ping Zhao; Yu-Chen Fan; Jian-Jun Zhang; Jing Zhao; Kai Wang (pp. 285-292).

► IL-17A and IL-22 participate in the pathogenesis of hepatitis B. ► IL-17A but not IL-22 could effectively suppress HBV replication. ► The over-expression of MxA and OAS was involved in the effect of IL-17A.Interleukin-17A (IL-17A) and interleukin-22 (IL-22), mainly secreted by interleukin-17-producing T help cells (Th17), are pleiotropic cytokines that regulate the biological responses of several target cells, including hepatocytes. Th17 frequency was reported to negatively correlate with plasma hepatitis B virus (HBV) DNA load in patients with HBV infection. Several studies have indicated that cytokines, such as IL-6 and IL-4, are involved in the noncytopathic suppression of HBV replication. We therefore hypothesized that IL-17A and IL-22 might have a potent suppressive effect on HBV replication. In our present study, we analyzed the suppressive effect of IL-17A and IL-22 on HBV replication in the hepatocellular carcinoma cell line HepG2.2.15. IL-17A did not inhibit the proliferation of HepG2.2.15 cells. It decreased the levels of HBV s antigen (HBsAg) and HBV e antigen (HBeAg) in culture medium and the levels of intracellular HBV DNA. By contrast, blockage of IL-17 receptor (IL-17R) increased the levels of HBsAg and extracellular HBV DNA in culture medium and the levels of intracellular HBV DNA. The expression of antiviral proteins, including myxovirus resistance A (MxA) and oligoadenylate synthetase (OAS), was enhanced by IL-17A. IL-22 and anti-human IL-22 receptor (IL-22R) antibody did not change any indexes. We demonstrated that IL-17A effectively suppressed HBV replication in a noncytopathic manner and the over-expression of MxA and OAS mRNA was involved in the suppression of HBV replication by IL-17A.

Keywords: Abbreviations; HBV; hepatitis B virus; HBeAg; hepatitis B e antigen; HBsAg; hepatitis B surface antigen; TH17; T helper 17; IL-17A; interleukin-17A; IL-22; Interleukin-22; Ab; antibody; MxA; myxovirus resistance A; OAS; oligoadenylate synthetase; ISFG3; IFN-stimulated gene factor3; STAT1; signal transducer and activator of transcription 1; STAT2; signal transducer and activator of transcription 2Interleukin-17A; Interleukin-22; Hepatitis B virus; Myxovirus resistance A; Oligoadenylate synthetase

A dual chicken IgY against rotavirus and norovirus by Ying-Chun Dai; Xu-Fu Zhang; Ming Tan; Pengwei Huang; Wen Lei; Hao Fang; Weiming Zhong; Xi Jiang (pp. 293-300).

► IgY against both RV and NoV were produced by immunizing chickens with a dual vaccine candidate P-VP8^∗. ► The resulting IgY blocked RV and NoV binding to their HBGA receptors. ► The IgY is also able to neutralize RV replication in cell culture condition. ► The P-VP8^∗ based IgY is a promising approach for passive immunization against both RV and NoV.Rotavirus (RV) and norovirus (NoV) are the two most important causes of viral gastroenteritis. While vaccine remains an effective prophylactic strategy, development of other approaches, such as passive immunization to control and treat clinical infection and illness of these two pathogens, is necessary. Previously we demonstrated that high titers of NoV-specific IgY were readily developed by immunization of chickens with the NoV P particles. In this study, we developed a dual IgY against both RV and NoV through immunization of chickens with a divalent vaccine comprising neutralizing antigens of both RV and NoV. This divalent vaccine, named P-VP8^∗ particle, is made of the NoV P particle as a carrier with the RV spike protein VP8^∗ as a surface insertion. Approximately 45mg of IgY were readily obtained from each yolk with high titers of anti-P particle and anti-VP8^∗ antibodies detected by ELISA, Western blot, HBGA blocking (NoV and RV) and neutralization (RV) assays. Reductions of RV replication were observed with viruses treated with the IgY before and after inoculation into cells, suggesting an application of the IgY as both prophylactic and therapeutic treatment. Collectively, our data suggested that the P-VP8^∗ based IgY could serve as a practical approach against both NoV and RV.

Keywords: Rotavirus; Norovirus; Diarrhea; Immunoglobulin Y (IgY); Passive immunization

Increased susceptibility of Cantagalo virus to the antiviral effect of ST-246® by Élida Santos-Fernandes; Cristiana O. Beltrame; Chelsea M. Byrd; Kara B. Cardwell; Laila C. Schnellrath; Maria Luiza G. Medaglia; Dennis E. Hruby; Robert Jordan; Clarissa R. Damaso (pp. 301-311).

► ST-246 is a potent inhibitor of Cantagalo virus replication in cell culture and in infected mice. ► Cantagalo virus has an increased susceptibility to the antiviral effect of ST-246 compared to other orthopoxviruses. ► ST-246 has a severe effect on the production of extracellular particles of Cantagalo virus. ► ST-246 prevents the formation of lesions induced by Cantagalo virus via tail scarification.Cantagalo virus (CTGV) is the etiologic agent of a pustular disease in dairy cows and dairy workers in Brazil with important economical and occupational impacts. Nevertheless, no antiviral therapy is currently available. ST-246 is a potent inhibitor of orthopoxvirus egress from cells and has proved its efficacy in cell culture and in animal models. In this work, we evaluated the effect of ST-246 on CTGV replication. Plaque reduction assays indicated that CTGV is 6–38 times more susceptible to the drug than VACV-WR and cowpox virus, respectively, with an EC₅₀ of 0.0086μM and a selective index of >11,600. The analysis of β-gal activity expressed by recombinant viruses in the presence of ST-246 confirmed these results. In addition, ST-246 had a greater effect on the reduction of CTGV spread in comet tail assays and on the production of extracellular virus relative to VACV-WR. Infection of mice with CTGV by tail scarification generated primary lesions at the site of scarification that appeared less severe than those induced by VACV-WR. Animals infected with CTGV and treated with ST-246 at 100mg/kg for 5days did not develop primary lesions and virus yields were inhibited by nearly 98%. In contrast, primary lesions induced by VACV-WR were not affected by ST-246. The analysis of F13 (p37) protein from CTGV revealed a unique substitution in residue 217 (D217N) not found in other orthopoxviruses. Construction of recombinant VACV-WR containing the D217N polymorphism did not lead to an increase in the susceptibility to ST-246. Therefore, it is still unknown why CTGV is more susceptible to the antiviral effects of ST-246 compared to VACV-WR. Nonetheless, our data demonstrates that ST-246 is a potent inhibitor of CTGV replication that should be further evaluated as a promising anti-CTGV therapy.

Keywords: Cantagalo virus; ST-246; Antiviral agent; Vaccinia virus; Poxvirus

Hepatitis B e antigen levels and response to peginterferon: Influence of precore and basal core promoter mutants by Milan J. Sonneveld; Vincent Rijckborst; Louwerens Zwang; Stefan Zeuzem; E. Jenny Heathcote; Krzysztof Simon; Roeland Zoutendijk; Ulus S. Akarca; Suzan D. Pas; Bettina E. Hansen; Harry L.A. Janssen (pp. 312-317).

Hepatitis B e antigen (HBeAg) levels may predict response to peginterferon (PEG-IFN) but are also influenced by presence of precore (PC) and core promoter (BCP) mutants.HBeAg was measured in 214 patients treated with PEG-IFN±lamivudine for 52weeks. Patients were classified at baseline as wildtype (WT) or non-WT (detectable PC/BCP mutants). Combined response (HBeAg loss with HBV DNA<2000IU/mL), HBeAg response (HBeAg loss with HBV DNA>2000IU/mL) or non-response was assessed at week78.Mean baseline HBeAg levels were 2.65logIU/mL in combined responders, 2.48 in non-responders and 2.24 in HBeAg responders ( p=0.034). Baseline HBeAg levels were not associated with combined response after stratification by WT/non-WT. Within the PEG-IFN monotherapy group ( n=104), patients with HBeAg<1logIU/mL at week24 had a higher probability of combined response (29% versus 12%, p=0.041). After stratification by WT/non-WT, WT patients with HBeAg<1logIU/mL at week24 had a probability of combined response of 78% (versus 19% in patients with >1logIU/mL, p<0.001), whereas no difference in response rates was observed in non-WT patients ( p=0.848).The relationship between HBeAg levels and response to PEG-IFN depends upon the presence of PC/BCP mutants. HBeAg levels should therefore not be routinely used to select patients for PEG-IFN, nor for monitoring of therapy.

Keywords: Abbreviations; HBeAg; Hepatitis B e Antigen; CHB; chronic hepatitis B; HBV; hepatitis B virus; HBsAg; hepatitis B surface antigen; ALT; alanine aminotransferase; ULN; upper limit of normal; AUC; area under the receiver-operating characteristic curveHBeAg; Peginterferon; Precore; Core promoter; Prediction of response; Immunomodulator

Passive immunization with a recombinant adenovirus expressing an HA (H5)-specific single-domain antibody protects mice from lethal influenza infection by Irina L. Tutykhina; Elena S. Sedova; Irina Y. Gribova; Tatiana I. Ivanova; Lev A. Vasilev; Marina V. Rutovskaya; Andrei A. Lysenko; Maxim M. Shmarov; Denis Y. Logunov; Boris S. Naroditsky; Sergei V. Tillib; Alexander L. Gintsburg (pp. 318-328).

► An adenoviral vector efficiently expresses anti-influenza virus-neutralizing single-domain antibodies. ► An adenovirus with single-domain antibodies gene efficiently prevents influenza virus infection. ► A single-domain antibodies efficiently prevent and treat influenza virus infection. ► A combination of single-domain antibodies and its gene expands the time of protection against influenza.One effective method for the prevention and treatment of influenza infection is passive immunization. In our study, we examined the feasibility of creating an antibody-based preparation with a prolonged protective effect against influenza virus. Single-domain antibodies (sdAbs) specific for influenza virus hemagglutinin were generated. Experiments in mouse models showed 100% survivability for both intranasal sdAbs administration 24h prior to influenza challenge and 24h after infection. sdAb-gene delivery by an adenoviral vector led to gene expression for up to 14days. Protection by a recombinant adenovirus containing the sdAb gene was observed in cases of administration prior to influenza infection (14d–24h). We also demonstrated that the single administration of a combined preparation containing sdAb DNA and protein expanded the protection time window from 14d prior to 48h after influenza infection. This approach and the application of a broad-spectrum sdAbs will allow the development of efficient drugs for the prevention and treatment of viral infections produced by pandemic virus variants and other infections.

Keywords: Abbreviations; Ab; antibody; mAb; monoclonal antibody; sdAb; single-domain antibody; rAd; recombinant adenovirus; Ad5; human adenovirus serotype 5; IAV; influenza A virus; HA; influenza virus hemagglutinin; HA1; membrane-distal globular domain of hemagglutinin; aHAsdAb; anti-hemagglutinin formatted sdAb; psdAb; prokaryotically (; E. coli; ) expressed aHAsdAb; esdAb; eukaryotically (adenoviral vector) expressed aHAsdAb; ILZ; isoleucine zipper; VNA; virus-neutralization assay; HIA; hemagglutination inhibition assay; WB; Western blotting; BALF; bronchoalveolar lavage fluid; NS; nasal swab; Cyk8; cytokeratin 8; PEG; polyethylene glycol; scFv; single-chain variable fragment; VHH; variable fragment of a camelid antibodyPassive immunization; Influenza virus; Single-domain antibody; Adenoviral vector

Recombinant duck enteritis virus works as a single-dose vaccine in broilers providing rapid protection against H5N1 influenza infection by Jinxiong Liu; Pucheng Chen; Yongping Jiang; Guohua Deng; Jianzhong Shi; Li Wu; Yuan Lin; Zhigao Bu; Hualan Chen (pp. 329-333).

► H5N1 avian influenza virus is endemic in several developing countries. ► Broilers (meat-chickens) are the major victims of the H5N1 infection in disease-endemic countries. ► We constructed a recombinant duck enteritis virus (DEV) expressing the HA gene of an H5N1 avian influenza virus. ► The recombinant DEV induced rapid and solid protection in broilers against H5N1 challenge after a single dose inoculation.Although vaccination is an important strategy for controlling H5N1 avian influenza virus infections, broilers (short-lived meat chickens) remain the major victims in disease-endemic countries. Inactivated vaccine usually requires 2–3weeks to establish solid protection, and recombinant vaccines, including the recombinant fowlpox virus vaccine and the recombinant Newcastle disease virus vaccine are affected by maternal antibodies against the vectors. These disadvantages compromise the protective efficacy of these vaccines in broilers. Here, we evaluated the safety and efficacy of a new recombinant duck enteritis virus that expresses the HA gene of an H5N1 virus (rDEV-re6) in specific-pathogen-free chickens and broilers. We found this new rDEV-re6 virus to be safe in chickens and to induce rapid and solid protection after a single dose. This virus may therefore serve as an ideal single-dose vaccine for broilers.

Keywords: Avian influenze; H5N1; Vaccine

Spray drying tenofovir loaded mucoadhesive and pH-sensitive microspheres intended for HIV prevention by Tao Zhang; Chi Zhang; Vivek Agrahari; James B. Murowchick; Nathan A. Oyler; Bi-Botti C. Youan (pp. 334-346).

► Tenofovir (anti-HIV microbicide) are loaded in pH-sensitive microspheres (MS) by spray drying. ► The MS release 91.7% of its payload in the presence of simulated human semen. ► The MS (1mg/ml) are noncytotoxic to vaginal cells and Lactobacillus crispatus. ► No statistically significant level of inflammatory cytokines are released by MS. ► Moreover, the percent mucoadhesion of MS is 2-fold higher than that of 1% HEC gel.To develop spray dried mucoadhesive and pH-sensitive microspheres (MS) based on polymethacrylate salt intended for vaginal delivery of tenofovir (a model HIV microbicide) and assess their critical biological responses.The formulation variables and process parameters are screened and optimized using a 2⁴⁻¹ fractional factorial design. The MS are characterized for size, zeta potential, yield, encapsulation efficiency, Carr’s index, drug loading, in vitro release, cytotoxicity, inflammatory responses and mucoadhesion.The optimal MS formulation has an average size of 4.73μm, zeta potential of −26.3mV, 68.9% yield, encapsulation efficiency of 88.7%, Carr’s index of 28.3 and drug loading of 2% (w/w). The MS formulation release 91.7% of its payload in the presence of simulated human semen. At a concentration of 1mg/ml, the MS are noncytotoxic to vaginal endocervical/epithelial cells and Lactobacillus crispatus when compared to control media. There is also no statistically significant level of inflammatory cytokine (IL1-α, IL-1β, IL-6, IL-8, and IP-10) release triggered by these MS. Their percent mucoadhesion is 2-fold higher than that of 1% HEC gel formulation.These data suggest the promise of using such MS as an alternative controlled microbicide delivery template by intravaginal route for HIV prevention.

Keywords: pH-sensitive; Mucoadhesive; Tenofovir; Spray drying; HIV/AIDS microbicide

Natural killer cell activity and function in chronic HCV-infected patients during peg interferon and ribavirin: Early effects of active substance use by Daphne M. Hotho; Kim Kreefft; Zwier M.A. Groothuismink; Harry L.A. Janssen; Robert J. de Knegt; André Boonstra (pp. 347-355).

► We examine the effect of morphine-derived products on NK cells in HCV patients. ► Response to IFN-α in vitro is similar between substance users and non-users. ► Early effects of IFN-based therapy are similar in substance users and non-users.In Western countries, chronic hepatitis C virus (HCV)-infection mostly affects former and active substance users. The effect of active substance use on interferon (IFN)-responsiveness and therapy efficacy is not well understood. In this study, we compared natural killer (NK) cell activity and function in healthy controls and chronic HCV-infected patients with and without active substance use, as well as the early effects of antiviral therapy with peg-IFN and ribavirin.No differences were observed between chronic HCV patients and healthy individuals in the number and frequencies of CD56^dim and CD56^bright NK cells. Also, IL-12/18-induced IFN-gamma production by NK cells was comparable between all groups, whereas the cytotoxic ability of NK cells (granzyme and CD107a levels) was more potent in HCV-infected patients as compared to healthy controls, and highest in non-substance users. Moreover, at baseline, the activation of NK cells was significantly lower in HCV-infected patients who used substances, when compared to healthy individuals. Therapy-induced viral load reduction assessed early at day 7 showed a similar decline in substance users and non-substance use HCV patients, with 25% substance users and 17% non-substance users testing HCV-RNA negative at day 7. Furthermore, early during IFN-based therapy, NK cells from HCV patients remained responsive to IFN, and only a minor decline in the degree of STAT-1 phosphorylation was observed irrespective of substance use. These findings were further supported by comparable in vitro p-STAT-1 induction in all three experimental groups.Despite subtle differences at baseline between healthy individuals and chronic HCV patients, we observed that active substance use in chronic HCV-infected patients did not affect the immune responsiveness to IFN early after start of treatment, and thus, we found no evidence – from an immunological point of view – that antiviral therapy of our cohort of HCV-infected patients with active substance use is less efficient.

Keywords: Abbreviations; IFN; interferon; PEG; pegylated; HCV; hepatitis C virus; IVDU; intravenous drug use; SVR; sustained virological response; NK; natural killerIFN-alpha; HCV; IDU; NK cells; Viral hepatitis

Resolution of the interaction mechanisms and characteristics of non-nucleoside inhibitors of hepatitis C virus polymerase by Johan Winquist; Eldar Abdurakhmanov; Vera Baraznenok; Ian Henderson; Lotta Vrang; U. Helena Danielson (pp. 356-368).

► The interaction details for HCV polymerase inhibitors were determined by an SPR biosensor. ► Tegobuvir did not interact with the enzyme, consistent with a requirement for activation. ► Filibuvir interacts in a single step while VX-222 inhibition involves induced fit. ► The induced fit mechanism of VX-222 gives it a longer residence time than filibuvir. ► Filibuvir and VX-222 interfere with the interaction between the polymerase and RNA.Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure–kinetic relationships. To enable the design of efficient allosteric drugs targeting the polymerase of hepatitis C virus (NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on its interaction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activity of VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5B inhibitors.

Keywords: HCV; NS5B; Inhibitor; Kinetics; Chemodynamics; Thermodynamics

In vitro inhibition of Japanese encephalitis virus replication by capsid-targeted virus inactivation by Ran Pang; Dan-ni He; Bin Zhou; Ke Liu; Jin Zhao; Xiao-min Zhang; Pu-yan Chen (pp. 369-375).

► CTVI have been extensively investigated against the Flavivirus and Pestivirus. ► It is firstly reported the feasibility of using CTVI against JEV infection. ► Expressed Cap-SNase could be use to efficiently inhibit JEV replication. ► CTVI approach will be applicable to JEV inhibition as a novel anti viral strategy.Japanese encephalitis virus (JEV) is a leading member of the mosquito-transmitted flavivirus family, and is mainly distributed in China, India and South East Asia, where it can cause the central nervous system disease with irreversible neurological damage in humans and animals. Few effective anti viral drugs are currently available against JEV infections. To explore the feasibility of using capsid-targeted viral inactivation (CTVI), as an anti viral strategy against JEV infection, a plasmid pcDNA-Cap-SNase was constructed for expressing a fusion protein of JEV capsid (Cap) and Staphylococcus aureus nuclease (SNase). Under G418 selection, a mammalian cell line BHK-21/Cap-SNase stably expressing Cap-SNase fusion proteins could be detected by rabbit antiserum against JEV and had good nuclease activity in degrading DNA or RNA. The viral titer from JEV-infected BHK-21/Cap-SNase cell line was reduced about 69.7% compared with that produced in control BHK-21 cells. It was clearly demonstrated that Cap-SNase fusion proteins could be use to efficiently inhibit JEV replication, resulting in a reduction of viral titer. Therefore, the CTVI approach might be applicable to JEV inhibition as a novel anti viral strategy.

Keywords: Capsid-targeted viral inactivation (CTVI); Japanese encephalitis virus (JEV); Capsid protein (Cap); Staphylococcus aureus nuclease (SNase); Anti viral strategy

Evaluation of antiherpetic activity of crude extract and fractions of Avicenna marina, in vitro by Mandana Behbahani; Mehrnaz Shanehsaz Zadeh; Hassan Mohabatkar (pp. 376-380).

► Herpes simplex virus 2 (HSV-2) is a harmful pathogen especially in highly susceptible individuals. ► Avicenna marina has been used to treat some diseases such as ulcers, rheumatism and small pox. ► Previously, the antiviral activity of A. marina has been investigated against poliovirus and herpes simplex virus type-1. ► In the present study loteolin and luteolin 7-O-methylether 3′-O-beta-d-glucoside (LMEG) isolated from A. marina.This study was carried out to check antiherpetic substances of crude methanol leaf extract of Avicenna marina and its column chromatographic fractions.Herpes simplex virus 2 (HSV-2) is a harmful pathogen especially in highly susceptible individuals.The antiherpetic activity of crude methanol extract and sub-fractions was performed in different concentrations (20, 2, 0.2, and 0.02μg/ml) by use of plaque-forming unit (PFU) assay and real time polymerase chain reaction (PCR) assay.The most active fraction analyzed by NMR contained luteolin 7-O-methylether 3′-O-beta-d-glucoside (LMEG). The other active fraction was detected by HPLC as luteolin. The apparent effective concentrations for 50% plaque reduction (EC50) of crude methanol extract, LMEG, luteolin and ACV were 10, 5, 16.6 and 2.97μg/ml, respectively. The three extracts showed no cytotoxic effect on Vero cell line at concentrations of 32μg/ml or below. According to the consequences of time-of-addition studies, antiherpetic compound LMEG exerted an inhibitory effect on the early stage of HSV-2 infection during which it was added.In conclusion, LMEG isolated from A. marina could probably inhibit HSV attachment to the cell membrane and its entry into the cell.

Keywords: Avicenna marina; HSV-2; Antiviral activity; Flavonoid; Real time PCR

Corrigendum to: “Rhinovirus infections and immunisation induce cross-serotype reactive antibodies to VP1” [Antiviral Res. 95(3) (2012) 193–201] by Gary R. McLean; Ross P. Walton; Shweta Shetty; Tamlyn J. Peel; Nasren Paktiawal; Tatiana Kebadze; Leila Gogsadze; Katarzyna Niespodziana; Rudolf Valenta; Nathan W. Bartlett; Sebastian L. Johnston (pp. 381-381).

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Antiviral Research (v.97, #3)