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Antiviral Research (v.97, #1)

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

Prelim II: Editorial Board (pp. ii).

Traditional Chinese herbal medicine as a source of molecules with antiviral activity by Ting Li; Tao Peng (pp. 1-9).

► Many traditional Chinese herbal medicines (TCHM) are used in China for the treatment of viral infections. ► These TCHM may contain drug-like molecules with antiviral activity. ► Novel antiviral compounds may potentially be identified in TCHM through activity-guided fractionation. ► Research on active molecules in TCHM is being aided by the development of large databases.Traditional Chinese herbal medicine (TCHM) is widely used in the prevention and treatment of viral infectious diseases. However, the operative mechanisms of TCHM remain largely obscure, mainly because of its complicated nature and the fragmented nature of research. In recent years, systematic methodologies have been developed to discover the active compounds in TCHM and to elucidate its underlying mechanisms. In this review, we summarize recent progress in TCHM-based antiviral research in China and other Asian countries. In particular, this review focuses on progress in targeting key steps in the viral replication cycle and key cellular components of the host defense system. Recent developments in centralized and standardized TCHM screening and databases are also summarized.

Keywords: Abbreviations; TCHM; traditional Chinese herbal medicine; TCM; traditional Chinese medicine; HIV; human immunodeficiency virus; HSV; herpes simplex virus (type 1 and 2); Flu; influenza; HBV; hepatitis B virus; HCV; hepatitis C virus; HCMV; human cytomegalovirus; EVs; enteroviruses; EV71; enterovirus 71; SARS-CoV; SARS coronavirus; NV; norovirus; FMDV; foot-and-mouth disease virus; AdV; adenovirus; PIV; parainfluenza virusTraditional Chinese herbal medicine; Antiviral therapy; Antiviral drugs; Activity-guided fractionation

An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs by Mirza Ibrišimović; Doris Kneidinger; Thomas Lion; Reinhard Klein (pp. 10-23).

► AmiRNAs can be utilized to inhibit adenovirus replication in vitro. ► Adenoviral vectors are an effective expression vehicle for anti-adenoviral amiRNAs. ► A combination of amiRNA and cidofovir results in additive effects.Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections ( i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

Keywords: Adenovirus; RNA interference; microRNA

Heterosubtypic protection against influenza A induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin by Zuzana Staneková; Irena Adkins; Martina Kosová; Jana Janulíková; Peter Šebo; Eva Varečková (pp. 24-35).

► Adenylate cyclase toxoid (CyaA-E5) is used to deliver influenza (IAV) HA2 to APCs. ► CyaA-E5–HA2 specifically targets and penetrates CD11b-expressing dendritic cells. ► This immunogen primes HA2_93–102, HA2_96–104 and HA2_170–178-specific T-cell response. ► CyaA-E5–HA2 elicits also broadly cross-reactive HA2-specific antibody response. ► CyaA-E5–HA2 immunization of mice is cross-protective against lethal IAV infection.The protective efficacy of currently available influenza vaccines is restricted to vaccine strains and their close antigenic variants. A new strategy to obtain cross-protection against influenza is based on conserved antigens of influenza A viruses (IAV), which are able to elicit a protective immune response. Here we describe a vaccination approach involving the conserved stem part of hemagglutinin, the HA2 subunit, shared by different HA subtypes of IAV. To increase its immunogenicity, a novel strategy of antigen delivery to antigen presenting cells (APCs) has been used. The HA2 segment (residues 23–185) was inserted into a genetically detoxified adenylate cyclase toxoid (CyaA-E5) which specifically targets and penetrates CD11b-expressing dendritic cells. The CyaA-E5–HA2 toxoid induced HA2_93–102, HA2_96–104 and HA2_170–178-specific and Th1 polarized T-cell responses, and also elicited strong broadly cross-reactive HA2-specific antibody response. BALB/c mice immunized with three doses of purified CyaA-E5–HA2 without any adjuvant recovered from influenza infection 2days earlier than the control mock-immunized mice. More importantly, immunized mice were protected against a lethal challenge with 2LD₅₀ dose of a homologous virus (H3 subtype), as well as against the infection with a heterologous (H7 subtype) influenza A virus. This is the first report on heterosubtypic protection against influenza A infection mediated by an HA2-based vaccine that can induce both humoral and cellular immune responses without the need of adjuvant.

Keywords: Abbreviations; IAV; influenza A virus; HA; hemagglutinin; HA2; the light chain of hemagglutinin; APCs; antigen presenting cells; CyaA; adenylate cyclase; CyaA-E5; adenylate cyclase toxoid; CyaA-E5–OVA; adenylate cyclase toxoid with inserted OVA epitope; CyaA-E5–HA2; adenylate cyclase toxoid with inserted HA2 epitopes; VN antibodies; virus neutralizing antibodies; CTL; cytotoxic T cells; A/Miss; influenza virus A/Mississippi/1/85 (H3N2); A/Chick; influenza virus A/Chicken Germany/34 (H7N1); LD; ₅₀; lethal dose of virus at which 50% of infected mice died; DCs; dendritic cells; PBS; phosphate buffered saline; EHA2; ectodomain of HA2 aa 23–185; NP; influenza A nucleoprotein; IFN; interferon Bordetella; adenylate cyclase toxoid; Influenza A infection; Cross-protection; Conserved epitopes; HA2 of influenza A hemagglutinin; Humoral and cell immune response

The changing face of hepatitis C in the new era of direct-acting antivirals by Vincent Soriano; Pablo Labarga; Jose V. Fernández-Montero; Jose M. Benito; Eva Poveda; Norma Rallon; Clara Sánchez; Eugenia Vispo; Pablo Barreiro (pp. 36-40).

► The arrival of direct-acting antivirals (DAA) is transforming the way hepatitis C virus (HCV) infection is managed. ► Continuous HCV replication is associated with extrahepatic manifestations. ► “ Test & treat strategies” for hepatitis C should be encouraged. ► First-generation DAA will soon be replaced with more potent, safer and convenient drugs. ► High cost of HCV therapies will result in progressive marginalization of HCV populations.The approval of the first protease inhibitors as treatment for hepatitis C virus (HCV) infection is rapidly transforming the way patients with chronic hepatitis C are managed. Treatment regimens are moving to combinations given for shortened periods, excluding poorly tolerated subcutaneous interferon, and providing rates of cure exceeding 75%. The recognition of HCV infection as a systemic disease, not limited to producing liver damage, in which extrahepatic complications play a major role as the cause of morbidity and mortality, is prompting the treatment of a growing number of HCV-infected individuals. However, new challenges are emerging, including the need to diagnose a substantial proportion of asymptomatic carriers, the risk of potentially harmful drug–drug interactions and the high cost of medications. The future will probably see a progressive marginalization of residual HCV populations, with increasing over-representation of illegal immigrants, alcohol abusers, intravenous drug users and the mentally disabled.

Keywords: Hepatitis C; Direct-acting antivirals; Human immunodeficiency virus; Telaprevir; Boceprevir; Antiviral therapy

Differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin A and baicalein in H5N1 influenza A virus-infected cells by Patchima Sithisarn; Martin Michaelis; Manfred Schubert-Zsilavecz; Jindrich Cinatl Jr. (pp. 41-48).

► 22 Flavonoids were examined for activity against H5N1 influenza A viruses. ► Biochanin A and baicalein exerted the highest potency. ► Biochanin A and baicalein interfered with H5N1 replication. ► Biochanin A and baicalein interfered with virus-induced cytokine expression. ► Biochanin A and baicalein differ in their molecular antiviral mechanisms.From a panel of 22 flavonoids, we identified six compounds (apigenin, baicalein, biochanin A, kaempferol, luteolin, naringenin) that inhibited influenza A nucleoprotein production in human lung epithelial (A549) cells infected with the highly pathogenic avian influenza H5N1 virus strain A/Thailand/Kan-1/04 in non-toxic concentrations. Baicalein ( IC₅₀: 18.79±1.17μM, selectivity index 5.82) and biochanin A ( IC₅₀ 8.92±1.87μM, selectivity index 5.60) were selected for further experiments. Both compounds reduced H5N1 infectious titres (baicalein 40μM: 29-fold reduction, biochanin A 40μM: 55-fold reduction after infection at MOI 0.01), virus-induced caspase 3 cleavage, nuclear export of viral RNP complexes, and enhanced the effects of the neuraminidase inhibitor zanamivir. Biochanin A and baicalein also inhibited the replication of the H5N1 strain A/Vietnam/1203/04. Time of addition experiments indicated that both compounds interfere with H5N1 replication after the adsorption period. Further mechanistic investigations revealed clear differences between these two flavonoids. Only baicalein interfered with the viral neuraminidase activity (39±7% inhibition at 100μM, the maximum concentration tested). In contrast to baicalein, biochanin A affected cellular signalling pathways resulting in reduced virus-induced activation of AKT, ERK 1/2, and NF-kB. Moreover, biochanin A inhibited the virus-induced production of IL-6, IL-8, and IP-10 while baicalein inhibited IL-6 and IL-8 production without affecting IP-10 levels. In primary human monocyte-derived macrophages, only baicalein but not biochanin A impaired H5N1 virus replication. Both flavonoids interfered with the H5N1-induced production of IL-6, IP-10, and TNF-α but not of IL-8 in macrophages. These findings indicate that closely related flavonoids can exert anti-H5N1 effects by different molecular mechanisms.

Keywords: H5N1; Biochanin A; Baicalein; Antiviral; Anti-inflammatory; Flavonoid

Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN) by Maria A. González-Carmona; Maria Quasdorff; Annabelle Vogt; Anja Tamke; Yildiz Yildiz; Per Hoffmann; Thomas Lehmann; Ralf Bartenschlager; Joachim W. Engels; Gerd A. Kullak-Ublick; Tilman Sauerbruch; Wolfgang H. Caselmann (pp. 49-59).

► 17mer Phosphorothioate modified ODN (tS-ODN) binding HCV5′NCR inhibit HCV translation. ► Conjugation of tS-ODN to taurocholate increases selective ODN uptake by hepatocytes. ► Taurocholate conjugated tS-ODNs enhance inhibition of HCV translation in liver cells.The 5′-noncoding region (5′NCR) of the HCV-genome comprises an internal ribosome entry site essential for HCV-translation/replication. Phosphorothioate oligodeoxynucleotides (tS-ODN) complementary to this region can inhibit HCV-translation in vitro. In this study, bile acid conjugated tS-ODN were generated to increase cell-selective inhibition of 5′NCR-dependent HCV-translation.Different bile acid conjugated tS-ODN complementary to the HCV5′NCR were selected for their inhibitory potential in an in vitro transcription/translation assay. To analyze OATP (organic anion transporting polypeptides)-selective uptake of bile acid conjugated ODN, different hepatoma cells were stably transfected with the OATP1B1-transporter and primary human hepatocytes were used. An adenovirus encoding the HCV5′NCR fused to the luciferase gene (Ad-GFP-NCRluc) was generated to quantify 5′NCR-dependent HCV gene expression in OATP-overexpressing hepatoma cells and in vivo.A 17mer phosphorothioate modified ODN (tS-ODN4_13) complementary to HCV5′NCR was able to inhibit 5′NCR-dependent HCV-translation in an in vitro transcription/translation test system by more than 90% and it was also effective in Huh7-cells containing the HCV subgenomic replicon. Conjugation to taurocholate (tS-ODN4_13T) significantly increased selective ODN uptake by primary human hepatocytes and by OATP1B1-expressing HepG2-cells compared to parental HepG2-cells. Correspondingly, tS-ODN4_13T significantly inhibited HCV gene expression in liver-derived OATP1B1-expressing HepG2- or CCL13-cells up to 70% compared to unconjugated tS-ODN and compared to mismatch taurocholate coupled tS-ODN. In vivo, tS-ODN4_13T showed also a trend to block 5′NCR-dependent HCV gene expression.The tested taurocholate conjugated 17mer antisense ODN complementary to HCV5′NCV showed an increased and selective uptake by hepatocytes and liver-derived cells through OATP-mediated transport resulting in enhanced specific inhibition of HCV gene expression in vitro and in vivo. Thus, this novel approach may represent a promising strategy to improve antisense approaches with ODN in the control of hepatitis C infection.

Keywords: Abbreviations; Ad; adenovirus vector; FITC; fluorescein isothiocyanate; GFP; green fluorescent protein; HCV; hepatitis C virus; MOI; multiplicity of infection; 5′NCR; 5′-noncoding region of HCV; IRES; internal ribosomal entry site; OATP; organic anion transporting polypeptide; ODN; oligodeoxynucleotide(s); RLU; relative light units; tS-ODN; phosphorothioate oligodeoxynucleotide; tS-ODN(T); taurocholate conjugated oligodeoxynucleotideAntisense oligodeoxynucleotides (ODN); Bile acid conjugated ODN; Hepatitis C virus (HCV); 5′ Noncoding region (5′NCR); Taurocholate conjugated ODN

Correlation between genotypic (V3 population sequencing) and phenotypic (Trofile ES) methods of characterizing co-receptor usage of HIV-1 from 200 treatment-naïve HIV patients screened for Study A4001078 by Simon Portsmouth; Srinivas Rao Valluri; Martin Däumer; Bernhard Thiele; Hernan Valdez; Marilyn Lewis; Charles Craig; Alexander Thielen; Ian James; James Demarest; Jayvant Heera (pp. 60-65).

► Assessment of tropism is needed to identify patients likely to respond to maraviroc. ► Concordance between tropism testing methods in screening samples was determined. ► Phenotypic and genotypic methods reported similar frequencies of R5 and non-R5 virus. ► Most samples had non-R5 HIV-1 RNA levels of <1 log₁₀ or ⩾4 log₁₀copies/mL. ► V3 genotyping is subject to fewer non-reportable results than phenotypic testing.Assessment of HIV-1 co-receptor usage is essential to identify patients who are likely to respond to maraviroc (MVC)-containing regimens. Co-receptor usage of plasma virus from all treatment-naïve patients screened for a MVC clinical trial was assessed using phenotypic and genotypic methodologies to evaluate concordance between testing methods and to assess the quantity of CXCR4-using (non-R5) virus in samples giving discordant results. Co-receptor usage was prospectively measured using the enhanced sensitivity Trofile assay (Trofile ES) to screen patients for enrollment in Study A4001078. Population and deep sequencing methodologies were utilized retrospectively to analyze all screening samples, with co-receptor usage determined using the geno2pheno algorithm. Concordance between methods was explored using descriptive statistics. The quantity of non-R5 virus in all samples was measured using deep sequencing. Trofile ES and matched genotype results were obtained for 199screening samples. Concordance of Trofile ES with population genotyping (5.75% false-positive rate [FPR]) and deep sequencing (3.5% FPR; 2% non-R5 threshold) was 91.7% and 89.6%, respectively. Population genotype data were available for all samples with non-reportable Trofile ES results; the distribution of co-receptor usage in this set was consistent with that in the overall population: 75% (12/16) R5 and 25% (4/16) non-R5. The majority of samples contained non-R5 plasma HIV-1 RNA estimated at either <1 log₁₀ (62.0%) or ⩾4 log₁₀ (30.5%) copies/mL; the absolute amount of detectable non-R5 virus remained stable between screening and baseline visits. Samples originally classified as non-R5 by Trofile ES but R5 by population sequencing had a relatively low absolute amount of non-R5 virus (mean 2.1%, median 0.1%). The determination of co-receptor usage using either Trofile ES or genotyping methodologies showed similar frequencies of R5 and non-R5 virus in this treatment-naïve study population. For both concordant and discordant samples, population sequencing appropriately identified R5 samples with low levels of non-R5-using virus.

Keywords: Abbreviations; ALT; alanine aminotransferase; AST; aspartate aminotransferase; ES; enhanced sensitivity; FPR; false-positive rate; g2p; geno2pheno; MVC; maraviroc; NR; non-reportable; QD; once daily; sff; Standard Flowgram FormatCXCR5; Genotype; HIV-1; Maraviroc; Phenotype

Inhibition of porcine reproductive and respiratory syndrome virus replication by flavaspidic acid AB by Qian Yang; Li Gao; Jianyong Si; Yipeng Sun; Jinhua Liu; Li Cao; Wen-hai Feng (pp. 66-73).

► Flavaspidic acid AB (FA-AB) inhibits PRRSV infection. ► FA-AB could partially inhibit PRRSV internalization and cell–cell spread. ► FA-AB could inhibit PRRSV replication. ► FA-AB could induce NF-α, INF-β, and IL1-β expression in PAM.Porcine reproductive and respiratory syndrome virus (PRRSV) represents a significant challenge to the swine industry worldwide. Current control strategies against PRRSV are still inadequate and there is an urgent need for new antiviral therapies. Flavaspidic acid AB (FA-AB) is a compound derived from Dryopteris crassirhizoma, a traditional antiviral Chinese medicine. Here, we first identified its anti-PRRSV activity through targeting multiple stages in PRRSV infection in vitro. Our studies demonstrated that FA-AB could inhibit the internalization and cell-to-cell spreading of PRRSV, but not block PRRSV binding to cells. By monitoring the kinetics of PRRSV replication, we showed that FA-AB significantly suppressed PRRSV replication when treatment was initiated 24h after virus infection. Furthermore, we confirmed that FA-AB was able to significantly induce IFN-α, IFN-β, and IL1-β expression in porcine alveolar macrophages, suggesting that induction of antiviral cytokines by FA-AB could contribute to FA-AB induced inhibition of PRRSV replication. In conclusion, we provide a foundation for the possibility to develop a new therapeutic agent to control PRRSV infection.

Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV); Flavaspidic acid AB (FA-AB); Alveolar macrophages; Therapeutic agent

Characterization of 8-hydroxyquinoline derivatives containing aminobenzothiazole as inhibitors of dengue virus type 2 protease in vitro by Huiguo Lai; G. Sridhar Prasad; Radhakrishnan Padmanabhan (pp. 74-80).

► A potent inhibitor of dengue protease with IC₅₀ of 0.91±0.05μM has been identified. ► The compound is an 8-hydroxyquinoline containing two aryl substitutions at 7 position. ► Kinetic analysis and molecular modeling support competitive mode of inhibition.Four serotypes of dengue virus (DENV1–4), mosquito-borne members of Flaviviridae family cause frequent epidemics causing considerable morbidity and mortality in humans throughout tropical regions of the world. There is no vaccine or antiviral therapeutics available for human use. In a previous study, we reported that compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of West Nile virus serine protease. In this study, we analyzed potencies of some compounds with (8-HQ)-aminobenzothiazole derivatives for inhibition of DENV2 protease in vitro. We identified analogs1–4 with 2-aminothiazole or 2-aminobenzothiazole scaffold with sub-micromolar potencies (IC₅₀) in the in vitro protease assays. The kinetic constant ( K_i) for the most potent 8-HQ-aminobenzothiazole inhibitor (compound1) with an IC₅₀ value of 0.91±0.05μM was determined to be 2.36±0.13μM. This compound inhibits the DENV2 NS2B/NS3pro by a competitive mode of inhibition.

Keywords: Dengue virus protease inhibitors; Molecular modeling; Molegro virtual docker; Kinetic analysis; Competitive mode of inhibition; Sub-micromolar IC; ₅₀

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Antiviral Research (v.97, #1)