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Antiviral Research (v.75, #2)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Insertions in the β3–β4 loop of reverse transcriptase of human immunodeficiency virus type 1 and their mechanism of action, influence on drug susceptibility and viral replication capacity by Dirk Eggink; Marleen C.D.G. Huigen; Charles A.B. Boucher; Matthias Götte; Monique Nijhuis (pp. 93-103).

Introduction of antiretroviral therapy combining protease and reverse transcriptase (RT) inhibitors has dramatically improved the quality of life and survival of patients infected with the human immunodeficiency virus (HIV). However, effective long-term therapy of HIV-infection has been severely hampered by the development of drug resistance. Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions in the target gene of the drug. Yet, occasionally gene insertions are being observed. The most commonly observed insertion is seen during substrate analogue RT inhibitor therapy and is selected in the β3–β4 loop of the RT enzyme. This flexible loop is located in the fingers subdomain of the enzyme and plays an important role in substrate binding. The acquisition of drug resistance related mutations or insertions might come at a price, which is reduced performance of the enzyme resulting in a diminished replication capacity of the virus. Various types of insertions have been described, and, in this review, we have summarized these data and discussed the mechanism of action of the RT inserts and their impact on both drug susceptibility and replication capacity.

Keywords: Human immunodeficiency virus; Insertion; Reverse transcriptase; Drug resistance

Correlation between breakdown of the blood–brain barrier and disease outcome of viral encephalitis in mice by Aaron L. Olsen; John D. Morrey; Donald F. Smee; Robert W. Sidwell (pp. 104-112).

Changes in the permeability of the blood–brain barrier (BBB) were evaluated in two mouse models of viral encephalitis. The ability of sodium fluorescein (NaFl) to cross the BBB from the serum into the central nervous system was assayed in animals inoculated with virulent strains of either Banzi or Semliki Forest viruses. To test the hypothesis that increases in BBB permeability were associated with poor disease outcome subsequent experiments measured BBB permeability in conjunction with treatment with the interferon inducer Ampligen (poly I:poly C₁₂U). A single intraperitoneal injection of Ampligen (1mg/kg) administered either 24h or 4–6h before, but not 24h after, virus inoculation with Banzi virus provided significant improvements in survival, viral brain titers, weight change and BBB permeability. In comparison, a similar treatment with Ampligen administered either 24h or 4–6h before inoculation with Semliki Forest virus was able to significantly improve weight change, and BBB permeability, but only animals receiving Ampligen 4–6h pre-virus showed a significantly improved mortality. In general, it was found that evaluation of BBB permeability was a more sensitive indicator of disease outcome and the antiviral efficacy Ampligen than either weight change or brain viral titers.

Keywords: Blood–brain barrier; Banzi virus; Semliki Forest virus; Viral encephalitis; Ampligen

Human monoclonal antibody against Hepatitis B virus surface antigen (HBsAg) by Yong-Won Shin; Kyung-Hwan Ryoo; Kwang-Won Hong; Ki-Hwan Chang; Jin-Seol Choi; Minyoung So; Pan-Kyung Kim; Jie-Young Park; Ki-Tae Bong; Se-Ho Kim (pp. 113-120).

Hepatitis B virus (HBV) is one of the main pathogens responsible for hepatitis and hepatocellular carcinoma. Human plasma-derived Hepatitis B immune globulin (HBIG) is being used for prophylactic and liver transplantation currently. However, it may be necessary to replace a HBIG with a recombinant one because of limited availability of human plasma with high anti-HBsAg antibody titer and possible contamination of human pathogens. A Chinese hamster ovary (CHO) cell line, HB-C7A, was established which produces a fully human IgG1 that binds HBsAg. The HB-C7A exhibits ∼2600units/mg of antibody. The affinity ( K_a) of HB-C7A is 1.1×10⁸M⁻¹ by Biacore analysis and estimated 6.7-fold higher than that of Hepabig^® (a plasma-derived HBIG from Green Cross Corp., Yongin, Korea) by competition ELISA. The HB-C7A recognizes the conformational “a” determinant of HBsAg and binds HBV particle more efficiently than the Hepabig^®. The HB-C7A binds to HBV-infected human liver tissue but does not bind to normal human tissues. This HB-C7A has several advantages compared to plasma-derived Hepabig^® such as activity, safety and availability.

Keywords: Human antibody; Hepatitis B virus (HBV); Hepatitis B virus surface antigen (HBsAg); Hepatitis B immune globulin (HBIG); Chinese hamster ovary (CHO) cell; Tissue cross-reactivity

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus by Márcia Cristina Livonesi; Ricardo Luiz Moro de Sousa; Soraya Jabur Badra; Luiz Tadeu Moraes Figueiredo (pp. 121-128).

Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses ( Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-α) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-α, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-α present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3h after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-α could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus.

Keywords: Interferon-alpha; Orthobunyavirus; Bunyaviridae; Plaque assay; Suckling mice

Inhibition of replication of primary HIV-1 isolates in huPBL-NOD/Scid mice by antibodies from HIV-1 infected patients by Sophia Steyaert; Leo Heyndrickx; Lieven Verhoye; Tine Vermoesen; Helen Donners; Katrien Fransen; Filip Van Wanzeele; Beatrijs Vandergucht; Guido Vanham; Geert Leroux-Roels; Peter Vanlandschoot (pp. 129-138).

Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.

Keywords: HIV-1; Primary virus; Neutralization; Antibodies; huPBL-NOD/Scid

In vitro resistance to interferon-alpha of hepatitis B virus with basic core promoter double mutation by Yan Wang; Lai Wei; Dong Jiang; Xu Cong; Ran Fei; Hongsong Chen; Jiang Xiao; Yu Wang (pp. 139-145).

The hepatitis B virus (HBV) genome basic core promoter (BCP) modulates HBeAg secretion at the transcriptional level. In addition to pre-core mutations, variations in the BCP are related to hepatitis B e antigen (HBeAg)-negative chronic hepatitis B. HBeAg-negative chronic hepatitis B patients show a lower sustained response to interferon (IFN). The aim of this study was to determine if there is a relationship between HBV BCP mutation and sensitivity of HBV to IFN-alpha in vitro. BCP mutations were introduced by site-directed mutagenesis and the entire genomes of wild-type and mutant HBV were transiently transferred into Huh7 cells by calcium phosphate transfection. With or without IFN-alpha, viral products in the culture medium and viral replication intermediates in the cytoplasm were detected 3 days after transfection. The amount of hepatitis B surface antigen (HBsAg) secreted by wild-type HBV and the BCP mutant was similar, while HBeAg secreted by the mutant was decreased by 35.4%. HBV particles and replication intermediates of the BCP mutant were increased. After IFN-alpha was added, HBeAg, HBV DNA and HBV replication intermediates decreased for both the wild-type HBV (by 25.7%, 31.8%, 29.8%, respectively) and the BCP mutant (by 8.4%, 27.4%, 10.1%, respectively). These data indicate that HBV harboring the BCP double mutation has stronger replication competence and lower sensitivity to IFN-alpha than wild-type.

Keywords: Abbreviations; HBeAg; hepatitis B e antigen; BCP; basic core promoter; HBV; hepatitis B virus; IFN; interferon; HBsAg; hepatitis B surface antigen; ORFs; open reading frames; nt; nucleotide; dNTPs; deoxynucleoside triphosphate; ELISA; enzyme-linked immunosorbent assay; pgRNA; pregenome RNA; CURS; core upstream regulatory sequences; NRE; negative regulatory elementHepatitis B virus; Basic core promoter; Mutation; Interferon-alpha

Adefovir dipivoxil treatment of lamivudine-resistant chronic hepatitis B by Chia-Yen Dai; Wan-Long Chuang; Ming-Yen Hsieh; Li-Po Lee; Jee-Fu Huang; Nai-Jen Hou; Zu-Yau Lin; Shinn-Cherng Chen; Ming-Yuh Hsieh; Liang-Yen Wang; Jun-Fa Tsai; Wen-Yu Chang; Ming-Lung Yu (pp. 146-151).

Adefovir dipivoxil (ADV)-resistant mutations have been identified in treating hepatitis B virus (HBV) infection. This study aimed to analyze the response, the incidence of ADV resistance and the virologic characteristics of ADV therapy. A total of 29 CHB patients with confirmed lamivudine (LAM)-resistant HBV were treated with ADV for more than 52 weeks. Serum HBV DNA, HBV genotypes and sequences of HBV polymerase reverse-transcriptase domain were determined. Rates for the biochemical response, HBeAg loss, HBeAg seroconversion and virologic response (<200copies/mL of HBV DNA) were 82.8, 23.5, 11.8, and 48.3%, respectively, at week 52 of treatment. Lower pre-treatment mean HBV DNA level was the only significant factor associated with negative HBV DNA after ADV therapy. Six (20.7%) patients had clearance of LAM-resistant YMDD variants with replacement by the wild type HBV at week 52. The rtN236T, rtA181V/T and rtI233V were not identified before ADV therapy and the genotypic mutation of rtN236T was detected in one (3.4%) patient. In conclusion, the 52-week ADV treatment for patients with LAM-resistant HBV variants significantly achieved normalization of ALT levels, reduced serum HBV DNA levels and induced HBeAg loss and seroconversion. The emergence of ADV-resistant mutations seemed rare at weeks 52.

Keywords: Adefovir dipivoxil; Lamivudine; Resistance; CHB; HBV

Suppression of virus replication via down-modulation of mitochondrial short chain enoyl-CoA hydratase in human glioblastoma cells by Megumi Takahashi; Eiji Watari; Eiji Shinya; Takako Shimizu; Hidemi Takahashi (pp. 152-158).

Several viruses have been demonstrated to be the etiologic agent in chronic progressive diseases, associated with persistence; however, major questions concerning the pathogenic mechanisms of viral persistence are still unanswered. With the aim of identifying host cellular proteins that may play a role in viral replication, we established long-term persistently infected human glioblastoma cell lines with mutant measles virus (MV) and analyzed the host proteins by two-dimensional gel electrophoresis (2-DE) with mass spectrometry. We observed significant down-modulation in the expression of mitochondrial short chain enoyl-CoA hydratase (ECHS), which catalyzes the β-oxidation pathway of fatty acid. Knockdown of this gene by a short interference RNA (siRNA) apparently impaired wild-type MV replication and the cytopathic effects (CPEs) of MV were significantly reduced in siRNA-transfected cells. These findings will shed light upon a new important notion for the interaction between virus replication and lipid metabolism in host cells and might provide a new strategy for virus control.

Keywords: Measles virus; Persistent infection; Mitochondrial short chain enoyl-CoA hydratase; β-Oxidation; Short interference RNA

Emergence of human immunodeficiency virus type 1 variants containing the Q151M complex in children receiving long-term antiretroviral chemotherapy by Shigeyoshi Harada; Rohan Hazra; Sadahiro Tamiya; Steven L. Zeichner; Hiroaki Mitsuya (pp. 159-166).

We examined 28 children with HIV-1 infection who were not responding to existing antiviral regimens and were enrolled into clinical trials conducted at the National Cancer Institute to receive salvage therapy. In 3 of the 28 patients (10.7%), the Q151M complex amino acid substitutions were identified. The three patients had received nucleoside reverse transcriptase inhibitor (NRTI) monotherapy and/or combination regimens with multiple NRTIs for 4.3–8.6 years prior to the study. Recombinant infectious clones generated by incorporating the RT-encoding region of HIV-1 isolated from patients’ plasma samples were highly resistant to zidovudine, didanosine and stavudine, while they were moderately resistant to lamivudine and tenofovir disoproxil fumarate (TDF). TDF-containing regimens reduced HIV-1 viremia in two of the three children carrying the Q151M complex. These data suggest that the Q151M could be prevalent in pediatric patients with long-term NRTI monotherapy and/or dual NRTI regimens and that HAART regimens containing TDF may be meritorious in such patients.

Keywords: Multi-dideoxynucleoside resistance; Reverse transcriptase; HIV-1; Q151M; Pediatric AIDS

Identification of Herpes TATT-binding protein by Lucjan S. Wyrwicz; Leszek Rychlewski (pp. 167-172).

The regulation of viral gene expression is a compilation of virus and host factors influencing the transcription machinery. In Epstein-Barr Virus (EBV) a distinct regulatory element utilizing the TATT-box was described. The motif is present in promoters of lytic cycle genes and resembles a crucial host genome motif (TATA-box). Since the binding specificity of eukaryotic proteins recognizing TATA-box (TBP) was determined and no specific preference for interaction with TATT motif was found, we performed a genome-wide fold recognition search to identify viral proteins potentially recognizing the TATT-box. By applying profile–profile comparisons and homology-based protein structure prediction we identified a protein of unknown function from Gammaherpesvirinae (BcRF1 of EBV) and their Betaherpesvirinae homologs (UL87 of CMV) as proteins encoding TBP fold. Although overall sequence identity is very low (circa 10%), the saddle-like fold and presence of important residues on a surface of DNA–protein interface marked both proteins as distantly related to TBP and permitted the characterization of a putative molecular basis of selective recognition of TATT-motif by BcRF1.

Keywords: Abbreviations; TBP; TATA-binding protein; PDB; Protein Data Bank Herpesviridae; Regulation of transcription; Promoter; Protein structure prediction; Bioinformatics

The emergence of adamantane resistance in influenza A(H1) viruses in Australia and regionally in 2006 by I.G. Barr; A.C. Hurt; N. Deed; P. Iannello; C. Tomasov; N. Komadina (pp. 173-176).

The adamantanes (amantadine and rimantadine) were the first antivirals licensed for use against influenza A viruses and have been used in some countries to control seasonal influenza. While increasing resistance of A(H3) viruses to this class of drug has been reported in recent years, only low levels of resistance were seen with A(H1) viruses until the 2005–2006 influenza season in the USA. In this study we analysed 101 human influenza A viruses isolated in 2006 that were referred to the WHO Collaborating Centre for Reference and Research in Melbourne, from Australia and the surrounding regions, for evidence of resistance to adamantanes. We found that whereas previously A(H1) resistant viruses were rare, 21.8% of the 2006 viruses had a resistant genotype. By comparison, 58.6% of influenza A(H3) viruses isolated in 2006 that were tested at the Centre, had a resistant genotype.

Keywords: Amantadine; Rimantadine; Matrix gene; Resistance; Australia; South East Asia

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Antiviral Research (v.75, #2)