Abstract
Viruses are obligatory intracellular parasites, whose replication depends on pathways and functions of the host cell. Consequently, it is difficult to define virus-specific functions as suitable targets for anti-infective therapy. However, significant progress has been made in the past 50 years towards the development of effective and specific antivirals. In particular, human immunodeficiency virus, hepatitis C virus, and hepatitis B virus, which cause chronic infections affecting millions of individuals world-wide, are a major focus of antiviral research. Initially, antivirals were mainly directed against virus-specific enzymes; more recently, drugs inhibiting the steps of virus entry or release have been developed. Rational approaches towards drug development, based on information about structure and function of viral proteins and molecular mechanisms of virus-host interactions, have become increasingly successful. Novel strategies currently explored in basic research or preclinical studies include approaches targeting host factors important for virus replication, the exploitation of the innate immune response system as well as the use of gene silencing strategies aimed at interfering with viral gene expression. Today, a number of effective virostatics targeting various viral replication steps are approved for treatment of important viral diseases. However, the use of these drugs is limited by the rapid development of antiviral resistance, which represents a central problem of current antiviral therapy.
Keywords: Antiviral Therapy, Severe Acute Respiratory Syndrome, Antiviral Drug, Severe Acute Respiratory Syndrome, Antiviral Resistance
Contributor Information
Hans-Georg Kräusslich, Email: hans-georg_kraeusslich@med.uni-heidelberg.de.
Ralf Bartenschlager, Email: ralf_bartenschlager@med.uni-heidelberg.de.
B. Müller, Email: Barbara_Mueller@med.uni-heidelberg.de
Hans-Georg Kräusslich, Email: hans-georg.kraeusslich@med.uni-heidelberg.de.
References
- Allaway GP. Development of Bevirimat (PA-457): first-in-class HIV maturation inhibitor. Retrovirology. 2006;3(Suppl 1):S8. doi: 10.1186/1742-4690-3-S1-S8. [DOI] [Google Scholar]
- Alymova IV, Taylor G, Portner A. Neuraminidase inhibitors as antiviral agents. Current drug targets. 2005;5:401–409. doi: 10.2174/156800505774912884. [DOI] [PubMed] [Google Scholar]
- Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science New York, N.Y. 2003;300:1763–1767. doi: 10.1126/science.1085658. [DOI] [PubMed] [Google Scholar]
- Baltimore D. Expression of animal virus genomes. Bacteriol Rev. 1971;35:235–241. doi: 10.1128/br.35.3.235-241.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baltimore D. Gene therapy. Intracellular immunization. Nature. 1988;335:395–396. doi: 10.1038/335395a0. [DOI] [PubMed] [Google Scholar]
- Bauer DJ. A history of the discovery and clinical application of antiviral drugs. Br Med Bull. 1985;41:309–314. doi: 10.1093/oxfordjournals.bmb.a072069. [DOI] [PubMed] [Google Scholar]
- Bauer DJ, Stvincent L, Kempe CH, Downie AW. Prophylactic treatment of small pox contacts with N-methylisatin beta-thiosemicarbazone (Compound 33t57, Marboran) Lancet. 1963;35:494–496. doi: 10.1016/S0140-6736(63)90230-7. [DOI] [PubMed] [Google Scholar]
- Berkhout B. RNA interference as an antiviral approach: targeting HIV-1. Curr Opin Mol Ther. 2004;6:141–145. [PubMed] [Google Scholar]
- Bieniasz PD. Late budding domains and host proteins in enveloped virus release. Virology. 2006;344:55–63. doi: 10.1016/j.virol.2005.09.044. [DOI] [PubMed] [Google Scholar]
- Bossart-Whitaker P, Carson M, Babu YS, Smith CD, Laver WG, Air GM. Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid. J Mol Biol. 1993;232:1069–1083. doi: 10.1006/jmbi.1993.1461. [DOI] [PubMed] [Google Scholar]
- Bourne CR, Finn MG, Zlotnick A. Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1. J Virol. 2006;80:11055–11061. doi: 10.1128/JVI.00933-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ. Identification of host proteins required for HIV infection through a functional genomic screen. Science. 2008;319:921–926. doi: 10.1126/science.1152725. [DOI] [PubMed] [Google Scholar]
- Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane resistance among influenza A viruses isolated early during the 2005–2006 influenza season in the United States. JAMA. 2006;295:891–894. doi: 10.1001/jama.295.8.joc60020. [DOI] [PubMed] [Google Scholar]
- Bunka DH, Stockley PG. Aptamers come of age — at last. Nat Rev. 2006;4:588–596. doi: 10.1038/nrmicro1458. [DOI] [PubMed] [Google Scholar]
- Damm EM, Pelkmans L. Systems biology of virus entry in mammalian cells. Cell Microbiol. 2006;8:1219–1227. doi: 10.1111/j.1462-5822.2006.00745.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Clercq E, Descamps J, De Somer P, Holy A. (S)-9-(2,3-Dihydroxypropyl)adenine: an aliphatic nucleoside analog with broad-spectrum antiviral activity. Science. 1978;5:563–565. doi: 10.1126/science.200.4341.563. [DOI] [PubMed] [Google Scholar]
- De Clercq E, Holy A. Acyclic nucleoside phosphonates: a key class of antiviral drugs. Nature reviews. 2005;4:928–940. doi: 10.1038/nrd1877. [DOI] [PubMed] [Google Scholar]
- Deres K, Schroder CH, Paessens A, Goldmann S, Hacker HJ, Weber O, Kramer T, Niewohner U, Pleiss U, Stoltefuss J, et al. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids. Science. 2003;299:893–896. doi: 10.1126/science.1077215. [DOI] [PubMed] [Google Scholar]
- Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer HJ. Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. Proc Natl Acad Sci USA. 1977;74:5716–5720. doi: 10.1073/pnas.74.12.5716. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eshleman SH, Mracna M, Guay LA, Deseyve M, Cunningham S, Mirochnick M, Musoke P, Fleming T, Glenn Fowler M, Mofenson LM, et al. Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012) Aids. 2001;15:1951–1957. doi: 10.1097/00002030-200110190-00006. [DOI] [PubMed] [Google Scholar]
- Eshleman SH, Hoover DR, Chen S, Hudelson SE, Guay LA, Mwatha A, Fiscus SA, Mmiro F, Musoke P, Jackson JB, et al. Nevirapine (NVP) resistance in women with HIV-1 subtype C, compared with subtypes A and D, after the administration of single-dose NVP. J Infect Dis. 2005a;192:30–36. doi: 10.1086/430764. [DOI] [PubMed] [Google Scholar]
- Eshleman SH, Hoover DR, Chen S, Hudelson SE, Guay LA, Mwatha A, Fiscus SA, Mmiro F, Musoke P, Jackson JB, et al. Resistance after single-dose nevirapine prophylaxis emerges in a high proportion of Malawian newborns. Aids. 2005b;19:2167–2169. doi: 10.1097/01.aids.0000194800.43799.94. [DOI] [PubMed] [Google Scholar]
- Flys TS, Chen S, Jones DC, Hoover DR, Church JD, Fiscus SA, Mwatha A, Guay LA, Mmiro F, Musoke P, et al. Quantitative analysis of HIV-1 variants with the K103N resistance mutation after single-dose nevirapine in women with HIV-1 subtypes A, C, and D. J Acquir Immune Defic Syndr. 2006;42:610–613. doi: 10.1097/01.qai.0000221686.67810.20. [DOI] [PubMed] [Google Scholar]
- Foy E, Li K, Sumpter R, Loo YM, Johnson CL, Wang C, Fish PM, Yoneyama M, Fujita T, Lemon SM, Gale M., Jr Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. PNAS. 2005;102:2986–2991. doi: 10.1073/pnas.0408707102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr, Haussinger D, Diago M, Carosi G, Dhumeaux D, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. New Engl J Med. 2002;347:975–982. doi: 10.1056/NEJMoa020047. [DOI] [PubMed] [Google Scholar]
- Galiana-Arnoux D, Dostert C, Schneemann A, Hoffmann JA, Imler JL. Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila. Nature immunology. 2006;7:590–597. doi: 10.1038/ni1335. [DOI] [PubMed] [Google Scholar]
- Gottwein E, Mukherjee N, Sachse C, Frenzel C, Majoros WH, Chi JT, Braich R, Manoharan M, Soutschek J, Ohler U, Cullen BR. A viral microRNA functions as an orthologue of cellular miR-155. Nature. 2007;450:1096–1099. doi: 10.1038/nature05992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, Guyomard C, Lucas J, Trepo C, Guguen-Guillouzo C. Infection of a human hepatoma cell line by hepatitis B virus. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:15655–15660. doi: 10.1073/pnas.232137699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein. J Virol. 2005;79:1613–1622. doi: 10.1128/JVI.79.3.1613-1622.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harris RS, Liddament MT. Retroviral restriction by APOBEC proteins. Nat Rev Immunol. 2004;4:868–877. doi: 10.1038/nri1489. [DOI] [PubMed] [Google Scholar]
- Haasnoot J, Berkhout B. RNA interference: its use as antiviral therapy. Handb Exp Pharmacol. 2006;173:117–150. doi: 10.1007/3-540-27262-3_7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamre D, Brownlee KA, Donovick R. Studies on the chemotherapy of vaccinia virus. II. The activity of some thiosemicarbazones. J Immunol. 1951;67:305–312. [PubMed] [Google Scholar]
- Hengel H, Koszinowski UH, Conzelmann KK. Viruses know it all: new insights into IFN networks. Trends Immunol. 2005;26:396–401. doi: 10.1016/j.it.2005.05.004. [DOI] [PubMed] [Google Scholar]
- Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995;373:123–126. doi: 10.1038/373123a0. [DOI] [PubMed] [Google Scholar]
- Hoofnagle JH, Seeff LB. Peginterferon and ribavirin for chronic hepatitis C. New Engl J Med. 2006;355:2444–2451. doi: 10.1056/NEJMct061675. [DOI] [PubMed] [Google Scholar]
- Jackson JB, Becker-Pergola G, Guay LA, Musoke P, Mracna M, Fowler MG, Mofenson LM, Mirochnick M, Mmiro F, Eshleman SH. Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission. Aids. 2000;14:F111–F115. doi: 10.1097/00002030-200007280-00001. [DOI] [PubMed] [Google Scholar]
- Johnson CL, Gale M., Jr CARD games between virus and host get a new player. Trends Immunol. 2006;27:1–4. doi: 10.1016/j.it.2005.11.004. [DOI] [PubMed] [Google Scholar]
- Kielian M, Rey FA. Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol. 2006;4:67–76. doi: 10.1038/nrmicro1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lai L, Han X, Chen H, Wei P, Huang C, Liu S, Fan K, Zhou L, Liu Z, Pei J, et al. Quaternary structure, substrate selectivity and inhibitor design for SARS 3C-like proteinase. Curr Pharm Des. 2006;12:4555–4564. doi: 10.2174/138161206779010396. [DOI] [PubMed] [Google Scholar]
- Lee EJ, Kantor R, Zijenah L, Sheldon W, Emel L, Mateta P, Johnston E, Wells J, Shetty AK, Coovadia H, et al. Breast-milk shedding of drug-resistant HIV-1 subtype C in women exposed to single-dose nevirapine. J Infect Dis. 2005;192:1260–1264. doi: 10.1086/444424. [DOI] [PubMed] [Google Scholar]
- Li F, Goila-Gaur R, Salzwedel K, Kilgore NR, Reddick M, Matallana C, Castillo A, Zoumplis D, Martin DE, Orenstein JM, et al. PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc Natl Acad Sci USA. 2003;100:13555–13560. doi: 10.1073/pnas.2234683100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li F, Wild C. HIV-1 assembly and budding as targets for drug discovery. Curr Opin Investig Drugs. 2005;6:148–154. [PubMed] [Google Scholar]
- Li XD, Sun L, Seth RB, Pineda G, Chen ZJ. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci USA. 2005;102:17717–17722. doi: 10.1073/pnas.0508531102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lilly F. Susceptibility to two strains of Friend leukemia virus in mice. Science. 1967;155:461–462. doi: 10.1126/science.155.3761.461. [DOI] [PubMed] [Google Scholar]
- Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, Dienstag JL, Heathcote EJ, Little NR, Griffiths DA, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology. 2003;125:1714–1722. doi: 10.1053/j.gastro.2003.09.033. [DOI] [PubMed] [Google Scholar]
- Luban J. Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J Virol. 2007;81:1054–1061. doi: 10.1128/JVI.01519-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ludwig S. Influenza viruses and MAP kinase cascades – novel targets for antiviral intervention. Signal Transduction. 2007;7:81–88. doi: 10.1002/sita.200600114. [DOI] [Google Scholar]
- Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. Peginterferon alfa-2b plus ribavirin compared with in-terferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet. 2001;358:958–965. doi: 10.1016/S0140-6736(01)06102-5. [DOI] [PubMed] [Google Scholar]
- Mansky LM, Temin HM. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol. 1995;69:5087–5094. doi: 10.1128/jvi.69.8.5087-5094.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matzen K, Elzaouk L, Matskevich AA, Nitzsche A, Heinrich J, Moelling K. RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides. Nat Biotechnol. 2007;25:669–674. doi: 10.1038/nbt1311. [DOI] [PubMed] [Google Scholar]
- Medawar PB, Medawar JS. Viruses. In: Medawar PB, Medawar JS, editors. Aristotle to zoos: a philosophical dictionary of biology. Cambridge, MA: Harvard University Press; 1983. p. 275. [Google Scholar]
- Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature. 2005;437:1167–1172. doi: 10.1038/nature04193. [DOI] [PubMed] [Google Scholar]
- Müller B, Kräusslich HG. Antiviral drugs. In: u. Rosenthal Offermanns., editor. Encyclopedic reference of molecular pharmacology. 2nd. Heidelberg: Springer Verlag; 2008. [Google Scholar]
- Münch J, Standker L, Adermann K, Schulz A, Schindler M, Chinnadurai R, hlmann PöS, Chaipan C, Biet T, Peters T, et al. Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide. Cell. 2007;129:263–275. doi: 10.1016/j.cell.2007.02.042. [DOI] [PubMed] [Google Scholar]
- Nisole S, Stoye JP, Saib A. TRIM family proteins: retroviral restriction and antiviral defence. Nat Rev Microbiol. 2005;3:799–808. doi: 10.1038/nrmicro1248. [DOI] [PubMed] [Google Scholar]
- Pelkmans L, Fava E, Grabner H, Hannus M, Habermann B, Krausz E, Zerial M. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature. 2005;436:78–86. doi: 10.1038/nature03571. [DOI] [PubMed] [Google Scholar]
- Petersen J, Dandri M, Mier W, Lütgehetmann M, Volz T, von Weizsäcker F, Haberkorn U, Fischer L, Pollok JM, Erbes B, Seitz S, Urban S. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat Biotechnol. 2008;26:335–341. doi: 10.1038/nbt1389. [DOI] [PubMed] [Google Scholar]
- Pornillos O, Garrus JE, Sundquist WI. Mechanisms of enveloped RNA virus budding. Trends Cell Biol. 2002;12:569–579. doi: 10.1016/S0962-8924(02)02402-9. [DOI] [PubMed] [Google Scholar]
- Ray N, Doms RW. HIV-1 coreceptors and their inhibitors. Current topics in microbiology and immunology. 2006;303:97–120. doi: 10.1007/978-3-540-33397-5_5. [DOI] [PubMed] [Google Scholar]
- Seth RB, Sun L, Chen ZJ. Antiviral innate immunity pathways. Cell research. 2006;16:141–147. doi: 10.1038/sj.cr.7310019. [DOI] [PubMed] [Google Scholar]
- Shapiro RL, Thior I, Gilbert PB, Lockman S, Wester C, Smeaton LM, Stevens L, Heymann SJ, Ndung'u T, Gaseitsiwe S, et al. Maternal single-dose nevirapine versus placebo as part of an antiretroviral strategy to prevent mother-to-child HIV transmission in Botswana. Aids. 2006;20:1281–1288. doi: 10.1097/01.aids.0000232236.26630.35. [DOI] [PubMed] [Google Scholar]
- Sieczkarski SB, Whittaker GR. Viral entry. Curr Top Microbiol Immunol. 2005;285:1–23. doi: 10.1007/3-540-26764-6_1. [DOI] [PubMed] [Google Scholar]
- Snell NJ. Ribavirin—current status of a broad spectrum antiviral agent. Expert opinion on pharmacotherapy. 2001;2:1317–1324. doi: 10.1517/14656566.2.8.1317. [DOI] [PubMed] [Google Scholar]
- Steeves R, Lilly F. Interactions between host and viral genomes in mouse leukemia. Ann Rev Genetics. 1977;11:277–296. doi: 10.1146/annurev.ge.11.120177.001425. [DOI] [PubMed] [Google Scholar]
- Sticht J, Humbert M, Findlow S, Bodem J, Muller B, Dietrich U, Werner J, Krausslich HG. A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol. 2005;12:671–677. doi: 10.1038/nsmb964. [DOI] [PubMed] [Google Scholar]
- Stockman LJ, Bellamy R, Garner P. SARS: systematic review of treatment effects. PLoS Med. 2006;3:e343. doi: 10.1371/journal.pmed.0030343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taylor JM, Quilty D, Banadyga L, Barry M. The vaccinia virus protein F1L interacts with Bim and inhibits activation of the pro-apoptotic protein Bax. The Journal of biological chemistry. 2006;281:39728–39739. doi: 10.1074/jbc.M607465200. [DOI] [PubMed] [Google Scholar]
- ter Brake O, Konstantinova P, Ceylan M, Berkhout B. Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther. 2006;14:883–892. doi: 10.1016/j.ymthe.2006.07.007. [DOI] [PubMed] [Google Scholar]
- von Laer D, Hasselmann S, Hasselmann K. Gene therapy for HIV infection: what does it need to make it work? J Gene Med. 2006;8:658–667. doi: 10.1002/jgm.908. [DOI] [PubMed] [Google Scholar]
- Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, A., Habermann A, Kräusslich HG, Mizokami M, Bartenschlager R, Liang TJ. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature medicine. 2005;11:791–796. doi: 10.1038/nm1268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang XH, Aliyari R, Li WX, Li HW, Kim K, Carthew R, Atkinson P, Ding SW. RNA interference directs innate immunity against viruses in adult Drosophila. Science. 2006;312:452–454. doi: 10.1126/science.1125694. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against viruses. Nature. 2001;411:834–842. doi: 10.1038/35081168. [DOI] [PubMed] [Google Scholar]
- Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, Lifson JD, Bonhoeffer S, Nowak MA, Hahn BH, et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature. 1995;373:117–122. doi: 10.1038/373117a0. [DOI] [PubMed] [Google Scholar]
- Wensing AM, van, de, Vijver DA, Angarano G, Asjo B, Balotta C, Boeri E, Camacho R, Chaix ML, Costagliola D, De Luca A, et al. Prevalence of drug-resistant HIV-1 variants in untreated individuals in Europe: implications for clinical management. J Infect Dis. 2005;192:958–966. doi: 10.1086/432916. [DOI] [PubMed] [Google Scholar]
- Wilkins C, Dishongh R, Moore SC, Whitt MA, Chow M, Machaca K. RNA interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature. 2005;436:1044–1047. doi: 10.1038/nature03957. [DOI] [PubMed] [Google Scholar]
- Zhang H, Zhao Q, Bhattacharya S, Waheed AA, Tong X, Hong A, Heck S, Curreli F, Goger M, Cowburn D, Freed EO, Debnath AK. A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J Mol Biol. 2008;378:565–580. doi: 10.1016/j.jmb.2008.02.066. [DOI] [PMC free article] [PubMed] [Google Scholar]
