Antiviral activity of Ribavirin nano-particles against measles virus

Engy M Ahmed; Samar M Solyman; Noha Mohamed; Abeer A Boseila; Amro Hanora

doi:10.14715/cmb/2018.64.9.4

Issue

Vol. 64 No. 9: Issue 9

Issue Published : July 19, 2018

The undersigned hereby assign all rights, included but not limited to copyright, for this manuscript to CMB Association upon its submission for consideration to publication on Cellular and Molecular Biology. The rights assigned include, but are not limited to, the sole and exclusive rights to license, sell, subsequently assign, derive, distribute, display and reproduce this manuscript, in whole or in part, in any format, electronic or otherwise, including those in existence at the time this agreement was signed. The authors hereby warrant that they have not granted or assigned, and shall not grant or assign, the aforementioned rights to any other person, firm, organization, or other entity. All rights are automatically restored to authors if this manuscript is not accepted for publication.

Antiviral activity of Ribavirin nano-particles against measles virus

https://doi.org/10.14715/cmb/2018.64.9.4

Engy M Ahmed

National Organization for Drug Control & Research (NODCR), Cairo, Egypt

Samar M Solyman

Microbiology &Immunology Department, College of Pharmacy, Suez Canal University, Ismailia, Egypt

Noha Mohamed

Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

Abeer A Boseila

National Organization for Drug Control & Research (NODCR), Cairo, Egypt

Amro Hanora

Microbiology &Immunology Department, College of Pharmacy, Suez Canal University, Ismailia, Egypt

Corresponding Author(s) : Amro Hanora

ahanora@yahoo.com

Cellular and Molecular Biology, Vol. 64 No. 9: Issue 9
Article Published : June 30, 2018

Abstract

Measles virus considers an important cause of child morbidity and mortality in some areas as Africa. Ribavirin's activity as a nucleoside analog can disclose the surprisingly broad spectrum action against several RNA viruses under laboratory cell culture conditions. The Current study aimed to investigate the antiviral activity of ribavirin Nano gold particles (AuNPs) against measles virus on vero cell line. Ribavirin- AuNPs was prepared, characterization and the cytotoxicity of ribavirin, AuNPs and ribavirin -AuNPs were tested on vero cells using MTT assay. Antiviral activiry of ribavirin, AuNPs and ribavirin- AuNPswere determined on vero cells using simultaneous, pre-infection and post-infection protocols. Results indicated safety of ribavirin and ribavirin-AuNPs on vero cells, there was a reduction by 78.1% when vero cells treated with ribavirin -AuNPs at 99.5µg/ml while, the viral reduction was 25.4% when ribavirin 500 µg /ml was used for the same viral concentration. Our results concluded that ribavirin - AuNPs had a higher antiviral activity with lower dose than ribavirin alone and the maximal activity showed when it used after the virus infection.

Keywords

Ribavirin Nano gold particles Measles virus Antiviral activity Vero cell line.

Ahmed, E. M., Solyman, S. M., Mohamed, N., Boseila, A. A., & Hanora, A. (2018). Antiviral activity of Ribavirin nano-particles against measles virus. Cellular and Molecular Biology, 64(9), 24–32. https://doi.org/10.14715/cmb/2018.64.9.4

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References

(1) Grifï¬n, D.E. Measles virus. In Fields Virology,. In Edited by D. M. Knipe, P.M.H., D. E. Grifï¬n, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. (ed), Philadelphia: Lippincott Williams & Wilkins 2001.
(2) Cathomen T, Mrkic B, Spehner D, Drillien R, Naef R, Pavlovic et al, A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain . The EMBO journal 1998; 17: 3899-3908.
(3) Nandy R, Handzel T, Zaneidou M, Biey J, Coddy R.Z, Perry R et al. Case-fatality rate during a measles outbreak in eastern Niger in 2003. Clin. Infect. Dis.: an official publication of the Infectious Diseases Society of America 2006; 42: 322-328.
(4) Grais R.F, Dubray C, Gerstl S, Guthmann J.P, Djibo A, Nargaye K.D et al. Unacceptably high mortality related to measles epidemics in Niger, Nigeria, and Chad. PLoS medicine 2007; 4: 16.
(5) Moss W.J. & Griffin D.E. Measles. Lancet (London, England) 2012; 379: 153-164.
(6) Bryce J, Boschi-Pinto C, Shibuya K. & Black R.E. WHO estimates of the causes of death in children. Lancet (London, England) 2005; 365: 1147-1152.
(7) Strebel P.M, Cochi S.L, Hoekstra E, Rota P.A, Featherstone D, Bellini W.J.et al. A world without measles. J INFECT DIS 2011; 204 Suppl 1, S1-3.
(8) Barskey A.E, Glasser J.W. & LeBaron C.W. Mumps resurgences in the United States: A historical perspective on unexpected elements. Vaccine 2009; 27: 6186-6195.
(9) World Health Organization. Global distribution of measles and rubella genotypes--update. Wkly Epidemiol Rec 2006; 81: 474-4
(10) Crumpacker C.S. Overview of ribavirin treatment of infection caused by RNA viruses. In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Academic Press, Inc., New York 1984; 33-37.
(11) Sidwell R.W. In vitro and in vivo inhibition of DNA viruses by ribavirin, In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Aca- demic Press, Inc., New York 1984; 19-32.
(12) Hruska J.F, Bernstein J.M, Douglas R.G, Jr. & Hall C.B. Effects of ribavirin on respiratory syncytial virus in vitro. Antimicrob. Agents Chemother.1980; 17: 770-775.
(13) Cummings K.J, Lee S.M, West E.S, Cid-Ruzafa J, Fein S.G, Aoki Y et al. Interferon and ribavirin vs interferon alone in the re-treatment of chronic hepatitis C previously nonresponsive to interferon: A meta-analysis of randomized trials. Jama 2001; 285: 193-199.
(14) Shulman N.R. Assessment of hematologic effects of ribavirin in humans. In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Aca- demic Press, Inc., New York 1984; 79-92.
(15) McCormick J.B, King I.J, Webb P.A, Scribner C.L, Craven R.B, Johnson K.M et al. Lassa fever. Effective therapy with ribavirin. N. Engl. J. Med.1986; 314: 20-26.
(16) Homma M, Matsuzaki Y, Inoue Y, Shibata M, Mitamura K, Tanaka N.et al. Marked elevation of erythrocyte ribavirin levels in interferon and ribavirin-induced anemia. Clin. Gastroenterol. Hepatol. 2004; 2: 337-339.
(17) Nimesh S, Manchanda R, Kumar R, Saxena A, Chaudhary P, Yadav V, et al. Preparation, characterization and in vitro drug release studies of novel polymeric nanoparticles. Int. J. Pharm 2006; 323: 146-152.
(18) Turkevich J, Steenson P.C, Hillier J,"A study of the nucleation and growth processes in the synthesis of colloidal gold". , Discuss. Faraday. Soc 1951; 11: 55-75.
(19) Frens G. "Particle size and sol stability in metal colloids". Colloid Polym Sci 1972; 250: 736–741.
(20) Pal S.L, Jana U, Manna PK, Mohanta G.P, & Manavalan. "Nanoparticle: An overview of preparation and characterization" Int. J. Appl. Pharm. Sci. Res 2011; 1: 228-234.
(21) Yasumura Y & Kawakita Y. Studies on SV40 in tissue culture – preliminary step for cancer research "in vitro. Article in Japanese1963; 21: 1201–1215.
(22) Cote R.J. Aseptic technique for cell culture. Curr Protoc Cell Biol 2001; Chapter 1, Unit 1.3.
(23) Takeuchi H, Baba M. & Shigeta S. An application of tetrazolium (MTT) colorimetric assay for the screening of anti-herpes simplex virus compounds. J. Virol. Methods 1991; 33: 61-71.
(24) Sieuwerts A, Klijn .J.G.M, Peters H.A, Foekens J.A. The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50 – values and cell survival. Eur J Clin Chem Clin Biochem1995; 33: 813-823.
(25) Chen T.h, kutty P. & Lowe L.E. Measles outbreak associated with an international youth sporting event in the united states2007. Pediatr. Infect. Dis. J 2010; 29: 794-800.
(26) Marini A.M, Ahmed I.B, Yahya M.D. & Yamin B.M. Optimization of cytotoxicity assay for estimating CD50 values of plant extracts and organometallic compounds. Malays Appl Biol 1998; 27: 63-67.
(27) Ogutcu H, Sokmen A, Sokmen M, Polissiou M, SerKedjieva J, Daferera D , et al. Bioactivities of the Various Extracts and Essential Oils of Saliva Limbata C.A. Mey. And Salvia sclareaL.Turk. J. Biol. 2008; 32: 181-192.
(28) Dragana S, Dragana M.C, Branka V.G, Draga S & Jelena K.V. Evaluation of antiviral activity of fractionated extracts of saga salvia officinalis L. (Lamiaceae). Arch.Biol.Sci. Belgrade 2008; 60: 421-429.
(29) Reed J. & Muench H. simple method of estimating fifty percent endpoints. Am J Hyg 1938; 27: 493-494?
(30) KoruKluoglu G, Liffick S, Guris D, Kobune F, Rota A.P, Bellini J.W et al. Genetic characterization of measles virus isolated in turkey during 2000-2001. Virol. J. 2005; 2: 58.
(31) DeAssis D.N , Mosqueira V. C, Vilela J.M, Andrade M.S. & Cardoso V.N. "Release profiles and morphological characterization by atomic force microscopy and photon correlation spectroscopy of 99m Technetium – fluconazole Nano capsules" Int J Pharm, 2008; 349: 152 – 160.
(32) Pangi Z, Beletsi A & Evangelatos K. "PEG-ylated nanoparticlesfor biological and pharmaceutical application". Adv. Drug Deliv. Rev 2003; 24: 403- 419.
(33) Abdelhalim M.A.K, Mady M.M, & Ghannam M.M. Physical Properties of Different Gold Nanoparticles: Ultraviolet-Visible and Fluorescence Measurements. J Nanomed Nanotechnol 2012; 3: 133.
(34) Foege W.H. & Foster S.O. Multiple antigen vaccine strategies in developing countries. Am. J. Trop. Med. Hyg. 1974; 23: 685-689.
(35) Halsey N.A, Boulos R, Mode F, Andre J, Bowman L, Yaeger R.G, et al. Response to measles vaccine in Haitian infants 6 to 12 months old. Influence of maternal antibodies, malnutrition, and concurrent illnesses. N. Engl. J. Med. 1985; 313: 544-549.
(36) Pal G. Effects of ribavirin on measles. J Indian Med Assoc 2011; 109: 666-667.
(37) Banks G. & Fernandez H. Clinical use of ribavirin in measles: a summarized review. In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Academic Press, Inc., New York, 1984; 203-209.
(38) Forni A.L, Schluger N.W. & and Roberts R.B. severe measles pneumonia in adults: an evaluation of clinical charactristic and therapy with intravenous ribavirin. Clin. Infect. Dis.1994; 19: 454-462.
(39) Hosoya M, Shigeta S, Nakamura K. & De Clercq E. Inhibitory effect of selected antiviral compounds on measles (SSPE) virus replication in vitro. Antiviral Res. 1989; 12: 87-97.
(40) Jen J, Laughlin M, Chung C, Heft S, Affrime M.B, Gupta S.K, Glue P. et al. (2002) Ribavirin dosing in chronic hepatitis C: application of population pharmacokinetic-pharmacodynamic models. Clin. Pharmacol. Ther. 2002; 72: 349-361.
(41) Takaki S, Tsubota A, Hosaka T, Akuta N, Someya T, Kobayashi M. et al. Factors contributing to ribavirin dose reduction due to anemia during interferon alfa2b and ribavirin combination therapy for chronic hepatitis C. ter Meulen, V. & Carter, M.J. (1984) Measles virus persistency and disease. Prog. Med. Virol. 2004; 30: 44-61.
(42) Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983; 65: 55-63.
(43) Andrighetti-Frohner C.R, Antonio R.V, Creczynski-Pasa T.B, Barardi C.R. & Simoes C.M. Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum. Mem. Inst. Oswaldo Cruz 2003; 98: 843-848.
(44) Vietinck A.J. & Vanden-Berghe D.A. Antiviral activity of3-methoxyflavones. Proceeding clinical biology research 1991; 213: 537-540.
(45) Simoes C.M, Amoros M. & Girre L. Mechanism of antiviral activity of triterpenoid saponins. Phytother Res 1999; 13: 323-328.
(46) Semple S.J, Pyke S.M, Reynolds G.D. & Flower R.L. In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus.
Antiviral Res. 2001; 49: 169-178.
(47) Li Y.L, Ma S.C, Yang Y.T, Ye S.M. & but P.P. Antiviral activities of flavonoids and organic acid from Trollius chinensis Bunge. J Ethnopharmacol 2002; 79: 365-368.
(48) Smee D.F, Morrison A.C, Barnard D.L. & Sidwell R.W. Comparison of colorimetric, fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds. J. Virol. Methods 2002; 106: 71-79.
(49) Ortac Ersoy E, Tanriover M.D, Ocal S, Ozisik L, Inkaya C. & Topeli A. Severe measles pneumonia in adults with respiratory failure: role of ribavirin and high-dose vitamin A. Clin Respir J 2016; 10: 673-675.
(50) Wray S.K, Gilbert B.E & Noall M.W. Mode of action of ribavirin: eï¬€ect of nucleotide pool alterations on inï¬‚uenza virus ribonucleoprotein synthesis, Noall MW, et al. Antiviral Res 1985; 5: 29-37.
(51) Thomas B, Beard S, Jin L, Brown K.E. & Brown D.W. Development and evaluation of a real-time PCR assay for rapid identification and semi-quantitation of measles virus. J. Med. Virol 2007; 79: 1587-1592.
(52) Hummel K.B, Lowe L, Bellini W.J. & Rota P.A. Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens. Virol. Methods 2006; 132: 166-173.
(53) Mackay I.M, Arden K.E. & Nitsche A. Real-time PCR in virology. Nucleic Acids Res 2002; 30: 1292-1305.
(54) Grifï¬n D. Measles virus. In: Fields BN, Knipe D.M, and Howley P.M, eds. Fields Virology. Philadelphia: Wolters Kluwer Health/ Lippincott Williams & Wilkins, 2007; Vol 2, 1551–1585.
(55) Williams D. The relationship between biomaterials and nanotechnology. Biomaterials 2008; 7: VoL. 193, 1515–1526.

References

(1) Grifï¬n, D.E. Measles virus. In Fields Virology,. In Edited by D. M. Knipe, P.M.H., D. E. Grifï¬n, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. (ed), Philadelphia: Lippincott Williams & Wilkins 2001.

(2) Cathomen T, Mrkic B, Spehner D, Drillien R, Naef R, Pavlovic et al, A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain . The EMBO journal 1998; 17: 3899-3908.

(3) Nandy R, Handzel T, Zaneidou M, Biey J, Coddy R.Z, Perry R et al. Case-fatality rate during a measles outbreak in eastern Niger in 2003. Clin. Infect. Dis.: an official publication of the Infectious Diseases Society of America 2006; 42: 322-328.

(4) Grais R.F, Dubray C, Gerstl S, Guthmann J.P, Djibo A, Nargaye K.D et al. Unacceptably high mortality related to measles epidemics in Niger, Nigeria, and Chad. PLoS medicine 2007; 4: 16.

(5) Moss W.J. & Griffin D.E. Measles. Lancet (London, England) 2012; 379: 153-164.

(6) Bryce J, Boschi-Pinto C, Shibuya K. & Black R.E. WHO estimates of the causes of death in children. Lancet (London, England) 2005; 365: 1147-1152.

(7) Strebel P.M, Cochi S.L, Hoekstra E, Rota P.A, Featherstone D, Bellini W.J.et al. A world without measles. J INFECT DIS 2011; 204 Suppl 1, S1-3.

(8) Barskey A.E, Glasser J.W. & LeBaron C.W. Mumps resurgences in the United States: A historical perspective on unexpected elements. Vaccine 2009; 27: 6186-6195.

(9) World Health Organization. Global distribution of measles and rubella genotypes--update. Wkly Epidemiol Rec 2006; 81: 474-4

(10) Crumpacker C.S. Overview of ribavirin treatment of infection caused by RNA viruses. In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Academic Press, Inc., New York 1984; 33-37.

(11) Sidwell R.W. In vitro and in vivo inhibition of DNA viruses by ribavirin, In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Aca- demic Press, Inc., New York 1984; 19-32.

(12) Hruska J.F, Bernstein J.M, Douglas R.G, Jr. & Hall C.B. Effects of ribavirin on respiratory syncytial virus in vitro. Antimicrob. Agents Chemother.1980; 17: 770-775.

(13) Cummings K.J, Lee S.M, West E.S, Cid-Ruzafa J, Fein S.G, Aoki Y et al. Interferon and ribavirin vs interferon alone in the re-treatment of chronic hepatitis C previously nonresponsive to interferon: A meta-analysis of randomized trials. Jama 2001; 285: 193-199.

(14) Shulman N.R. Assessment of hematologic effects of ribavirin in humans. In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Aca- demic Press, Inc., New York 1984; 79-92.

(15) McCormick J.B, King I.J, Webb P.A, Scribner C.L, Craven R.B, Johnson K.M et al. Lassa fever. Effective therapy with ribavirin. N. Engl. J. Med.1986; 314: 20-26.

(16) Homma M, Matsuzaki Y, Inoue Y, Shibata M, Mitamura K, Tanaka N.et al. Marked elevation of erythrocyte ribavirin levels in interferon and ribavirin-induced anemia. Clin. Gastroenterol. Hepatol. 2004; 2: 337-339.

(17) Nimesh S, Manchanda R, Kumar R, Saxena A, Chaudhary P, Yadav V, et al. Preparation, characterization and in vitro drug release studies of novel polymeric nanoparticles. Int. J. Pharm 2006; 323: 146-152.

(18) Turkevich J, Steenson P.C, Hillier J,"A study of the nucleation and growth processes in the synthesis of colloidal gold". , Discuss. Faraday. Soc 1951; 11: 55-75.

(19) Frens G. "Particle size and sol stability in metal colloids". Colloid Polym Sci 1972; 250: 736–741.

(20) Pal S.L, Jana U, Manna PK, Mohanta G.P, & Manavalan. "Nanoparticle: An overview of preparation and characterization" Int. J. Appl. Pharm. Sci. Res 2011; 1: 228-234.

(21) Yasumura Y & Kawakita Y. Studies on SV40 in tissue culture – preliminary step for cancer research "in vitro. Article in Japanese1963; 21: 1201–1215.

(22) Cote R.J. Aseptic technique for cell culture. Curr Protoc Cell Biol 2001; Chapter 1, Unit 1.3.

(23) Takeuchi H, Baba M. & Shigeta S. An application of tetrazolium (MTT) colorimetric assay for the screening of anti-herpes simplex virus compounds. J. Virol. Methods 1991; 33: 61-71.

(24) Sieuwerts A, Klijn .J.G.M, Peters H.A, Foekens J.A. The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50 – values and cell survival. Eur J Clin Chem Clin Biochem1995; 33: 813-823.

(25) Chen T.h, kutty P. & Lowe L.E. Measles outbreak associated with an international youth sporting event in the united states2007. Pediatr. Infect. Dis. J 2010; 29: 794-800.

(26) Marini A.M, Ahmed I.B, Yahya M.D. & Yamin B.M. Optimization of cytotoxicity assay for estimating CD50 values of plant extracts and organometallic compounds. Malays Appl Biol 1998; 27: 63-67.

(27) Ogutcu H, Sokmen A, Sokmen M, Polissiou M, SerKedjieva J, Daferera D , et al. Bioactivities of the Various Extracts and Essential Oils of Saliva Limbata C.A. Mey. And Salvia sclareaL.Turk. J. Biol. 2008; 32: 181-192.

(28) Dragana S, Dragana M.C, Branka V.G, Draga S & Jelena K.V. Evaluation of antiviral activity of fractionated extracts of saga salvia officinalis L. (Lamiaceae). Arch.Biol.Sci. Belgrade 2008; 60: 421-429.

(29) Reed J. & Muench H. simple method of estimating fifty percent endpoints. Am J Hyg 1938; 27: 493-494?

(30) KoruKluoglu G, Liffick S, Guris D, Kobune F, Rota A.P, Bellini J.W et al. Genetic characterization of measles virus isolated in turkey during 2000-2001. Virol. J. 2005; 2: 58.

(31) DeAssis D.N , Mosqueira V. C, Vilela J.M, Andrade M.S. & Cardoso V.N. "Release profiles and morphological characterization by atomic force microscopy and photon correlation spectroscopy of 99m Technetium – fluconazole Nano capsules" Int J Pharm, 2008; 349: 152 – 160.

(32) Pangi Z, Beletsi A & Evangelatos K. "PEG-ylated nanoparticlesfor biological and pharmaceutical application". Adv. Drug Deliv. Rev 2003; 24: 403- 419.

(33) Abdelhalim M.A.K, Mady M.M, & Ghannam M.M. Physical Properties of Different Gold Nanoparticles: Ultraviolet-Visible and Fluorescence Measurements. J Nanomed Nanotechnol 2012; 3: 133.

(34) Foege W.H. & Foster S.O. Multiple antigen vaccine strategies in developing countries. Am. J. Trop. Med. Hyg. 1974; 23: 685-689.

(35) Halsey N.A, Boulos R, Mode F, Andre J, Bowman L, Yaeger R.G, et al. Response to measles vaccine in Haitian infants 6 to 12 months old. Influence of maternal antibodies, malnutrition, and concurrent illnesses. N. Engl. J. Med. 1985; 313: 544-549.

(36) Pal G. Effects of ribavirin on measles. J Indian Med Assoc 2011; 109: 666-667.

(37) Banks G. & Fernandez H. Clinical use of ribavirin in measles: a summarized review. In R. A. Smith, V. Knight, and J. A. D. Smith (ed.), Clinical applications of ribavirin. Academic Press, Inc., New York, 1984; 203-209.

(38) Forni A.L, Schluger N.W. & and Roberts R.B. severe measles pneumonia in adults: an evaluation of clinical charactristic and therapy with intravenous ribavirin. Clin. Infect. Dis.1994; 19: 454-462.

(39) Hosoya M, Shigeta S, Nakamura K. & De Clercq E. Inhibitory effect of selected antiviral compounds on measles (SSPE) virus replication in vitro. Antiviral Res. 1989; 12: 87-97.

(40) Jen J, Laughlin M, Chung C, Heft S, Affrime M.B, Gupta S.K, Glue P. et al. (2002) Ribavirin dosing in chronic hepatitis C: application of population pharmacokinetic-pharmacodynamic models. Clin. Pharmacol. Ther. 2002; 72: 349-361.

(41) Takaki S, Tsubota A, Hosaka T, Akuta N, Someya T, Kobayashi M. et al. Factors contributing to ribavirin dose reduction due to anemia during interferon alfa2b and ribavirin combination therapy for chronic hepatitis C. ter Meulen, V. & Carter, M.J. (1984) Measles virus persistency and disease. Prog. Med. Virol. 2004; 30: 44-61.

(42) Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983; 65: 55-63.

(43) Andrighetti-Frohner C.R, Antonio R.V, Creczynski-Pasa T.B, Barardi C.R. & Simoes C.M. Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum. Mem. Inst. Oswaldo Cruz 2003; 98: 843-848.

(44) Vietinck A.J. & Vanden-Berghe D.A. Antiviral activity of3-methoxyflavones. Proceeding clinical biology research 1991; 213: 537-540.

(45) Simoes C.M, Amoros M. & Girre L. Mechanism of antiviral activity of triterpenoid saponins. Phytother Res 1999; 13: 323-328.

(46) Semple S.J, Pyke S.M, Reynolds G.D. & Flower R.L. In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus.

Antiviral Res. 2001; 49: 169-178.

(47) Li Y.L, Ma S.C, Yang Y.T, Ye S.M. & but P.P. Antiviral activities of flavonoids and organic acid from Trollius chinensis Bunge. J Ethnopharmacol 2002; 79: 365-368.

(48) Smee D.F, Morrison A.C, Barnard D.L. & Sidwell R.W. Comparison of colorimetric, fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds. J. Virol. Methods 2002; 106: 71-79.

(49) Ortac Ersoy E, Tanriover M.D, Ocal S, Ozisik L, Inkaya C. & Topeli A. Severe measles pneumonia in adults with respiratory failure: role of ribavirin and high-dose vitamin A. Clin Respir J 2016; 10: 673-675.

(50) Wray S.K, Gilbert B.E & Noall M.W. Mode of action of ribavirin: eï¬€ect of nucleotide pool alterations on inï¬‚uenza virus ribonucleoprotein synthesis, Noall MW, et al. Antiviral Res 1985; 5: 29-37.

(51) Thomas B, Beard S, Jin L, Brown K.E. & Brown D.W. Development and evaluation of a real-time PCR assay for rapid identification and semi-quantitation of measles virus. J. Med. Virol 2007; 79: 1587-1592.

(52) Hummel K.B, Lowe L, Bellini W.J. & Rota P.A. Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens. Virol. Methods 2006; 132: 166-173.

(53) Mackay I.M, Arden K.E. & Nitsche A. Real-time PCR in virology. Nucleic Acids Res 2002; 30: 1292-1305.

(54) Grifï¬n D. Measles virus. In: Fields BN, Knipe D.M, and Howley P.M, eds. Fields Virology. Philadelphia: Wolters Kluwer Health/ Lippincott Williams & Wilkins, 2007; Vol 2, 1551–1585.

(55) Williams D. The relationship between biomaterials and nanotechnology. Biomaterials 2008; 7: VoL. 193, 1515–1526.

Author biographies is not available.