Mimiviruses: Replication, Purification, and Quantification

Jônatas Santos Abrahão1, Graziele Pereira Oliveira1, Lorena Christine Ferreira da Silva2, Ludmila Karen dos Santos Silva2, Erna Geessien Kroon2, Bernard La Scola3

1 These authors contributed equally to this work, 2 Instituto de Ciências Biológicas, Departamento de Microbiologia, Laboratório de Vírus, Universidade Federal de Minas Gerais, Belo Horizonte‐Minas Gerais, Brazil, 3 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Aix‐Marseille Université, Marseille, France
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14G.1
DOI:  10.1002/cpmc.2
Online Posting Date:  May, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity. The discovery of these viruses rekindled discussions about their origin and evolution, and the genetic and structural complexity opened up a new field of study. Here, we describe some methods utilized for mimiviruses replication, purification, and titration. © 2016 by John Wiley & Sons, Inc.

Keywords: mimiviruses; purification; replication; titration

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Mimivirus Production
  • Basic Protocol 2: Mimivirus Purification
  • Basic Protocol 3: Mimivirus Titer Calculation by end‐Point Dilution Assay
  • Support Protocol 1: Growth and Passing of Acanthamoeba Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Mimivirus Production

  Materials
  • Viral samples to be produced (a previously titrated seed pool; see protocol 3)
  • Acanthamoeba castellanii culture grown in tissue culture flasks (see protocol 4Support Protocol)
  • Peptone‐yeast extract with glucose (PYG) medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Incubator (28° to 32°C)
  • 150‐cm2 cell culture flasks
  • Refrigerated low‐speed table‐top centrifuge
  • 50‐ml conical centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • 37°C water bath
  • Inverted microscope

Basic Protocol 2: Mimivirus Purification

  Materials
  • Viral samples to be purified (see protocol 1)
  • 24% sucrose solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe, or Page's amoeba saline [PAS; see recipe])
  • Dounce homogenizer, glass, with tight pestle
  • 50‐ml conical centrifuge tubes
  • Refrigerated low‐speed table‐top centrifuge
  • 1.2‐μm pore syringe filter
  • Ultracentrifuge (Sorvall Combi OTD or equivalent)
  • Beckman ultracentrifuge polypropylene tubes
  • 0.6‐ml microcentrifuge tubes

Basic Protocol 3: Mimivirus Titer Calculation by end‐Point Dilution Assay

  Materials
  • Viral samples to be quantified
  • Acanthamoeba castellanii culture grown in tissue culture flasks (see protocol 4Support Protocol)
  • Peptone‐yeast extract with glucose (PYG) medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe, or Page's amoeba saline [PAS; see recipe])
  • Incubator (28°C)
  • 96‐well plates
  • 75‐cm2 tissue culture flasks
  • Transfer pipet
  • 1.5‐ml microcentrifuge tubes (for serial dilutions of virus)
  • Inverted microscope

Support Protocol 1: Growth and Passing of Acanthamoeba Culture

  Materials
  • Acanthamoeba castellanii cells (ATCC; ATCC 30234 and ATCC 30010)
  • Peptone‐yeast extract with glucose (PYG) medium (see recipe)
  • Ice
  • Incubator (28°C)
  • 25‐, 75‐, and 150‐cm2 tissue culture flasks
  • Neubauer chamber and glass cover (for cell counting)
  • 1.5‐ml microcentrifuge tubes
  • 50‐ml tubes
  • Inverted microscope
NOTE: Preparation of A. castellanii cultures: All solutions and equipment coming into contact with A. castellanii cultures must be sterile, and aseptic technique should be used accordingly.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Abrahão, J.S., Dornas, F.P., Silva, L.C., Almeida, G.M., Boratto, P.V.M., Colson, P., La Scola, B., and Kroon, E.G. 2014a. Acanthamoeba polyphaga mimivirus and other giant viruses: An open field to outstanding discoveries. Virol. J. 11:120. doi: 10.1186/1743‐422X‐11‐120.
  Abrahão, J.S., Boratto, P.V.M., Dornas, F.P., Silva, L.C., Campos, R.K., Almeida, G.M., Kroon, E.G., and La Scola, B. 2014b. Growing a giant: Evaluation of the virological parameters for mimivirus production. J. Virol. Methods 207:6‐11. doi: 10.1016/j.jviromet.2014.06.001.
  Andrade, K.R., Boratto, P.V.M., Rodrigues, F.P., Silva, L.C., Dornas, F.P., Pilotto, M.R., La Scola, B., Almeida, G.M., Kroon, E.G., and Abrahão, J.S. 2015. Oysters as hot spots for mimivirus isolation. Arch. Virol. 160:477‐82. doi: 10.1007/s00705‐014‐2257‐2.
  Arslan, D., Legendre, M., Seltzer, V., Abergel, C., and Claverie, J. 2011. Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. Proc. Natl. Acad. Sci. U.S.A. 108:17486‐17491. doi: 10.1073/pnas.1110889108.
  Boughalmi, M., Saadi, H., Pagnier, I., Colson, P., Fournous, G., Raoult, D., and La Scola, B. 2012. High‐throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment. Environ. Microbiol. 53:344‐353. doi: 10.1111/1462‐2920.12068.
  Bousbia, S., Papazian, L., Saux, P., Forel, J.M., Auffray, J.P., Martin, C., Raoult, D., and La Scola, B. 2013. Serologic prevalence of amoeba‐associated microorganisms in intensive care unit pneumonia patients. PLoS One 8:e58111. doi: 10.1371/journal.pone.0058111.
  Boyer, M., Madoui, M.A., Gimenez, G., La Scola, B., and Raoult, D. 2010. Phylogenetic and phyletic studies of informational genes in genomes highlight existence of a 4 domain of life including giant viruses. PLoS One 5:e15530. doi: 10.1371/journal.pone.0015530.
  Campos, R.K., Boratto, P.V., Assis, F.L., Aguiar, E.R.G.R., Silva, L.C.F., Albarnaz, J.D., Dornas, F.P., Trindade, G.S., Ferreira, P.C.P., Marques, J.T., Roberts, C., Raoult, D., Kroon, E.G., La Scola, B., and Abrahao, J.S. 2014. Samba virus: A novel mimivirus from a giant rain forest, the Brazilian Amazon. Virol. J. 11:95. doi: 10.1186/1743‐422X‐11‐95.
  Claverie, J.M., Grzela, R., Lartigue, A., Bernadac, A., Nitsche, S., Vacelet, J., Ogata, H., and Abergel, C. 2009. Mimivirus and Mimiviridae: Giant viruses with an increasing number of potential hosts, including corals and sponges. J. Invertebr. Pathol. 101:172‐180. doi: 10.1016/j.jip.2009.03.011.
  Colson, P., La Scola, B., and Raoult, D. 2013. Giant viruses of amoebae as potential human pathogens. Intervirology 56:376‐385. doi: 10.1159/000354558.
  Dornas, F.P., Rodrigues, F.P., Boratto, P.V., Silva, L.C., Ferreira, P.C., Bonjardim, C.A., Trindade, G.S., Kroon, E.G., La Scola, B., and Abrahao, J.S. 2014a. Mimivirus circulation among wild and domestic mammals, Amazon Region, Brazil. Emerg. Infect. Dis. 20:469‐472. doi: 10.3201/eid2003.131050.
  Dornas, F.P., Silva, L.C., de Almeida, G.M., Campos, R.K., Boratto, P.V., Franco‐Luiz, A.P., La Scola, B., Ferreira, P.C., Kroon, E.G., and Abrahão, J.S. 2014b. Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: Implications for virus detection and isolation. PLoS One 9:e87811. doi: 10.1371/journal.pone.0087811.
  Ghedin, E., and Claverie, J.M. 2005. Mimivirus relatives in the Sargasso sea. Virol. J. 2:62. doi: 10.1186/1743‐422X‐2‐62.
  Ghigo, E., Kartenbeck, J., Lien, P., Pelkmans, L., Capo, C., Mege, J.L., and Raoult, D. 2008. Ameobal pathogen mimivirus infects macrophages through phagocytosis. PLoS Pathog. 4:e1000087. doi: 10.1371/journal.ppat.1000087.
  Khan, M., La Scola, B., Lepidi, H., and Raoult, D. 2007. Pneumonia in mice inoculated experimentally with Acanthamoeba polyphaga mimivirus. Microb. Pathog. 42:56‐61. doi: 10.1016/j.micpath.2006.08.004.
  King, A.M.Q., Adams, M.J., Carstens, E.B., and Lefkowitz, E.J. 2012. Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, New York.
  Kusnetsov, Y.G., Xiao, C., Sun, S., Raoult, D., Rossmann, M., and McPherson, A. 2010. Atomic force microscopy investigation of the giant mimivirus. Virology 404:127‐137. doi: 10.1016/j.virol.2010.05.007.
  La Scola, B., Marrie, T.J., Auffray, J.P., and Raoult, D. 2005. Mimivirus in pneumonia patients. Emerg. Infect. Dis. 11:449‐452. doi: 10.3201/eid1103.040538.
  La Scola, B., Audic, S., Robert, C., Jungang, L., de Lamballerie, X., Drancourt, M., Birtles, R., Claverie, J.M., and Raoult, D. 2003. A giant virus in amoebae. Science 299:2033. doi: 10.1126/science.1081867.
  Legendre, M., Arslan, D., Abergel, C., and Claverie, J.M. 2012. Genomics of Megavirus and the elusive fourth domain of Life. Commun. Integr. Biol. 5:102‐106. doi: 10.4161/cib.18624.
  Marciano‐Cabral, F. and Cabral, G. 2003. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Ver. 16:273‐307. doi: 10.1128/CMR.16.2.273‐307.2003.
  Monier, A., Larsen, J.B., Sandaa, R.A., Bratbak, G., Claverie, J.M., and Ogata, H. 2008. Marine mimivirus relatives are probably large algal viruses. Virol. J. 5:12. doi: 10.1186/1743‐422X‐5‐12.
  Moreira, D., and Brochier‐Armanet, C. 2008. Giant viruses, giant chimeras: The multiple evolutionary histories of mimivirus genes. BMC Evol. Biol. 8:12. doi: 10.1186/1471‐2148‐8‐12.
  Mutsafi, Y., Shimoni, E., Shimon, A., and Minsky, A. 2013. Membrane assembly during the infection cycle of the giant mimivirus. PLoS Pathog. 9:5. doi: 10.1371/journal.ppat.1003367.
  Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., and Claverie, J.M. 2004. The 1.2‐megabase genome sequence of mimivirus. Science 306:1344‐1350. doi: 10.1126/science.1101485.
  Raoult, D., and Forterre, P. 2008. Redefining viruses: Lessons from mimivirus. Nat. Rev. Microbiol. 6:315‐319. doi: 10.1038/nrmicro1858.
  Raoult, D., Renesto, P., and Brouqui, P. 2006. Laboratory infection of a technician by mimivirus. Ann. Intern. Med. 144:702‐703. doi: 10.7326/0003‐4819‐144‐9‐200605020‐00025.
  Reed, L.J., and Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27:493‐497.
  Saadi, H., Pagnier, I., Colson, P., Cherif, J.K., Beji, M., Boughalmi, M., Azza, S., Armstrong, N., Robert, C., Fournous, G., La Scola, B., and Raoult, D. 2013a. First isolation of Mimivirus in a patient with pneumonia. Clin. Infect. Dis. 57:127‐134. doi: 10.1093/cid/cit354.
  Saadi, H., Reteno, D.G., Colson, P., Aherfi, S., Minodier, P., Pagnier, I., Raoult, D., and La Scola, B. 2013b. Shan virus: A new mimivirus isolated from the stool of a Tunisian patient with pneumonia. Intervirology 56:424‐429. doi: 10.1159/000354564.
  Santos Silva, L.K., Arantes, T.S., Andrade, K.R., Lima Rodrigues, R.A., Miranda Boratto, P.V., de Freitas Almeida, G.M., Kroon, E.G., La Scola, B., Clemente, W.T., and Santos Abrahão, J. 2015. High positivity of mimivirus in inanimate surfaces of a hospital respiratory‐isolation facility, Brazil. Clin. Virol. 66:62‐65. doi: 10.1016/j.jcv.2015.03.008.
  Shah, N., Hülsmeier, A.J., Hochhold, N., Neidhart, M., Gay, S., and Hennet, T. 2013. Exposure to mimivirus collagen promotes arthritis. J. Virol. 88:838‐845. doi: 10.1128/JVI.03141‐13.
  Silva, L.C., Almeida, G.M., Oliveira, D.B., Dornas, F.P., Campos, R.K., La Scola, B., Ferreira, P.C., Kroon, E.G., and Abrahao, J.S. 2013. A resourceful giant: APMV is able to interfere with the human type I interferon system. Microbes Infect. 16:187‐195. doi: 10.1016/j.micinf.2013.11.011.
  Williamson, S.J., Allen, L.Z., Lorenzi, H.A., Fadrosh, D.W., Brami, D., Thiagarajan, M., McCrow, J.P., Tovchigrechko, A., Yooseph, S., and Venter, J.C. 2012. Metagenomic exploration of viruses throughout the Indian Ocean. PLoS One 7:e42047. doi: 10.1371/journal.pone.0042047.
  Xiao, C., Chipman, P.R., Battisti, A.J., Bowman, V.D., Renesto, P., Raoult, D., and Rossmann, M.G. 2005. Cryo‐electron microscopy of the giant mimivirus. J. Mol. Biol. 353:493‐496. doi:10.1016/j.jmb.2005.08.060.
  Yamada, T. 2011. Giant viruses in the environment: Their origins and evolution. Curr. Opin. Virol. 1:58‐62. doi: 10.1016/j.coviro.2011.05.008.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library