Dengue Virus: Isolation, Propagation, Quantification, and Storage

Freddy Medina1, Juan Francisco Medina1, Candimar Colón1, Edgardo Vergne1, Gilberto A. Santiago1, Jorge L. Muñoz‐Jordán1

1 Centers for Disease Control and Prevention, Division of Vector Borne Infectious Diseases, Dengue Branch, San Juan, Puerto Rico
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15D.2
DOI:  10.1002/9780471729259.mc15d02s27
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV‐1, ‐2, ‐3, and ‐4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single‐stranded RNA genome. Clinical manifestations of dengue can have a wide range of outcomes varying from a mild febrile illness to a life‐threatening condition. New techniques have largely replaced the use of DENV isolation in disease diagnosis. However, virus isolation still serves as the gold standard for detection and serotyping of DENV and is common practice in research and reference laboratories where clinical isolates of the virus are characterized and sequenced, or used for a variety of research experiments. Isolation of DENV from clinical samples can be achieved in mammalian and mosquito cells or by inoculation of mosquitoes. The experimental methods presented here describe the most common procedures used for the isolation, serotyping, propagation, and quantification of DENV. Curr. Protoc. Microbiol. 27:15D.2.1‐15D.2.24. © 2012 by John Wiley & Sons, Inc.

Keywords: dengue; infection; titration; plaque assay; flow cytometry; mosquito inoculation; immunofluorescence

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Dengue Virus Isolation from Diagnostic Samples in C6/36 Cells
  • Basic Protocol 2: Dengue Virus Inoculation of Mosquitoes In Vivo
  • Basic Protocol 3: Immunofluorescence Assays
  • Basic Protocol 4: Titration of Dengue Virus by Plaque Assay
  • Alternate Protocol 1: Titration of Dengue Virus by Flow Cytometry
  • Basic Protocol 5: Dengue Virus Propagation
  • Basic Protocol 6: Purification of Dengue Virus by Sucrose Gradients
  • Support Protocol 1: Growth and Splitting of C6/36 Cells
  • Support Protocol 2: Growth and Splitting of Vero Cells
  • Support Protocol 3: Freezing C6/36 and Vero Cells
  • Support Protocol 4: Thawing Frozen C6/36 and Vero Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Dengue Virus Isolation from Diagnostic Samples in C6/36 Cells

  Materials
  • C6/36 cells (see protocol 8)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium ( appendix 2A)
  • 0.05% trypsin‐EDTA: 0.05% (w/v) trypsin in 1 mM EDTA
  • DMEM‐5 and DMEM‐2 media (see reciperecipes)
  • 7.5% (w/v) sodium bicarbonate (Gibco, cat. no. 25080‐094)
  • Fetal bovine serum (FBS)
  • Incubator (33°C)
  • 16 × 125‐mm screw‐capped tissue culture tubes
  • Slanted tube racks, 5° and 20° angles
  • 0.02‐µm × 13‐mm filter (optional)
  • Pillow
  • Transfer pipet

Basic Protocol 2: Dengue Virus Inoculation of Mosquitoes In Vivo

  Materials
  • Adult Aedes aegypti or Toxorhynchites amboinensis mosquitoes (15 per sample plus 20 for controls)
  • DENV‐containing sample (e.g., serum, plasma, or cell culture supernatant from infected cells)
  • 10% (w/v) sucrose in dH 2O
  • Acetone, chilled to –20°C
  • BA‐1 diluent (see recipe)
  • Borosilicate glass tubing (internal diameter 0.4 mm; outside diameter 0.7 to 1.0 mm; 30 in. length)
  • Bunsen burner
  • Jeweler's forceps
  • Rubber stamp with lines marked 1 mm apart
  • Injection apparatus [see Rosen and Gubler ( ) for assembly details]
    • Metal holder for glass needle
    • Plastic tubing [internal diameter 3/16 in., wall thickness 1/16 in., outside diameter 5/16 in. (7.9 mm)]
    • 50‐ml syringe
    • 3‐way stopcock
  • Dissecting (stereo) microscope
  • 28°C incubator
  • Cotton balls
  • Scalpel
  • Poly‐lysine–treated glass slides
  • 2.0 ml microcentrifuge tubes, snap‐cap
  • 4.5‐mm premium‐grade copper or stainless steel BBs
  • TissueLyser mixer mill or vortex

Basic Protocol 3: Immunofluorescence Assays

  Materials
  • 0.005% (w/v) poly‐lysine
  • C6/36 DENV‐infected cells (Basic Protocol protocol 11 or protocol 65)
  • Acetone
  • Primary FITC‐conjugated polyclonal antibody
  • Phosphate‐buffered saline (PBS) without calcium or magnesium ( appendix 2A)
  • 90% (v/v) glycerol in PBS
  • Primary monoclonal antibodies (mAbs) for all four DENV serotypes (for use in indirect method)
    • DENV‐1: 15F3‐1 (purified from hybridoma ATCC #HB‐47)
    • DENV‐2: 3H5‐1 (purified from hybridoma ATCC #HB‐46)
    • DENV‐3: 5D4‐11 (purified from hybridoma ATCC #HB‐49)
    • DENV‐4: 1H10‐6 (purified from hybridoma ATCC #HB‐48)
  • Human sera (for use in indirect method)
  • Secondary FITC‐conjugated anti‐mouse antibody (for use in indirect method)
  • Teflon‐masked 12‐well slides
  • Pillow
  • Transfer pipet
  • Coplin jar
  • 37°C incubator
  • Humidified chamber (e.g., plastic box containing moistened paper towels)
  • Slide coverslip
  • Fluorescence microscope

Basic Protocol 4: Titration of Dengue Virus by Plaque Assay

  Materials
  • Vero cells ( protocol 9)
  • M199‐5 medium (see recipe)
  • 10 mg/ml gentamicin stock solution
  • 7.5% (w/v) sodium bicarbonate stock solution (Gibco, cat. no. 25080‐094)
  • SeaKem ME agarose
  • 2× Ye‐Lah overlay medium (see recipe)
  • 30% (v/v) FBS in PBS (see appendix 2A for PBS recipe)
  • 3.2% (v/v) neutral red in PBS (see appendix 2A for PBS recipe)
  • Hemacytometer
  • 6‐well plates
  • Light microscope
  • 37°C incubator with 10% CO 2
  • 45°C water bath

Alternate Protocol 1: Titration of Dengue Virus by Flow Cytometry

  Materials
  • Vero cells ( protocol 9)
  • 106 pfu/ml DENV stock (Basic Protocols protocol 11, protocol 22, protocol 65, or protocol 76)
  • M199‐0 and ‐5 (see reciperecipes)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium (see appendix 2A)
  • 1× Hanks balanced salt solution (HBSS) (optional) (see appendix 2A)
  • 0.05% trypsin‐EDTA: 0.05% (w/v) trypsin in 1 mM EDTA
  • 10% (v/v) FBS in PBS
  • BD Cytofix/Cytoperm (BD Biosciences, cat. no. 554722)
  • BD Cytoperm/Cytowash (BD Biosciences, cat. no. 554723)
  • Pan‐DENV fluorophore‐conjugated mAb
    • 4G2 (purified from hybridoma ATCC #HB‐112) or
    • 2H2 (purified from hybridoma ATCC #HB‐114)
  • 24‐well plates
  • 37°C incubator
  • Aspirator
  • Light microscope
  • 96‐well V‐bottom plates
  • Plate adaptors for centrifuge
  • Multichannel pipettor
  • Parafilm (optional)
  • Microtiter tubes
  • Flow cytometer and analysis software

Basic Protocol 5: Dengue Virus Propagation

  Materials
  • C6/36 cells ( protocol 8)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium
  • DMEM‐0, ‐2 (see recipe)
  • FBS
  • 75‐cm2 seal‐cap tissue culture flasks
  • 33°C incubator
  • Light microscope
  • 15‐ml sterile conical polypropylene centrifuge tubes
  • Millipore Centricon Plus‐20 (UFC2BHK08) or Centricon Plus‐70 (UFC710008) (optional)

Basic Protocol 6: Purification of Dengue Virus by Sucrose Gradients

  Materials
  • 20% (w/v) sucrose in dH 2O
  • Virus supernatant ( protocol 6)
  • DMEM‐30 (see recipe)
  • Disposable bottle‐top 0.2‐µm SFCA membrane filter unit
  • Polyallomer 25 × 89‐mm ultracentrifuge tubes (Beckman)
  • Ultracentrifuge with SW‐28 Beckman rotor

Support Protocol 1: Growth and Splitting of C6/36 Cells

  Materials
  • C6/36 cells (ATCC #CRL‐1660)
  • DMEM‐5 (see recipe), 33°C
  • Phosphate‐buffered saline (PBS) without calcium or magnesium (see appendix 2A), 33°C
  • 0.05% trypsin‐EDTA: 0.05% (w/v) trypsin in 1 mM EDTA
  • 33°C incubator
  • 15‐ml conical centrifuge tubes
  • 75‐cm2 seal‐cap tissue culture flasks

Support Protocol 2: Growth and Splitting of Vero Cells

  Materials
  • Vero cells (ATCC #CCL‐81)
  • M199‐5 (see recipe)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium (see appendix 2A)
  • 0.05% trypsin‐EDTA: 0.05% (w/v) trypsin in 1 mM EDTA
  • 150‐cm2 seal‐cap tissue culture flasks
  • 37°C incubator

Support Protocol 3: Freezing C6/36 and Vero Cells

  Materials
  • C6/36 cells ( protocol 8) or Vero cells ( protocol 9)
  • DMEM‐5 medium (for C6/36 cells) or M199‐5 medium (for Vero cells) with 5% (v/v) DMSO
  • Hemacytometer
  • Cryovials
  • Freezing container (Mr. Frosty)

Support Protocol 4: Thawing Frozen C6/36 and Vero Cells

  Materials
  • Cryovial of C6/36 or Vero cells frozen at −70°C and stored in liquid nitrogen ( protocol 10)
  • DMEM‐5 medium (for C6/36 cells) or M199‐5 medium (for Vero cells) with 5% DMSO
  • 15‐ml conical test tube
  • 25‐cm2 tissue culture flask
  • Incubator (33°C for C6/36 cells or 37°C for Vero cells)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 15.D0.1 Photograph sequence demonstrating how to construct a mosquito inoculation needle out of a fine borosilicate tube. The tube is placed in the flame; as soon as it becomes malleable, it is pulled apart (A and B). Needle tip refinement is achieved under a stereomicroscope by using forceps to break the closed end of the tube (C and D). The needle is then stamped with the measuring guides (E) and attached to the syringe system in the stereomicroscope (F).
  •   FigureFigure 15.D0.2 Photograph sequence of an intrathoracic inoculation of a Toxorhynchites amboinensis mosquito. A virus inoculum of 0.34 µl is injected through the soft cuticle between the sclerites of the mosquito (A). The inoculated mosquito is removed from the microscope and placed in a container for incubation using the needle's grasp (B). Inoculated mosquitoes are stored at 28°C for 14 days to obtain optimal virus replication (C).
  •   FigureFigure 15.D0.3 Photograph sequence of an intrathoracic inoculation of an Aedes aegypti mosquito. A virus inoculum of 0.17 µl is injected into the membrane anterior to the mesepisternum of the mosquito (A). The inoculated mosquito is removed from the microscope and placed in a container for incubation using the needle's grasp (B). Inoculated mosquitoes are stored at 28°C for 14 days to obtain optimal virus replication (C).
  •   FigureFigure 15.D0.4 Example of DENV plaque assay. Vero cells were infected with serial 1:10 dilutions of dengue virus strain 16681 in duplicate. Plaques were stained with 3.2% neutral red solution 5 days after infection, and visualized 24 hr later for counting.
  •   FigureFigure 15.D0.5 Titration of dengue virus by flow cytometry. Vero cells were infected with serial 1:10 dilutions of dengue virus strain 16681 for 24 hr. Cell fluorescence was measured on a BD FACS Calibur and data analysis was conducted using BD Cell Quest software to determine the percent of dengue‐positive cells (see upper‐right‐hand corner of each graph).

Videos

Literature Cited

Literature Cited
   Gubler, D.J. 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11:480‐496.
   Gubler, D.J., Suharyono, W., Tan, R., Abidin, M., and Sie, A. 1981. Viraemia in patients with naturally acquired dengue infection. Bull. World Health Organ. 59:623‐630.
   Gubler, D.J., Kuno, G., Sather, G.E., Velez, M., and Oliver, A. 1984. Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Am. J. Trop. Med. Hyg. 33:158‐165.
   Guzman, M.G. and Kouri, G. 2002. Dengue: an update. Lancet Infect Dis. 2:33‐42.
   Halstead, S.B. 1970. Observations related to pathogenesis of dengue hemorrhagic fever. VI. Hypotheses and discussion. Yale J. Biol. Med. 42:350‐362.
   Halstead, S.B. 1988. Pathogenesis of dengue: challenges to molecular biology. Science 239:476‐481.
   Holmes, E.C. and Twiddy, S.S. 2003. The origin, emergence and evolutionary genetics of dengue virus. Infect. Genet. Evol. 3:19‐28.
   Igarashi, A. 1978. Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses. J. Gen. Virol. 40:531‐544.
   Kuno, G. and Oliver, A. 1989. Maintaining mosquito cell lines at high temperatures: effects on the replication of flaviviruses. In Vitro Cell Dev. Biol. 25:193‐196.
   Kuno, G., Gubler, D.J., Velez, M., and Oliver, A. 1985. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses. Bull. World Health Organ. 63:279‐286.
   Lambeth, C.R., White, L.J., Johnston, R.E., and de Silva, A.M. 2005. Flow cytometry‐based assay for titrating dengue virus. J. Clin. Microbiol. 43:3267‐3272.
   Libraty, D.H., Young, P.R., Pickering, D., Endy, T.P., Kalayanarooj, S., Green, S., Vaughn, D.W., Nisalak, A., Ennis, F.A., and Rothman, A.L. 2002. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J. Infect. Dis. 186:1165‐1168.
   McElroy, K.L., Santiago, G.A., Lennon, N.J., Birren, B.W., Henn, M.R., and Muñoz‐Jordán, J.L. 2011. Endurance, refuge, and reemergence of dengue virus type 2, Puerto Rico, 1986‐2007. Emerg. Infect. Dis. 17:64‐71.
   Mukhopadhyay, S., Kuhn, R.J., and Rossmann, M.G. 2005. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3:13‐22.
   OhAinle, M., Balmaseda, A., Macalalad, A.R., Tellez, Y., Zody, M.C., Saborío, S., Nuñez, A., Lennon, N.J., Birren, B.W., Gordon, A., Henn, M.R., and Harris, E. 2011. Dynamics of dengue disease severity determined by the interplay between viral genetics and serotype‐specific immunity. Sci. Transl. Med. 3:114ra128.
   Rico‐Hesse, R. 2003. Microevolution and virulence of dengue viruses. Adv. Virus Res. 59:315‐341.
   Rico‐Hesse, R., Harrison, L.M., Salas, R.A., Tovar, D., Nisalak, A., Ramos, C., Boshell, J., de Mesa, M.T., Nogueira, R.M., and da Rosa, A.T. 1997. Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 230:244‐251.
   Rosen, L. 1981. The use of Toxorhynchites mosquitoes to detect and propagate dengue and other arboviruses. Am. J. Trop. Med. Hyg. 30:177‐183.
   Rosen, L. and Gubler, D. 1974. The use of mosquitoes to detect and propagate dengue viruses. Am. J. Trop. Med. Hyg. 23:1153‐1160.
   Rosen, L., Roseboom, L.E., Gubler, D.J., Lien, J.C., and Chaniotis, B.N., 1985. Comparative susceptibility of mosquito species and strains to oral and parenteral infection with dengue and Japanese encephalitis viruses. Am. J. Trop. Med. Hyg. 34:603‐615.
   Sukhavachana, P., Nisalak, A., and Halstead, S.B. 1966. Tissue culture techniques for the study of dengue viruses. Bull. World Health Organ. 35:65‐66.
   Vaughn, D.W., Green, S., Kalayanarooj, S., Innis, B.L., Nimmannitya, S., Suntayakorn, S., Endy, T.P., Raengsakulrach, B., Rothman, A.L., Ennis, F.A., and Nisalak, A. 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J. Infect. Dis. 181:2‐9.
   Vorndam, V. and Kuno, G. 1997. Laboratory diagnosis of dengue virus infections. In Dengue and Dengue Hemorrhagic Fever (D.J. Gubler, ed.) pp. 313‐333. CAB International, London, United Kingdom.
   Wang, W.K., Chao, D.Y., Kao, C.L., Wu, H.C., Liu, Y.C., Li, C.M., Lin, S.C., Ho, S.T., Huang, J.H., and King, C.C. 2003. High levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: Implications for pathogenesis. Virology 305:330‐338.
   Westaway, E.G. and Blok, J. 1997. Taxonomy and evolutionary relationships of flaviviruses. In Dengue and Dengue Hemorrhagic Fever (D.J. Gubler, ed.) pp. 147‐173. CAB International, London, United Kingdom.
   World Health Organization. 2009. Dengue: Guidelines for diagnosis, treatment, prevention and control. World Health Organization and the Special Programme for Research and Training in Tropical Diseases, Geneva.
   World Health Organization, 2010. Impact of Dengue. http://www.who.int/csr/disease/dengue/impact/en/index.html.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library