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Chapter 10.5 : Viral Culture: Isolation of Viruses in Cell Cultures

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Viral Culture: Isolation of Viruses in Cell Cultures, Page 1 of 2

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Abstract:

Viruses can only replicate inside the cells of a susceptible cell type. For the diagnostic culture of human pathogenic viruses from clinical specimens, monolayer cultures of mammalian cells are generally used. These methods detect the presence of infectious virus particles: they will not detect inactivated viral particles incapable of infecting a cell that may be detected by antigen or nucleic acid detection methods. Diagnostic viral culture laboratories may use a variety of methods, including conventional tube cultures and rapid centrifugation-enhanced culture in either shell vials or multiwell plates, or a combination of these systems. Several variables can influence the virus susceptibility of cell cultures and therefore their sensitivity for virus detection. These include the cell culture type, cell age or passage number, the confluence of the monolayer, the culture medium and additives, the number of tubes or vials inoculated, the volume of the inoculum, the inoculation and incubation conditions, and the method and reagent used for isolate detection or identification. Procedures in this chapter are largely reproduced from those in the previous edition ( ) with updates and edits.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Figures

Image of Figure 10.5–1
Figure 10.5–1

Inoculation of tube and shell vial cell cultures for the detection of cytopathic and hemadsorbing viruses. RSV, respiratory syncytial virus.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–2
Figure 10.5–2

Observation of inoculated monolayers: CPE and HAd. Rarely, cells exhibiting minimal CPE revert to a normal appearance. Subpassage or reinoculation may be helpful in this instance. RSV, respiratory syncytial virus.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–3
Figure 10.5–3

Observation of inoculated cell cultures: toxicity, microbial contamination, and atypical changes. RSV, respiratory syncytial virus.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 1)
Figure 10.5–4 (part 1)

Primary rhesus monkey kidney cell cultures. Original magnification, ×200. (A) Uninoculated confluent monolayer; (B) influenza virus type B-infected culture showing granular cytopathic changes produced by some influenza virus strains; (C) culture infected with influenza virus type A, showing HAd of guinea pig RBCs; (D) cellular rounding and syncytium formation produced by measles virus; (E) culture infected with coxsackievirus type B3 showing scattered rounded refractile cells frequently referred to as enterovirus-like CPE; (F) CPE produced by echovirus type 11 (courtesy of CDC).

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 2)
Figure 10.5–4 (part 2)

Low-passage-number human neonatal kidney cell cultures. Original magnification, ×200. (A) Uninoculated confluent monolayer. (B) Early focus of enlarged rounded cells produced by HSV type 2. (C) Extensive involvement of the monolayer by the rapidly progressive CPE of HSV type 2. (D) Focal area of CPE produced by VZV. Progression of cytopathic changes produced by this virus is slower than with infection by HSV. (E) Culture infected with adenovirus type 3 showing the tightly overlapped rounded cells frequently referred to as grapelike clusters. (F) Latticed arrangement of cells that may result from infection by adenoviruses. (G) Generalized rounding and extensive monolayer destruction caused by echovirus type 6. (H) Early cytopathic changes produced by coxsackievirus type B3. (I) Nonspecific type of CPE observed with infection by reoviruses. Monolayer shows cellular rounding, degeneration, and lifting from the glass surface.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 3)
Figure 10.5–4 (part 3)

A-549 cells. Original magnification, ×200. (A) Uninoculated culture showing overgrowth of the monolayer typical of rapidly growing continuous cell lines; (B) early focus of CPE produced by HSV type 2 against a background of uninfected confluent cells; (C) advanced CPE of HSV type 2; (D) early CPE of adenovirus type 3; (E) advanced CPE of adenovirus type 3.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 4)
Figure 10.5–4 (part 4)

Primary rabbit kidney cell cultures. Original magnification, ×200. (A) Uninoculated confluent monolayer; (B) rapidly progressive CPE produced by HSV type 2.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 5)
Figure 10.5–4 (part 5)

HEp-2 cell cultures. Original magnification, ×200. (A) Uninoculated confluent monolayer; (B) CPE produced by respiratory syncytial virus showing numerous syncytia formed as a result of cell fusion; (C) syncytium formation resulting from infection with measles virus.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 6)
Figure 10.5–4 (part 6)

Diploid human lung fibroblast cultures. Magnification, ×200. (A) Uninoculated confluent monolayer. (B) Advanced CPE produced by HSV type 2 (courtesy of CDC). (C) Focal area of CPE produced by VZV (courtesy of CDC). (D) Advanced CPE of VZV. (E) Focal area of CPE produced by CMV. This CPE usually progresses slowly. (F) Large focal area of late CPE produced by CMV. (G) Late CPE produced by adenovirus (courtesy of CDC). (H) CPE of echovirus type 11 starting at the monolayer edge. (I) Late CPE of echovirus type 11 showing complete involvement of the monolayer. (J) Focal area of replicating A-549 cells that have cross-contaminated an MRC-5 monolayer.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–4 (part 7)
Figure 10.5–4 (part 7)

HSV type 1-infected ELVIS HSV cells (blue cells, appear black in this photo, ×200) stained 16 h after infection. ELVIS (enzyme-linked virus-inducible system) HSV cells are genetically engineered BHK cells in which HSV proteins induce the intracellular accumulation of the reporter enzyme β-galactosidase, which is then histochemically detected. Provided by Diagnostic Hybrids, Inc. Reprinted from Isenberg HD (ed). 1998. ASM Press, Washington, DC.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–5
Figure 10.5–5

HAd with guinea pig RBCs. (Top) Uninoculated primary rhesus monkey kidney cells; (bottom) primary rhesus monkey kidney cells infected with parainfluenza virus type 3. Magnification, ×100.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–6
Figure 10.5–6

HAd procedure. NDV, Newcastle disease virus.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–7
Figure 10.5–7

Isolate identification: slide preparation.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–8
Figure 10.5–8

Isolate identification: IF.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–9
Figure 10.5–9

(A) LLC-MK cells infected with parainfluenza virus (magnification, ×200). (B) Adenovirus culture showing nuclear and cytoplasmic staining (magnification, ×400). (C) Perinuclear and cytoplasmic staining characteristic of HSV-infected cells (magnification, ×400). (D) Brilliant oval nuclei of human fibroblasts infected with CMV after being stained with a MAb to CMV early nuclear protein (magnification, ×200). (E) MRC-5 cells stained after 72 h with a mixture of MAbs directed against intermediate-early nuclear and late cytoplasmic antigens (×200). (F) VZV in human fibroblast culture (magnification, ×400). (G) LLC-MK cells infected with influenza A virus (×200). (H) HEp-2 cells infected with respiratory syncytial virus (×200). (I) Echovirus type 4-infected BGMK cells stained with pan enterovirus blend of MAbs (×200). (J) Echovirus type 4-infected BGMK cells stained using echovirus type 4 typing reagent (×200). (K) Influenza A- and influenza B-infected LLC-MK cells stained with SimulFluor influenza A/influenza B reagent and observed by using filters for FITC. Influenza A-infected cells (arrows) stain apple-green; influenza B-infected cells are yellow-orange. (L) Same field as panel K viewed with a tetramethyl rhodamine isocyanate/rhodamine filter. Influenza B-infected cells appear hot pink to red (×200); arrows indicate the location of influenza A-infected cells observed in panel K but which are not visible with this filter system. I, J, K, L: Provided by Chemicon International.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–10
Figure 10.5–10

Shell vial processing.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–11
Figure 10.5–11

Shell vial cultures, IF.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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Image of Figure 10.5–A1
Figure 10.5–A1

Viral titration with serial 10-fold dilutions.

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
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References

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Tables

Generic image for table
Table 10.5–1

Viral culture characteristics

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5
Generic image for table
Table 10.5–A2

Hemagglutination characteristics of representative viruses

Citation: Leber A. 2016. Viral Culture: Isolation of Viruses in Cell Cultures, p 10.5.1-10.5.39. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch10.5

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