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

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Abstract:

Viral culture laboratories generally use a combination of tube and shell vial cultures. A number of variables can influence the sensitivity of viral cultures, including cell culture type, age and confluence of the monolayer, the number of tubes or vials inoculated, the inoculation and incubation conditions, and the method and reagent used for isolate detection or identification.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-1

Inoculation of tube and shell vial cell cultures for the detection of cytopathic and hemadsorbing viruses (see Table 10.5-1 ). RSV, respiratory syncytial virus.

CMV antigenemia or molecular assays (e.g., PCR) have largely supplanted cell culture for detecting CMV in blood samples. When using shell vial cultures for recovery of CMV from blood, inoculate three vials with approximately 1.5 × 10leukocytes per vial. It has been reported that a total of at least 4 × 10leukocytes is recommended for the sensitive detection of CMV viremia using human lung fibroblasts ( ); concentrations of >2 × 10cells per vial are not generally recommended because of toxicity.

Eagle minimal essential medium (EMEM) supplemented with 2% heat-inactivated FBS is a medium suitable for all or most viral cultures performed in the diagnostic setting with mono layered cell cultures; serum-free EMEM or other serum-free cell culture medium isused by many laboratories for the isolation of influenza and parainfluenza viruses. Medium containing trypsin is recommended for influenza virus cultures when using MDCK cells (see Table 10.2-1).

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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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.

See Fig. 10.5-3.

Viral viability may decline rapidly with incubation in the absence of cells, particularly with labile viruses (e.g., RSV, CMV).

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.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.

In rare cases, specimen toxicity may be so great that two or three subpassages are required. Unlike viral CPE, toxicity may sometimes be observed at 3 to 4 h after inoculation.

Viral viability may decline rapidly with incubation in the absence of cells, particularly with labile viruses (e.g., respiratory syncytial virus, CMV).

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-4a
Figure 10.5-4a

(part 1) Primary rhesus monkey kidney cell cultures. Original magnification, ×200. (A) Uninoculated confluent monolayer; (B)influenza virus typeB-infected culture showing granular cytopathic changes produced by some influenza virusstrains; (C) culture infected within fluenza virus typeA, 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: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-4b

(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: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-4c

(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: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-4d

(part 3) A-549 cells. Original magnification, ×200. (A) Uninoculated culture showing over growth 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: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-4e
Figure 10.5-4e

(part 4) Primary rabbit kidney cell cultures. Original magnification, ×200. (A) Uninoculated confluent monolayer; (B) rapidly progressive CPE produced by HSV type 2.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-4f

(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: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-4g

(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 f echovirus type 11 starting at the monolayer edge. (I) Late CPE of echo virus type 11 showing complete involvement of the monolayer. (J) Focal area of replicating A-549 cells that have inadvertently cross-contaminated an MRC-5 monolayer.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-4h
Figure 10.5-4h

(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 f echovirus type 11 starting at the monolayer edge. (I) Late CPE of echo virus type 11 showing complete involvement of the monolayer. (J) Focal area of replicating A-549 cells that have inadvertently cross-contaminated an MRC-5 monolayer.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-4

(part 7) HSV type 1-infected EL VIS HSV cells (black cells in this photo, ×200) stained 16 h after infection. EL VIS (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 H. D. Isenberg (ed.). 1998. ASM Press, Washington, D.C.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.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: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-6
Figure 10.5-6

Had procedure. NDV, Newcastle disease virus.

Agglutinated RBCs may be observed in the residual fluid (hemagglutination) or by incubating cell culture fluid with a suspension of appropriate RBCs ( Appendix 10.5-5 ).

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-7
Figure 10.5-7

Isolate identification: slide preparation.

This method is rapid but may yield higher levels of nonspecific staining than observed when the monolayers or packed cells are washed before spotting.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-8

Isolate identification: IF.

When removing slides from cold storage, allow them to equilibrate to room temperature before opening the storage container.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-9a
Figure 10.5-9a

(A) LLC-MK2 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-MK2 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 panenterovirus blend of MAbs (×200). Provided by Chemicon International. (J) Echovirus type 4-infected BGMK cells stained using echovirus type 4 typing reagent (×200). Provided by Chemicon International. (K) Influenza A- and influenza B-infected LLC-MKcells stained with SimulFluor influenza A/influenza B reagent and observed using filters for FITC. Influenza A-infected cells (arrows) stain apple-green; influenza B-infected cells are yellow-orange. Provided by Chemicon International. (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. Provided by Chemicon International.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Figure 10.5-9b

(A) LLC-MK2 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-MK2 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 panenterovirus blend of MAbs (×200). Provided by Chemicon International. (J) Echovirus type 4-infected BGMK cells stained using echovirus type 4 typing reagent (×200). Provided by Chemicon International. (K) Influenza A- and influenza B-infected LLC-MKcells stained with SimulFluor influenza A/influenza B reagent and observed using filters for FITC. Influenza A-infected cells (arrows) stain apple-green; influenza B-infected cells are yellow-orange. Provided by Chemicon International. (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. Provided by Chemicon International.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-9c
Figure 10.5-9c

(A) LLC-MK2 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-MK2 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 panenterovirus blend of MAbs (×200). Provided by Chemicon International. (J) Echovirus type 4-infected BGMK cells stained using echovirus type 4 typing reagent (×200). Provided by Chemicon International. (K) Influenza A- and influenza B-infected LLC-MKcells stained with SimulFluor influenza A/influenza B reagent and observed using filters for FITC. Influenza A-infected cells (arrows) stain apple-green; influenza B-infected cells are yellow-orange. Provided by Chemicon International. (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. Provided by Chemicon International.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-10
Figure 10.5-10

Shell vial processing.

The sensitivity of shell vial cultures is influenced by the length of incubation, and each laboratory must determine appropriate staining timepoints. Typically, shell vial incubation times range from 18 to 36 h (e.g., CMV, HSV) to up to 5 days (e.g., VZV).

Complete monolayer destruction may also be caused by a rapid CPE.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-11
Figure 10.5-11

Shell vial cultures, IF.

When removing vials from storage, allow them to equilibrate to room temperature before opening the storage container.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Image of Figure 10.5-A1
Figure 10.5-A1

Viral titration with serial 10-fold dilutions.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
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Tables

Generic image for table
Table 10.5-1a

Viral culture characteristics

Certain viruses require specialized culture techniques or are noncultivatable. Refer to Table 10.5-A2 .

Time generally required for majority of isolates to produce CPE or HAd.

Neutralization assay involves mixing the isolate with antiserum containing neutralizing antibodies and, after a brief incubation, inoculating the cell cultures with the virus-antibody mixture(s). Neutralization by an antibody is determined by the failure to develop CPE, thereby establishing viral identity ( Appendix 10.5-2 ).

RSV, respiratory syncytial virus.

See Fig. 10.5-4 , parts 1 to 6.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
Generic image for table
Table 10.5-1b

Viral culture characteristics

Certain viruses require specialized culture techniques or are noncultivatable. Refer to Table 10.5-A2 .

Time generally required for majority of isolates to produce CPE or HAd.

Neutralization assay involves mixing the isolate with antiserum containing neutralizing antibodies and, after a brief incubation, inoculating the cell cultures with the virus-antibody mixture(s). Neutralization by an antibody is determined by the failure to develop CPE, thereby establishing viral identity ( Appendix 10.5-2 ).

RSV, respiratory syncytial virus.

See Fig. 10.5-4 , parts 1 to 6.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
Generic image for table
Table 10.5-1c

Viral culture characteristics

Certain viruses require specialized culture techniques or are noncultivatable. Refer to Table 10.5-A2 .

Time generally required for majority of isolates to produce CPE or HAd.

Neutralization assay involves mixing the isolate with antiserum containing neutralizing antibodies and, after a brief incubation, inoculating the cell cultures with the virus-antibody mixture(s). Neutralization by an antibody is determined by the failure to develop CPE, thereby establishing viral identity ( Appendix 10.5-2 ).

RSV, respiratory syncytial virus.

See Fig. 10.5-4 , parts 1 to 6.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
Generic image for table
Table 10.5-1d

Viral culture characteristics

Certain viruses require specialized culture techniques or are noncultivatable. Refer to Table 10.5-A2 .

Time generally required for majority of isolates to produce CPE or HAd.

Neutralization assay involves mixing the isolate with antiserum containing neutralizing antibodies and, after a brief incubation, inoculating the cell cultures with the virus-antibody mixture(s). Neutralization by an antibody is determined by the failure to develop CPE, thereby establishing viral identity ( Appendix 10.5-2 ).

RSV, respiratory syncytial virus.

See Fig. 10.5-4 , parts 1 to 6.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
Generic image for table
Table 10.5-A1

Calculation of TCID50 by the Reed-Muench method ( )

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
Generic image for table
Table 10.5-A2

Problems associated with interpretation of the neutralization test

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5
Generic image for table
Table 10.5-A3

Hemagglutination characteristics of representative viruses

RT, room temperature.

Depends on virus type.

Citation: Garcia L. 2010. Viral Culture: Isolation of Viruses in Cell Cultures, p 51-91. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.5

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