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Chapter 7 : Primary Isolation of Viruses

Authors: Marie L. Landry1, Diane Leland2
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Affiliations: 1: Yale University, Departments of Laboratory Medicine and Internal Medicine (Infectious Diseases) New Haven, CT 06520; 2: Indiana University School of Medicine, Department of Pathology and Laboratory Medicine Indianapolis, IN 46202
Content Type: Reference
Publication Year: 2016

Category: Clinical Microbiology

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Abstract:

Viruses are obligate intracellular parasites and thus are propagated using living cells in the form of cultured cells, embryonated hen's eggs, or laboratory animals. Culture has long been considered the “gold standard” for viral diagnosis because it secures an isolate for further analysis, is more “open-minded” than methods that target single agents, and allows the unexpected or even novel agent to be recovered. In practice, use of specialized cell culture systems, embryonated eggs, and laboratory animals is confined to research or major public health reference laboratories, with cell cultures in monolayers the sole isolation system utilized in routine diagnostic laboratories. The past two decades have seen conventional cell culture methods supplemented or even replaced by more rapid and targeted cell culture methods. Rapid culture methods can be performed by less experienced personnel, with less labor, and with results reported within 1 to 5 days of inoculation.

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
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Primary Isolation of Viruses
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Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
/content/10.1128/9781555819156.ch07.ch7fig1
FIGURE 1

Uninfected cell cultures and cell cultures showing CPE of viruses commonly isolated. (A) Uninfected A549 cells, (B) HSV-2 in A549, (C) adenovirus in A549, (D) uninfected MRC-5 fibroblasts, (E) CMV in MRC-5, (F) rhinovirus in MRC-5, (G) uninfected RhMK, (H) enterovirus in RhMk, (I) influenza A in RhMk, (J) uninfected HEp-2, (K) RSV in HEp-2, and (L) monkey virus contaminant in RhMk. ×85. (Photos by permission, Leland and Ginocchio [ ].)

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10.1128/9781555819156/9156ch7fig1.gif
Image of FIGURE 1
FIGURE 1

Uninfected cell cultures and cell cultures showing CPE of viruses commonly isolated. (A) Uninfected A549 cells, (B) HSV-2 in A549, (C) adenovirus in A549, (D) uninfected MRC-5 fibroblasts, (E) CMV in MRC-5, (F) rhinovirus in MRC-5, (G) uninfected RhMK, (H) enterovirus in RhMk, (I) influenza A in RhMk, (J) uninfected HEp-2, (K) RSV in HEp-2, and (L) monkey virus contaminant in RhMk. ×85. (Photos by permission, Leland and Ginocchio [ ].)

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
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FIGURE 2

Hemadsorption of guinea pig red blood cells by parainfluenza virus in monkey kidney (MK) cells. (A) Uninfected MK cells; (B) specific hemadsorption in parainfluenza infected MK cells; (C) nonspecific hemadsorption seen with aged red blood cells in uninfected cell cultures. Modified from Hsiung et al. ( ). (Reprinted from last edition.)

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Image of FIGURE 2
FIGURE 2

Hemadsorption of guinea pig red blood cells by parainfluenza virus in monkey kidney (MK) cells. (A) Uninfected MK cells; (B) specific hemadsorption in parainfluenza infected MK cells; (C) nonspecific hemadsorption seen with aged red blood cells in uninfected cell cultures. Modified from Hsiung et al. ( ). (Reprinted from last edition.)

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
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FIGURE 3

Centrifugation culture: detection of CMV immediate early antigens in infected nuclei at 16 to 24 hours postinoculation (immunofluorescence stain). (Reprinted from last edition.)

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Image of FIGURE 3
FIGURE 3

Centrifugation culture: detection of CMV immediate early antigens in infected nuclei at 16 to 24 hours postinoculation (immunofluorescence stain). (Reprinted from last edition.)

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
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FIGURE 4

R-mix cells in shell vial format. (A) Stained with respiratory virus screen reagent at day 1 postinoculation; (B) identified as influenza A by spotting shell vial cells onto a multiwell slide and staining with individual antibodies (immunofluorescence stain). Photos courtesy of Diagnostic Hybrids, Inc. (Reprinted from last edition.)

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10.1128/9781555819156/9156ch7fig4.gif
Image of FIGURE 4
FIGURE 4

R-mix cells in shell vial format. (A) Stained with respiratory virus screen reagent at day 1 postinoculation; (B) identified as influenza A by spotting shell vial cells onto a multiwell slide and staining with individual antibodies (immunofluorescence stain). Photos courtesy of Diagnostic Hybrids, Inc. (Reprinted from last edition.)

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
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FIGURE 5

Detection of HSV in ELVIS cells. Blue cells positive for HSV infection (X-Gal stain). Photo courtesy of Diagnostic Hybrids, Inc. (Reprinted from last edition.)

10.1128/9781555819156/9156ch7fig5_thmb.gif
10.1128/9781555819156/9156ch7fig5.gif
Image of FIGURE 5
FIGURE 5

Detection of HSV in ELVIS cells. Blue cells positive for HSV infection (X-Gal stain). Photo courtesy of Diagnostic Hybrids, Inc. (Reprinted from last edition.)

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
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Tables

Primary Isolation of Viruses
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Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
/content/10.1128/9781555819156.ch07.ch7tab1
TABLE 1

Types of cell cultures commonly used in a clinical virology laboratory

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TABLE 1

Types of cell cultures commonly used in a clinical virology laboratory

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
/content/10.1128/9781555819156.ch07.ch7tab2
TABLE 2

Conventional cell cultures for viruses commonly isolated in a clinical virology laboratory

Generic image for table
TABLE 2

Conventional cell cultures for viruses commonly isolated in a clinical virology laboratory

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
/content/10.1128/9781555819156.ch07.ch7tab3
TABLE 3

Supplies and equipment needed for isolation of viruses in cell culture

Generic image for table
TABLE 3

Supplies and equipment needed for isolation of viruses in cell culture

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7
/content/10.1128/9781555819156.ch07.ch7tab4
TABLE 4

Mixed cell culture methods used in clinical laboratories

Generic image for table
TABLE 4

Mixed cell culture methods used in clinical laboratories

Citation: Landry M, Leland D. 2016. Primary Isolation of Viruses, p 79-93. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch7

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