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Chapter 3 : Primary Isolation of Viruses

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Abstract:

Despite the increasing availability of viral antigen and nucleic acid tests, culture methods remain important, especially in the hospital setting. This chapter focuses on the isolation methods currently used in routine clinical laboratories. Recent innovations in monolayer cell culture include the use of mixtures of two or three different cell lines and genetically altered cells to enhance sensitivity or facilitate detection of virus infection. The materials needed for the isolation of viruses in cell culture are those necessary for the safe handling and inoculation of cell cultures, maintenance and observation of cell cultures, and preservation and storage of clinical specimens and virus isolates. Virus-induced cytopathic effect (CPE) must be distinguished from nonspecific CPE caused by toxicity of specimens, contamination with bacteria or fungi, or old cells. The rapid diagnosis of viral infections is important in patient management. One of the most significant contributions to rapid diagnosis in the clinical laboratory has been the application of centrifugation cultures to viral diagnosis. To get the best results from primary isolation in cell culture, healthy cell cultures susceptible to a spectrum of viruses are essential. In addition, conventional culture should be used for lower respiratory tract and tissue biopsy samples to detect additional or unsuspected viruses. While molecular methods are essential for optimal detection of viruses in spinal fluid and to monitor viral load in blood, virus isolation continues to play an important role in viral diagnosis and patient management, especially when performed locally.

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
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Image of FIGURE 1
FIGURE 1

Examples of characteristic CPE of different viruses. (1) Uninfected RhMK cells; (2) poliovirus CPE in RhMK cells; (3) influenza B virus CPE in RhMK cells; (4) uninfected HEp-2 cells; (5) adenovirus CPE in HEp-2 cells; (6) RSV CPE in HEp-2 cells; (7) uninfected HDFs; (8) rhinovirus CPE in HDFs; (9) CMV CPE in HDFs.

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
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Image of FIGURE 2
FIGURE 2

Cell sensitivity and rate of progression of CPE of two herpesviruses: HSV-1 and human CMV (HCMV). (A) Uninfected WI-38 cells; (B) extensive HSV-1 CPE in WI-38 cells, 2 days post-inoculation; (C) CMV in WI-38 cells, 1 week postinoculation; (D) uninfected RK cells; (E) extensive HSV-1 CPE in RK cells, 1 day postinoculation; (F) absence of CMV CPE in RK cells, 2 weeks postinoculation. (Modified from Hsiung et al., 1994.)

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
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FIGURE 3

Differential sensitivity of cell cultures to enteroviruses. (A) Uninfected RhMK cells; (B) poliovirus-infected RhMK cells showing advanced CPE; (C) echovirus-infected RhMK cells showing advanced CPE; (D) uninfected HEp-2 cells; (E) poliovirus-infected HEp-2 cells showing advanced CPE; (F) echovirus-infected HEp-2 cells showing absence of CPE. (Reprinted from Hsiung et al., 1994, with permission of the publisher.)

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
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Image of FIGURE 4
FIGURE 4

Hemadsorption of guinea pig RBC by parainfluenza virus in MK cells. (A) Uninfected MK cells; (B) specific hemadsorption in parainfluenza virus-infected MK cells; (C) nonspecific hemadsorption seen with aged RBC in uninfected cell cultures. (Modified from Hsiung et al., 1994.)

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
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Tables

Generic image for table
TABLE 1

Types of cell cultures commonly used in a clinical virology laboratory

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
Generic image for table
TABLE 2

Variation in sensitivity of conventional cell cultures to infection by viruses commonly isolated in a clinical virology laboratory

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
Generic image for table
TABLE 3

Supplies and equipment needed for isolation of viruses in cell culture

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
Generic image for table
TABLE 4

Time to virus detection by conventional and centrifugation culture a

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
Generic image for table
TABLE 5

Newer cell culture methods used in clinical laboratories

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3
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TABLE 6

Cell culture susceptibility for enteroviruses

Citation: Landry M. 2009. Primary Isolation of Viruses, p 36-63. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch3

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