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Chapter 13 : Diagnosis of Viral Infections*
Authors:
Guy Boivin,
Tony Mazzulli,
Martin Petric,
Michel Couillard
Content Type: Reference
Publication Year:
2009
Category: Viruses and Viral Pathogenesis
Book DOI:
10.1128/9781555815981
Chapter DOI:
10.1128/9781555815981.ch13
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Diagnosis of Viral Infections*, Page 1 of 2
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Abstract:
The traditional epidemiological and academic reasons for diagnosis of viral infections have been expanded by rapid, often quantitative assays that can impact on therapeutic management and public health decisions. All methods used for viral isolation require living cells because viruses are obligate intracellular parasites. Historically, the systems used to isolate viruses of medical importance consisted of laboratory animals, embryonated eggs, and cultured cells. In the past two decades, safe and effective antiviral drugs have been developed for the treatment of many acute and chronic viral infections. In most diagnostic laboratories, electron microscopy (EM) for the diagnosis of viral infections has been supplanted by other methods, but EM remains an important and often rapid method for detecting viruses in clinical samples. Viral serologic assays contribute significantly to the indirect diagnosis of acute, recent, or chronic viral infections and are used widely for determining the immune status of a person or group of individuals with regard to a specific virus or to verify the immune response to vaccination. A variety of methods are available for serodiagnosis of viral infections. Failure to establish an accurate serologic diagnosis frequently results from the inability to submit an adequate pair of serum samples.
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
Figures
Diagnosis of Viral Infections*
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Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig01
FIGURE 1
CPE induced by some viruses. Row A shows uninfected cell lines, including HEp-2 cells (
1
), RD cells (
2
), fibroblasts (
3
), and Vero cells (
4
). Row B shows the same cell lines infected with RSV (
1
), enterovirus (
2
), CMV (
3
), and HSV (
4
).
10.1128/9781555815981/f0268-01_thmb.gif
10.1128/9781555815981/f0268-01.gif
FIGURE 1
CPE induced by some viruses. Row A shows uninfected cell lines, including HEp-2 cells ( 1 ), RD cells ( 2 ), fibroblasts ( 3 ), and Vero cells ( 4 ). Row B shows the same cell lines infected with RSV ( 1 ), enterovirus ( 2 ), CMV ( 3 ), and HSV ( 4 ).
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig02
FIGURE 2
CMV UL97 mutations associated with ganciclovir resistance. CMV UL97 conserved regions are represented by shaded boxes. Numbers under the boxes indicate the positions (codon numbers) of these conserved regions. Vertical bars indicate the presence of amino acid substitutions, while the hatched box indicates a region (codons 590 to 607) in which diverse deletions (from 1 to 17 codons) have been reported.
10.1128/9781555815981/f0270-01_thmb.gif
10.1128/9781555815981/f0270-01.gif
FIGURE 2
CMV UL97 mutations associated with ganciclovir resistance. CMV UL97 conserved regions are represented by shaded boxes. Numbers under the boxes indicate the positions (codon numbers) of these conserved regions. Vertical bars indicate the presence of amino acid substitutions, while the hatched box indicates a region (codons 590 to 607) in which diverse deletions (from 1 to 17 codons) have been reported.
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig03
FIGURE 3
PCR. The strands of the target DNA are separated by heating (melting), and on cooling they anneal with the complementary primers present in excess. The thermostable DNA polymerase extends the primers, forming two double-stranded DNA molecules. On subsequent heating, the strands separate and each anneals with the complementary primer. The cycling of temperature among melting, annealing, and primer extension is repeated multiple times, and the number of product strands is doubled with each cycle. (Reprinted from reference
141a
with permission.)
10.1128/9781555815981/f0275-01_thmb.gif
10.1128/9781555815981/f0275-01.gif
FIGURE 3
PCR. The strands of the target DNA are separated by heating (melting), and on cooling they anneal with the complementary primers present in excess. The thermostable DNA polymerase extends the primers, forming two double-stranded DNA molecules. On subsequent heating, the strands separate and each anneals with the complementary primer. The cycling of temperature among melting, annealing, and primer extension is repeated multiple times, and the number of product strands is doubled with each cycle. (Reprinted from reference 141a with permission.)
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig04
FIGURE 4
RVP assay (Luminex Molecular Diagnostics Inc.). Identification of the target-specific primer extension (TSPE) reaction that has been captured on the microbead is achieved through the oligonucleotide tag. Sorting of the microbeads occurs in the Luminex 100 flow cell instrument, which identifies colored beads with one laser and a phycoerythrin signal on the attached extended amplicon with a second laser.
10.1128/9781555815981/f0276-01_thmb.gif
10.1128/9781555815981/f0276-01.gif
FIGURE 4
RVP assay (Luminex Molecular Diagnostics Inc.). Identification of the target-specific primer extension (TSPE) reaction that has been captured on the microbead is achieved through the oligonucleotide tag. Sorting of the microbeads occurs in the Luminex 100 flow cell instrument, which identifies colored beads with one laser and a phycoerythrin signal on the attached extended amplicon with a second laser.
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig05
FIGURE 5
Real-time PCR (TaqMan process). In this thermocycling reaction, the internal probe, which is conjugated to the fluorescent dye (F) and the quencher dye (Q), hybridizes with the denatured target DNA. When these two dyes are present in close proximity on the probe, the fluorescence of the F dye is quenched. When the new strand being synthesized as an extension of the terminal primers reaches the probe, it is digested by the 5′ exonuclease activity of the thermostable polymerase, liberating the F dye and resulting in the generation of a fluorescent signal. (Reprinted from reference
141a
with permission.)
10.1128/9781555815981/f0276-02_thmb.gif
10.1128/9781555815981/f0276-02.gif
FIGURE 5
Real-time PCR (TaqMan process). In this thermocycling reaction, the internal probe, which is conjugated to the fluorescent dye (F) and the quencher dye (Q), hybridizes with the denatured target DNA. When these two dyes are present in close proximity on the probe, the fluorescence of the F dye is quenched. When the new strand being synthesized as an extension of the terminal primers reaches the probe, it is digested by the 5′ exonuclease activity of the thermostable polymerase, liberating the F dye and resulting in the generation of a fluorescent signal. (Reprinted from reference 141a with permission.)
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig06
FIGURE 6
Strand displacement amplification. This isothermal amplification assay is based on four requirements, namely, primers which include an upstream restriction endonuclease site (BsoB1), the cognate enzyme (BsoB1), a DNA polymerase lacking 5′ exonuclease activity, and nucleoside triphosphates, of which one has been modified to contain an alpha-thiol group (dCTPαS). In this reaction, the target DNA anneals with the primers and is converted to a double-stranded form by the polymerase. The restriction endonuclease introduces a cleavage in the primer sequence, and the polymerase synthesizes a new strand from this site and displaces the existing strand. The restriction endonuclease is not able to cut the newly synthesized strands because of the modified nucleotides and can only introduce cuts in the sites present in the primers. Restriction site-bearing primers are designed to anneal to sequences of both strands of the target DNA, resulting in an exponential synthesis of displaced strands. (Adapted from reference
141a
with permission.)
10.1128/9781555815981/f0277-01_thmb.gif
10.1128/9781555815981/f0277-01.gif
FIGURE 6
Strand displacement amplification. This isothermal amplification assay is based on four requirements, namely, primers which include an upstream restriction endonuclease site (BsoB1), the cognate enzyme (BsoB1), a DNA polymerase lacking 5′ exonuclease activity, and nucleoside triphosphates, of which one has been modified to contain an alpha-thiol group (dCTPαS). In this reaction, the target DNA anneals with the primers and is converted to a double-stranded form by the polymerase. The restriction endonuclease introduces a cleavage in the primer sequence, and the polymerase synthesizes a new strand from this site and displaces the existing strand. The restriction endonuclease is not able to cut the newly synthesized strands because of the modified nucleotides and can only introduce cuts in the sites present in the primers. Restriction site-bearing primers are designed to anneal to sequences of both strands of the target DNA, resulting in an exponential synthesis of displaced strands. (Adapted from reference 141a with permission.)
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig07
FIGURE 7
Ligase chain reaction. This reaction includes two pairs of primers, each pair annealing to one strand of heat-denatured target DNA with a gap of 2 to 7 nucleotides between. The primers are designed to bind so that the gap between them consists of a single nucleotide type. The reaction also contains the relevant nucleotide triphosphates, a thermostable DNA polymerase, and a thermostable DNA ligase. Once the gap is filled by the polymerase, the ligase joins the last nucleotide to the downstream primer. The temperature is then raised to denature the product and then lowered to allow further primer binding. The cycle is repeated so that additional primer pairs can be ligated. By having the upstream primer labeled at the 5′ end with biotin (B) and the downstream primer labeled at the 3′ end with a fluorescent label, the products can be captured on a solid phase and tested for the presence of the fluorescent label. (Reprinted from reference
141a
with permission.)
10.1128/9781555815981/f0277-02_thmb.gif
10.1128/9781555815981/f0277-02.gif
FIGURE 7
Ligase chain reaction. This reaction includes two pairs of primers, each pair annealing to one strand of heat-denatured target DNA with a gap of 2 to 7 nucleotides between. The primers are designed to bind so that the gap between them consists of a single nucleotide type. The reaction also contains the relevant nucleotide triphosphates, a thermostable DNA polymerase, and a thermostable DNA ligase. Once the gap is filled by the polymerase, the ligase joins the last nucleotide to the downstream primer. The temperature is then raised to denature the product and then lowered to allow further primer binding. The cycle is repeated so that additional primer pairs can be ligated. By having the upstream primer labeled at the 5′ end with biotin (B) and the downstream primer labeled at the 3′ end with a fluorescent label, the products can be captured on a solid phase and tested for the presence of the fluorescent label. (Reprinted from reference 141a with permission.)
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig08
FIGURE 8
NASBA. This procedure is particularly well adapted to the detection of RNA. The reaction mixture consists of one primer which contains the sequence for the T7 RNA polymerase promoter at its 5′ end, a second primer at the downstream end of the sequence to be amplified, T7 RNA polymerase, reverse transcriptase, and RNase H as well as the ribo- and deoxyribonucleotide triphosphates. When the primer containing the T7 promoter anneals to the target RNA, the reverse transcriptase synthesizes the complementary DNA strand and the RNA portion of this duplex is digested by the RNase H. After binding of the downstream primer, the reverse transcriptase synthesizes a double-stranded DNA with a T7 promoter at one end which serves as a template for the T7 RNA polymerase, which synthesizes approximately 1,000 copies of antisense RNA in a promoter-dependent manner. This RNA can be further reverse transcribed to double-stranded DNA which can serve as a template on which the RNA polymerase can synthesize multiple antisense RNA copies. (Reprinted from reference
141a
with permission.)
10.1128/9781555815981/f0278-01_thmb.gif
10.1128/9781555815981/f0278-01.gif
FIGURE 8
NASBA. This procedure is particularly well adapted to the detection of RNA. The reaction mixture consists of one primer which contains the sequence for the T7 RNA polymerase promoter at its 5′ end, a second primer at the downstream end of the sequence to be amplified, T7 RNA polymerase, reverse transcriptase, and RNase H as well as the ribo- and deoxyribonucleotide triphosphates. When the primer containing the T7 promoter anneals to the target RNA, the reverse transcriptase synthesizes the complementary DNA strand and the RNA portion of this duplex is digested by the RNase H. After binding of the downstream primer, the reverse transcriptase synthesizes a double-stranded DNA with a T7 promoter at one end which serves as a template for the T7 RNA polymerase, which synthesizes approximately 1,000 copies of antisense RNA in a promoter-dependent manner. This RNA can be further reverse transcribed to double-stranded DNA which can serve as a template on which the RNA polymerase can synthesize multiple antisense RNA copies. (Reprinted from reference 141a with permission.)
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig09
FIGURE 9
Branched-chain DNA (bDNA) assay of first (A) and third (B) generations. Target DNA hybridizes to capture probes linked to a solid phase. A short oligonucleotide called the label extender hybridizes with complementary sequences on the target DNA and with either the amplifier oligonucleotide shown in panel A or a longer oligonucleotide called the preamplifier that contains multiple repeat sequences, shown in panel B. Amplifier oligonucleotides then hybridize to the alkaline phosphatase-linked probes shown in panel A as well as the preamplifier sites shown in panel B. Alkaline phosphatase is detected by standard reagents for the enzyme.
10.1128/9781555815981/f0279-01_thmb.gif
10.1128/9781555815981/f0279-01.gif
FIGURE 9
Branched-chain DNA (bDNA) assay of first (A) and third (B) generations. Target DNA hybridizes to capture probes linked to a solid phase. A short oligonucleotide called the label extender hybridizes with complementary sequences on the target DNA and with either the amplifier oligonucleotide shown in panel A or a longer oligonucleotide called the preamplifier that contains multiple repeat sequences, shown in panel B. Amplifier oligonucleotides then hybridize to the alkaline phosphatase-linked probes shown in panel A as well as the preamplifier sites shown in panel B. Alkaline phosphatase is detected by standard reagents for the enzyme.
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13fig10
FIGURE 10
Diagram depicting the typical IgM and IgG antibody responses following primary viral infection, reactivation, or reinfection. During primary infection, IgM appears within several days after onset of symptoms, peaks at 7 to 10 days, and normally declines to undetectable levels within 1 to 3 months. Following natural viral infection or successful immunization, IgG antibodies appear several days after the production of IgM, reach higher levels than IgM, and can persist for years, even lifelong, in lower quantities. During reactivation or exogenous reinfection, an anamnestic response in IgG antibodies will occur and an IgM response may or may not be observed.
10.1128/9781555815981/f0282-01_thmb.gif
10.1128/9781555815981/f0282-01.gif
FIGURE 10
Diagram depicting the typical IgM and IgG antibody responses following primary viral infection, reactivation, or reinfection. During primary infection, IgM appears within several days after onset of symptoms, peaks at 7 to 10 days, and normally declines to undetectable levels within 1 to 3 months. Following natural viral infection or successful immunization, IgG antibodies appear several days after the production of IgM, reach higher levels than IgM, and can persist for years, even lifelong, in lower quantities. During reactivation or exogenous reinfection, an anamnestic response in IgG antibodies will occur and an IgM response may or may not be observed.
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
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Tables
Diagnosis of Viral Infections*
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Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab01
TABLE 1
Susceptibility of cell culture types to commonly isolated human viruses
a
TABLE 1
Susceptibility of cell culture types to commonly isolated human viruses a
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab02
TABLE 2
Times required to detect viruses in cell culture
a
TABLE 2
Times required to detect viruses in cell culture a
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab03
TABLE 3
IC50 cutoffs for HSV and CMV antiviral resistance using PRA
TABLE 3
IC50 cutoffs for HSV and CMV antiviral resistance using PRA
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab04
TABLE 4
Cytological changes associated with selected viral infections
TABLE 4
Cytological changes associated with selected viral infections
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab05
TABLE 5
Detection of viral antigens by DFA staining
TABLE 5
Detection of viral antigens by DFA staining
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab06
TABLE 6
Utility of serologic determinations in clinical virology
TABLE 6
Utility of serologic determinations in clinical virology
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab07
TABLE 7
Examples of viruses for which IgM serologic determinations are useful and commercial reagents and/or kits are available
TABLE 7
Examples of viruses for which IgM serologic determinations are useful and commercial reagents and/or kits are available
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13
/content/10.1128/9781555815981.ch13.ch13tab08
TABLE 8
Interpretation of results for virus-specific antibodies in clinical virology
TABLE 8
Interpretation of results for virus-specific antibodies in clinical virology
Citation:
Boivin G, Mazzulli T, Petric M, Couillard M.
2009.
Diagnosis of Viral Infections*,
p 265-294.
In
Richman D, Whitley R, Hayden F (ed),
Clinical Virology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815981.ch13