Chapter 15 : Diagnosis of Viral Infections

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The clinical virology laboratory is an important and leading component of general microbiology that provides significant benefits to patient care. The traditional epidemiologic 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. This development is the result of many advances in diagnostic virology including improvement in cell culture (shell vial assays, mixed cell cultures, genetically engineered cell lines), availability of specific reagents such as monoclonal antibodies and, most importantly, the introduction of molecular techniques mostly based on polymerase chain reaction (PCR), which allow the sensitive and rapid detection of slowly growing or uncultivable viruses. The impact of the latter procedure is illustrated by the recent identification of several respiratory viruses including the human metapneumovirus (1), multiple coronaviruses including severe acute respiratory syndrome (SARS) (2) and Middle East respiratory syndrome (MERS) (3), human bocavirus (4), etc.

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of FIGURE 1

Cytopathic effects induced by some viruses. Panel A shows uninfected cell lines including HEp-2 ( ), RD ( ), fibroblast ( ), and Vero ( ) cells. Panel B shows the same cell lines infected with respiratory syncytial virus ( ), enterovirus ( ), cytomegalovirus ( ), and herpes simplex virus ( ).

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of FIGURE 2

Cytomegalovirus (CMV) UL97 mutations associated with ganciclovir resistance. CMV UL97 conserved regions are represented by shaded boxes. Numbers under the boxes indicate the positions (codons no.) of these conserved regions. Vertical bars indicate the presence of amino acids substitutions while the hatched box indicates a region (codons 590−607) in which diverse deletions (from 1 to 17 codons) have been reported.

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of FIGURE 3

Polymerase chain reaction. 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 between melting, annealing, and primer extension is repeated multiple times and the number of product strands is doubled with each cycle. (Reprinted from reference ( ) with permission.)

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of FIGURE 4

The RVP assay (Luminex Corporation, Austin, TX). Identification of the target-specific primer extension reaction that had 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. TSPE: target-specific primer extension.

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of 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 resulting in the generation of a fluorescent signal. (Reprinted from reference ( ) with permission.)

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of 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 nucleotide 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. (Reprinted from reference ( ) with permission.)

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of 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 additional primer pairs can be ligated. By having the upstream primer labeled at the 5′-end with biotin (B) and the downstream primer at the 3′-end with a fluorescent label, the products can be captured on a solid phase and tested for presence of the fluorescent label. (Reprinted from reference ( ) with permission.)

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of FIGURE 8

Nucleic acid sequence-based amplification (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 the 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 that 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 ( ) with permission.)

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of 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 A or a longer oligonucleotide called the preamplifier that contains multiple repeat sequences shown in B. Amplifier oligonucleotides then hybridize to the alkaline phosphatase (AP)-linked probes shown in A as well as the preamplifier sites shown in B. Alkaline phosphatase is detected by standard reagents for the enzyme.

Citation: Boivin G, Mazzulli T, Petric M. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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Image of 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 after successful immunization, IgG antibodies appear several days after the production of IgM, reaches higher levels than IgM, and can persist for years, even life-long, 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. 2017. Diagnosis of Viral Infections, p 291-319. In Richman D, Whitley R, Hayden F (ed), Clinical Virology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819439.ch15
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