Diagnosis of viral infections

image of Diagnosis of viral infections
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Viral infections of small animals are common and frequently represent an important cause of disease.Although precise diagnosis is not always necessary, it may be required to determine appropriate therapy and prognosis, and to give advice about the potential for disease in other susceptible animals sharing the same environment. This chapter deals with virus detection, antibody detection, feline viruses and canine viruses. The section concludes with case examples.

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28.1 Cytopathic effects (cpe). (a) Feline calicivirus (FCV) cpe (in tissue culture) manifests as cell rounding and shrinkage (arrowed). (b) Feline herpesvirus (FHV) cpe (in tissue culture) manifests as ballooning of cells and long strands of cellular material (arrowed). (c) Feline immunodeficiency virus (FIV) cpe (in lymphocytes in liquid co-culture) manifests as ballooning of infected cells (arrowed). (d) Cowpox virus causes intracytoplasmic inclusion bodies (arrowed). (Haematoxylin and eosin stain). (e) FHV cpe induces syncytium formation and intranuclear inclusion bodies (arrow shows inclusion body within a syncytium) (Haematoxylin and eosin stain). (a, © Susan Dawson; c, Courtesy of O Jarrett; d, Courtesy of M Bennett; e, Courtesy of RM Gaskell)
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28.2 Viral antigen detection by (a–b) direct and (c–d) indirect methods. The antigen to be detected is in red. In direct antigen detection, the antigen is already immobilized and can be detected either (a) by a labelled laboratory antibody or (b) by an unlabelled primary laboratory antibody which itself is detected by a secondary labelled antibody. In indirect antigen detection (sometimes called antigen capture), the antigen is first immobilized using an antibody to a solid surface. This immobilized antigen can then be detected either by (c) a labelled laboratory antibody or (d) by an unlabelled primary laboratory antibody which itself is detected by a secondary labelled antibody.
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28.3 Immunofluorescence of FeLV antigens in a peripheral blood smear, showing green positive fluorescence in leucocytes and platelets. (Courtesy of O Jarrett)
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28.4 Immunoperoxidase method. Granulomatous inflammation of the intestinal serosa in a cat with feline infectious peritonitis, showing a central area of necrosis surrounded by macrophages expressing viral antigen. (Courtesy of A Kipar)
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28.5 Examples of rapid immunomigration technology used for the detection of (a) FeLV p27 antigen and (b) FIV antibodies. (Based on pictures kindly provided by Synbiotics Europe)
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28.6 Polymerase chain reaction. Reactions occur in a solution containing the template DNA, the DNA polymerase enzyme and nucleotides. The template is double-stranded DNA, shown as two black lines with the nucleotides at each end specified; 3' and 5' refer to the different chemical ends of the DNA, and are shown because DNA polymerase extends the sequence only in the 5' to 3' direction. (a) Denaturation: the DNA template is heated, so the two strands separate, allowing access by the primers and enzyme. (b) Primer annealing: at a lower temperature, specifically designed primers (short sequences of DNA, shown in red) complementary to the DNA sequence flanking the area of interest bind (anneal) to their target sequence on the new single-stranded template DNA. (c) Primer extension: DNA polymerase creates a complementary DNA strand (shown in blue) from the primer in a 5' to 3' direction. The result is double-stranded DNA that is a copy of the template DNA. These cycles are repeated and the number of DNA copies increases exponentially. (d) Results of a typical PCR (there is one sample present in each lane): lanes 1–6 are positive; lanes 7 and 8 are negative; lanes 9 and 10 are positive controls; lane 11 is the negative control; lane 12 contains a molecular weight marker to identify the size of the amplified DNA product.
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28.7 Histopathology. Lesions can sometimes be used to provide a diagnosis for viral infections. (a) Cowpox lesion in a cat. The arrow indicates an intracytoplasmic inclusion. (Haematoxylin and eosin stain; original magnification X400). (b) Infectious canine hepatitis, showing adenoviral inclusions (arrowed) in infected hepatocytes. (Haematoxylin and eosin stain; original magnification X1000) (a, Courtesy of M Bennett; b, Courtesy of A Kipar)
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28.8 Electron microscopy. Although often difficult to find, most viruses have a characteristic morphology that can allow a diagnosis to be made. (a) Cowpox virus with typical orthopoxvirus morphology. (b) Feline herpesvirus particle showing capsid morphology and envelope. (c) Canine parvovirus particles in a faecal sample. (a, Courtesy of M Bennett; b, Courtesy of RM Gaskell; c, ©Alan Radford)
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28.9 Haemagglutination inhibition. The presence of specific antibodies to a virus is detected by the inhibition of red cell agglutination. Where agglutination occurs, cells are clumped and remain in suspension; where agglutination is inhibited, cells settle to form a pellet. Wells 1, 4 and 5 are positive (agglutination is inhibited). Wells 2 and 3 are negative (no inhibition of agglutination). If this were a haemagglutination test, the opposite results would be reported. (Courtesy of DD Addie)
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28.10 Detection of antibody using bound antigen. Antigen from the virus is immobilized on a solid surface to bind or ‘capture’ any virus-specific antibodies present in the test sample. Unbound, non-specific antibodies are removed by washing. Subsequently, the presence of bound specific antibody is detected with a second, species-specific, antibody that reacts with any antibody from that species, i.e. anti-dog IgG or anti-cat IgG. This second antibody incorporates a marker system that allows its binding to be detected. This may be a directly visible product (e.g. fluorescein or colloidal gold particles) or, more commonly, an enzyme that catalyses a colour reaction with its substrate (e.g. peroxidase and alkaline phosphatase). The same format is essentially used for ELISA, immunofluorescence, Western blot and RIM tests.
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28.11 Immunofluorescence (IF). (a) Typical IF reaction. (b) Cells infected with feline calicivirus (FCV) showing typical FCV cytopathic effect (cpe), including rounding and shrinkage of numerous infected cells (arrowed), surrounding expanding holes in the cell monolayer (*). (c) Same slide as (b) showing specific fluorescence in FCV-positive cells. (d–e) Positive IF reactions for (d) feline immunodeficiency virus and (e) feline coronavirus. (bc, © Alan Radford; d, Courtesy of O Jarrett; e, Courtesy of M Bennett)
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28.12 A typical Western blot reaction. The viral proteins are disassembled and separated on a gel according to their molecular weight. Specific anti-viral antibodies from the patient’s serum (black) are used as a target for subsequent antibody detection by the secondary antibody system (blue with black spiked star).

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