Lecture 4: Diagnosis Flashcards
What are some examples of CPE that can be visualized?
Inclusión bodies, and syncytia
Syncytia = multiple cells that become one
Procedures for diagnosing viral infections
-cytologic/histologic examination
_electron microscope
-detection of viral proteins and genomes
-serology
Many viruses within a family cannot be distinguished without more specific methods, why?
Because many viruses cause similar CPE
cytology on clinical samples is usually the preferred method of surgical pathology rather than a diagnostic viral laboratories
Cytology = cell culture = rarely used due to lack of specificity and safety concerns = methods are versatile and cheap
Viral plaques
Similar to bacterial colony
-each plaque originated from a single virus that infected a single cell then spread directly cell-to-cell
Preparation and staining of clinical samples for CPE
-tzanck smear: view CPE in clinical specimens: the CPE = “cowdry bodies”. Clinical specimens = HHV 1/2/3 (HHV-3=VZV)
-papanicolaou (pap smear): cytological changes in epithelial cells
-lung biopsy: (CMV)
Can syncytia discriminate between viruses?
No
Syncytia is observed from viruses grown in cell culture
-Tzanck smear (HSV, VZV)
-Lung biopsy (CMV)
Which viruses have inclusion bodies found in the nucleus and the cytoplasm?
CMV and Measles
Which viruses have inclusion bodies found in the nucleus?
HSV, VZV, CMV, measles, adeno, parvo
Electron microscopy
For viruses that cannot be grown in cell culture
VIRUS FAMILY MAY BE DETERMINED
-too expensive, time consuming
Common methodologies used to detect viral proteins
-What do these methods rely on?
-direct vs. indirect?
These techniques rely on antibodies
Can all be indirect vs. direct
-immunofluorescene microscopy:
-Elisa:
-western blot
-lateral flow immunochromatographic assay
-heterophile antibody test
with these techniques we can provide a definitive identification/diagnosis using the viral proteins to check for specificity/sensitivity of the virus
Direct immunofluorescence
-detect what?
Uses only primary antibody directly attached to the fluorophore. Detect antigen
Indirect immunofluorescence
-detect?
Use of 2 or more antibodies. The second is attached to the flourophore. Detect antibodies
Elisa
(Enzyme-linked immunosorbent assay)
-a colorimetric assay
-the intensity of the color is directly related to the amount of material present
quantitative analysis
Western blot
Separates proteins via electrophoresis
In a solid matrix rather than in liquid
Lateral flow assay
RAPID
-pregnancy test
-rapid COVID test
=Involves capillary action
=a control line = shows whether test is viable = will always bind to antigen to show test is working
=when enough bind… color will appear
Heterophile antibody test
Takes advantage of rare cross-reactive antibodies
-example antibodies produced from EBV will cross react with RBC from sheep or horse therefore showing agglutination and a positive heterophile antibody test
Serology
Inherent to antibody-antigen interactions
-USE THE SAME BASIC TECHNIQUES = ELISA, WESTERN BLOT, ETC.
Specifically detects
-IgM - useful to indicate acute infections, but disappear quickly - (largest)
-IgG - prior exposure to a pathogen
IgM vs IgG
IgG: appear later and stay for life (indicates prior exposure)
IgM: appear first during acute infections and disappear
Neutralization assays
Neutralizing the antibodies are BLOCKING the virus from entering host cells
=if the antibodies neutralize, then less viruses enter
*useful for *
-identifying viruses that are highly related
-if you have immunity from the virus
quantitative analysis = b/c the more antibodies the more protected
Techniques for detection of viral genomes
-PCR
-RT-PCR
-Biofire Film Array
PCR technique
-denature
-annealing
-elongation
PCR first and then RT-PCR for specific viruses
PCR is specific to DNA
Biofire Film Array
Can detect many pathogens simultaneously from a single sample
