Lecture 4: Diagnosis

Question 1

What are some examples of CPE that can be visualized?

Answer 1

Inclusión bodies, and syncytia
Syncytia = multiple cells that become one

Question 2

Procedures for diagnosing viral infections

Answer 2

-cytologic/histologic examination
_electron microscope
-detection of viral proteins and genomes
-serology

Question 3

Many viruses within a family cannot be distinguished without more specific methods, why?

Answer 3

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

Question 4

Viral plaques

Answer 4

Similar to bacterial colony
-each plaque originated from a single virus that infected a single cell then spread directly cell-to-cell

Question 5

Preparation and staining of clinical samples for CPE

Answer 5

-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)

Question 6

Can syncytia discriminate between viruses?

Answer 6

No
Syncytia is observed from viruses grown in cell culture
-Tzanck smear (HSV, VZV)
-Lung biopsy (CMV)

Question 7

Which viruses have inclusion bodies found in the nucleus and the cytoplasm?

Answer 7

CMV and Measles

Question 8

Which viruses have inclusion bodies found in the nucleus?

Answer 8

HSV, VZV, CMV, measles, adeno, parvo

Question 9

Electron microscopy

Answer 9

For viruses that cannot be grown in cell culture
VIRUS FAMILY MAY BE DETERMINED
-too expensive, time consuming

Question 10

Common methodologies used to detect viral proteins
-What do these methods rely on?
-direct vs. indirect?

Answer 10

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

Question 11

Direct immunofluorescence
-detect what?

Answer 11

Uses only primary antibody directly attached to the fluorophore. Detect antigen

Question 12

Indirect immunofluorescence
-detect?

Answer 12

Use of 2 or more antibodies. The second is attached to the flourophore. Detect antibodies

Question 13

Elisa
(Enzyme-linked immunosorbent assay)

Answer 13

-a colorimetric assay
-the intensity of the color is directly related to the amount of material present
quantitative analysis

Question 14

Western blot

Answer 14

Separates proteins via electrophoresis
In a solid matrix rather than in liquid

Question 15

Lateral flow assay

Answer 15

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

Question 16

Heterophile antibody test

Answer 16

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

Question 17

Serology

Answer 17

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

Question 18

IgM vs IgG

Answer 18

IgG: appear later and stay for life (indicates prior exposure)
IgM: appear first during acute infections and disappear

Question 19

Neutralization assays

Answer 19

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

Question 20

Techniques for detection of viral genomes

Answer 20

-PCR
-RT-PCR
-Biofire Film Array

Question 21

PCR technique

Answer 21

-denature
-annealing
-elongation
PCR first and then RT-PCR for specific viruses
PCR is specific to DNA

Question 22

Biofire Film Array

Answer 22

Can detect many pathogens simultaneously from a single sample