Week 5 Module 3

Question 1

Diagnostic Virology

Answer 1

-In clinical medicine, if pathogen presence is suspected patient samples are collected aseptically and then analyzed. Collection can be from respiratory tract, stool
samples, blood (serology), cerebrospinal fluid etc.

Question 2

What is Specificity?

Answer 2

– ability to recognize a single pathogen (out of a possible mix).

Question 3

What is Sensitivity?

Answer 3

– lowest quantity of pathogen or pathogen Product (protein) detectable through laboratory means.

Question 4

What is the challenge with diagnostic virology?

Answer 4

-The challenge with diagnostic virology is that viruses can’t be cultured directly on medium as they require a host cell. Still, several methods have been developed

Question 5

What is Agglutination?

Answer 5

-Agglutination is clumping of blood cells, and this is a way antibodies can be detected in presence of antibodies blood will clump and if not it will smear

Question 6

Microscopic examination

Answer 6

-electron microscopy typically (high powered microscope) mostly likely transmission electron microscopy (TEM); Used to determine morphology of any virus particles.
- may be used for viruses causing gastroenteritis, or HPV
-may be used as confirmatory test

Question 7

What are the advantages of Microscopic examination?

Answer 7

• no pre-bias as to identity
  -sample preparation and technique quick so rapid diagnosis

Question 8

What are the disadvantages of Microscopic examination?

Answer 8

• expensive and not always available
  -minimum titer (concentration) required for detection
  -some viruses do not have a distinct morphology
  -Electron microscopy is not selective, and looks at whole sample

Question 9

What is Immune electron microscopy (IEM)?

Answer 9

– method where reference antiserum added to sample in hopes that if Abs to virus present, virions will agglutinate or clump

-need to suspect particular type of virus ahead of time; might guess possibilities from stool sample

-has been used for Norovirus detection

Question 10

What are the techniques used in Diagnostic Virology?

Answer 10

-Transmission electron microscope, Immune electron microscopy (IEM), ELISA, Immunofluorescence, Rapid tests, Serology tests, PCR (q, RT), and Cell culturing of viruses

Question 11

What are Enzyme Immunoassays?

Answer 11

It is a method to detect viral antigens.

-antigen binds to Ab which binds to enzyme

-substrate added for enzyme; When substrate converted to product, colour produced
-similar idea to Western blot but done in microtiter plate vs membrane; Faster than Western blot though
-various formats exist including ELISA (enzyme-linked immunosorbent assay)

Question 12

What does the secondary antibody do?

Answer 12

-Secondary antibody amplifies the signal, allows colour detection to be easier to see

Question 13

What kind of well plate does ELISA use?

Answer 13

-ELISA typically done in a 96 well plate, in every well you can have an antigen from virus or an antibody that binds to the antigen

Question 14

What is Immunofluorescence?

Answer 14

-staining technique that detects viral antigen
-Sample from patient put on slide, add fluorescent dye

Question 15

What are Rapid Tests?

Answer 15

-still immunoassays but reagents adhered to solid support to allow quick, portable, small size test

-There’s a control antibody which is the housekeeping protein, and there’s a T which is antibody against capsid (ex. COVID-19)

Question 16

What’s a limitation of rapid tests?

Answer 16

Limit of sensitivity on rapid tests, there’s a threshold level of how much viral antigen present to produce a positive result

Question 17

What is Viral Serology?

Answer 17

Serology – Detection of antibodies against viral antigens produced in blood sera after viral infection.

-Blood serum Isolation from Blood sample
-whole blood collected, then allowed to clot (RT for 15 – 30 min)
-sample centrifuged and supernatant (serum) taken

Question 18

What are Serology tests?

Answer 18

-typically need to compare antibody titers in acute serum (take while illness is symptomatic) vs. convalescent (after symptoms subside)
-looking for 4x increase in IgM and IgG titers in acute vs. convalescent samples
-if compare to reference convalescent sample (from different person) can help in diagnosis
-if using paired samples (same patient), only useful after patient is recovering and is therefore, confirmatory test

Question 19

What is IgM?

Answer 19

-An antibody that may be attached to the surface of a B cell or secreted into the blood. Responsible for early stages of immunity

Question 20

What is IgG?

Answer 20

-An antibody secreted by plasma cells in the blood. Able to cross the placenta into the fetus.

Question 21

What is Nucleic Acid Detection?

Answer 21

-identify viral DNA/RNA (genome) or RNA (from transcription) includes:
-PCR
-qPCR (quantitative PCR) also called real-time PCR (more quantitative)
-RT-PCR (Reverse transcription PCR) to detect viruses with RNA genomes or viral transcripts

Question 22

What are the Advantages of Nucleic Acid Detection?

Answer 22

-highly sensitive and accurate
-fairly quick results

Question 23

What are the Disadvantages of Nucleic Acid Detection?

Answer 23

• can require specific primers i.e. virus sequence known in nature
  -possibility of false positive if contamination, (but likelihood
  decreasing with advancing technology)
  -not as fast as ‘on-site’ rapid tests

Question 24

What is Cell Culture of Viruses?

Answer 24

-trying to isolate virions and then infect animal cell culture that is
known to be permissive to a wide array of viruses
-infectious virus may induce visible cytopathic effects (CPEs)
e.g. ballooning of cells or formation of syncytia (multinucleate
cells due to cell fusion)

Question 25

What are the Advantages of Cell Culture of Viruses?

Answer 25

-can propagate viral material and then carry out
microscopic examination and or/ antigen/nucleic acid detection
-modern methods allow different cell lines to be grown in same
tube; If add antibody cocktail can look for multiple viruses at
same time

Question 26

What are the Disadvantages of Cell Culture of Viruses?

Answer 26

-cell line used can strongly influence results; Primary cell lines e.g. monkey kidney cells are most permissive but most expensive to obtain and grow poorly in culture
-some viruses grow poorly in culture
-can take days or weeks for CPEs to appear
-sensitive to contamination

Question 27

What are Antivirals?

Answer 27

• An antiviral drug should block viral replication completely to
  stop infection and prevent drug-resistant mutants from arising.
  -Goal is to create antiviral that has selective toxicity for
  virus i.e. affects mechanism unique to virus.

-Antivirals can be broadly classified into two categories: Antiretrovirals, and Other Antivirals

Question 28

What’s a disadvantage to antivirals?

Answer 28

-it’s more difficult to develop effective antivirals than other antimicrobials because drugs that
affect virus may also affect the host cell

Question 29

Antiretrovirals?

Answer 29

-Include: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptases inhibitors (NNRTIs), protease inhibitors, fusion inhibitors

Question 30

Other Antivirals?

Answer 30

-Broad spectrum antivirals include: fusion inhibitors, inhibitors of endocytosis pathway, next generation nucleotide, anti-herpesvirus drugs, and anti-influenza drugs

Question 31

What is Nucleoside reverse transcriptase inhibitors (NRTIs)?

Answer 31

-analogs of nucleosides (thymidine or guanosine); When incorporated into cDNA, they prevent further elongation because
of chemical nature e.g. dideoxy derivatives that lack the 3’ OH
-e.g. AZT against HIV

Question 32

What is Non-nucleoside reverse transcriptase inhibitors (NNRTIs)?

Answer 32

-targets reverse transcriptase enzyme directly includes: Efavirenz (EFV), Nevirapine (NVP), and Delavirdine (DLV)

Question 33

What are Protease Inhibitors?

Answer 33

-They bind and inhibit proteases necessary for cleavage of viral polyprotein; Interfere with virus maturation ex. Ritanovir, Saquinavir etc. All protease inhibitors end in “vir”

Question 34

What are Fusion Inhibitors?

Answer 34

-prevents fusion (attachment) of retrovirus (specifically HIV) with enfuvirtide

Question 35

Fusion inhibitors can also be broad spectrum antivirals, how do they work?

Answer 35

Fusion inhibitors affect common ancillary host molecules e.g. sialic acid, glycosaminoglycans etc.
-such molecules are involved non-specific interactions but are necessary for many viruses to enter cell

Question 36

What are inhibitors of endocytosis pathway?

Answer 36

-entry of many viruses involves first acidification of endocyticial
vesicle to allow fusion with virus and then endosomal trafficking (ER, Golgi etc.)
-chemicals could block the acidification step and/or progression
from early to late endosome

Question 37

What are next generation nucleotide?

Answer 37

-next-generation nucleotide, nucleoside analogs,
-drugs that affect host pathway exploited in virus maturation

Question 38

What are Anti-Herpesvirus drugs?

Answer 38

-includes inhibitors specifically of herpesvirus encoded DNA Pol
or viral encoded thymidine kinase; Both interfere with virus replication