Virology IV - Diagnostics

Question 1

what type of sample should be submitted for viral diagnostics

Answer 1

sample from the site of shedding
- fluids: blood, milk, lavage fluid, genital discharge
- feces
- swabs: buccal or nasal cavities, eyes, skin
- tissues: post mortem only

Question 2

how should viral samples be transported

Answer 2

refrigerated or frozen

Question 3

what are 4 diagnostic methods for viruses

Answer 3

1. histopathology
2. virion/viral nucleic acid detection (PCR, antigen ELISA, EM)
3. virus isolation (culture)
4. serology (use IgM vs IgG antibody ELISA)

Question 4

what diagnostics are most commonly used in parvovirus

Answer 4

1. fecal antigen ELISA
   - detects CPV antigen
   - low sensitivity due to intermittent shedding
2. hemagglutination of feces
   - detects CPV antigen
3. PCR
   - detects CPV DNA in feces/tissues
4. virus isolation (difficult and slow)

Question 5

steps of PCR detection

Answer 5

1. fecal swab
2. add to tube with liquid
3. filter to remove large particles
4. treat with nuclease - chops up host nucleic acid (virus protected by capsid)
5. chemical extraction - removes capsid
6. add qPCR reactants (probe, primers, DNA polymerase)
7. run PCR to detect fluorescence

**must know viral genome in order to add correct primers

Question 6

CPV treatment

Answer 6

supportive care + antibiotics to kill opportunistic enterobacteria

NO CPV specific antivirals exist

Question 7

how do you differentiate CPV DNA on PCR from infection vs vaccination

Answer 7

must use SEQUENCING
1. extract nucleic acid from samples using same methods as regular PCR
2. amplify and sequence using sequencing agents (labeled nucleotides, primers, DNA poly)
3. run gel electrophoresis
4. labeled terminating nucleotides provide DNA sequence

sequence specific probes selectively detect LAV or naturally acquired CPV DNA based on which primer was used

Question 8

how can you determine infection vs vaccination DNA without sequencing

Answer 8

use regular PCR and determine if DNA load was high or low

high load: most likely naturally infected
low load: most likely vaccinated w/ LAV

Question 9

what are the cause of most zoonotic viral diseases

Answer 9

RNA viruses

Question 10

what are the cause of most human pandemics

Answer 10

RNA viruses

Question 11

why do RNA viruses contribute so much to zoonotic and pandemic viral infections

Answer 11

because they are highly mutable

RNA polymerases are unable to error correct leading to highly error prone replication

Question 12

how does mutation increase chances of viral survival

Answer 12

allows for infection of new hosts or greater transmissibility in existing host

Question 13

what does the rate of mutation depend on

Answer 13

1. nucleic acid type (DNA vs RNA)
2. genome size

RNA virus > DNA virus > host cells

Question 14

how did West Nile virus spread to the US

Answer 14

point mutation in amino acid at NS3249 causing increased viremia

higher viremia = greater concentration reaching shedding site

Question 15

what species was first affected by WNV

Answer 15

american crows