Virology

Question 1

What can be detected in virology?

Answer 1

1. Infectious Virus
   o virus isolation & EM
2. Protein components
   o ANTIGENS on the virus
   e.g. p24 antigen in HIV
3. Genetic components
   o DNA or RNA
4. Host response
   o Antibody OR cell responses

Question 2

What are different METHODS of diagnosing?

Answer 2

o Cell culture & EM
(not used commonly now as replaced by PCR)

o PCR
- detects specific sequences of DNA

o Antibody detection
- serology (EIA = enzyme innunoassay)

o Antigen detection
- serology (EIA, IF = immunofluorescence)

o Quantification (of antibody or antigen)
o Serotyping (i.e. in HIV)

o Genome detection

• quantification of genomes (viral load)
• genome sequencing (genotyping & antiviral resistance testing)

Question 3

Limitations of laboratory tests?

Answer 3

ALL assays give rise to FALSE NEGATIVES & FALSE POSITIVES results

Question 4

Sensitivity?

Answer 4

Test’s ability to:

CORRECTLY identify POSITIVE samples

Question 5

Specificity?

Answer 5

Test’s ability to:

CORRECTLY identify NEGATIVE samples

Question 6

Typical samples used?

Answer 6

o Throat swab, nasopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), ET (endotracheal tube) secretions
- use PCR

o Stools

• rotavirus, adenovirus & norovirus
• antigen detection (EIA) OR PCR

o Urine

• BK virus & adenovirus
• PCR

o CSF

• herpes viruses & enteroviruses
• PCR

o Blood (clotted)
 - serology (antibody detection)

o Blood (EDTA)
 - PCR/viral load testing

o Saliva
- serology/PCR e.g. measles

Question 7

How is serology used to detect HIV, HepA, HBV, HCV, CMV & EBV, VZV, MMR and Parvovirus B19?

Answer 7

HIV = Antibody and p24 antigen

HepA = IgM & IgG

HBV = surface Ag/Ab, eAg/Ab, core Ab, core IgM

HCV = antibody +/- core antigen

CMV & EBV = IgM & IgG

VZV = IgG

MMR = IgM & IgG

Parovirus = IgM and IgG

Question 8

Difference between IgM & IgG?

Answer 8

IgM is a marker of RECENT INFECTION

IgG is created later in host response & lasts longer!

Both are created in acute phase of dieases BUT:
o IgG levels rise higher and plateau (lifelong)
o IgM levels peak early and drop (3months)

Question 9

Antibody avidity testing?

Answer 9

Allows for POSITIVE IgM result confirmation!

Early on in infection:
o avidity is LOW
o gets better over a PERIOD OF MONTHS as the antibodies mature

i.e. if have a HIGH antibody avidity, unlikely infection occurred in last 3 months

Question 10

Avidity?

Answer 10

Strength with which ANTIBODIES bind to a SPECIFIC ANTIGEN

Question 11

How often is virus isolation & EM used?

Answer 11

RARELY!

Virus isolation in CELL CULTURE is
o SLOW
o time-consuming
BUT useful for phenotypic antiretroviral susceptibility testing (e.g. HSV)

Viruses TOO SMALL to be seen by light microscrope so EM used
- sample types include stool and vesicle fluid

Question 12

How does HIV serology work?

Answer 12

4th generation EIA - Ab + p24 Ag detection

All REACTIVE samples go onto confirmatory testing in a 2nd assay
- to exclude non-specific reactivity (false positives)

Confirmed positives undergo TYPING (HIV 1 vs 2)

Question 13

What happens after HIV serology?

Answer 13

Repeat blood sample & EDTA blood

for the HIV VIRAL LOAD - for genotyping & baseline resistance testing

Question 14

Point-of-care testing?

Answer 14

Exists outside of lab (small equipment)

Question 15

How is immunofluorescence used (IF)?

Answer 15

Useful for DIRECT detection of:
o VIRAL ANTIGENS in clinical samples
e.g. respiratory viruses

o rapid & inexpensive
BUT
o SUBJECTIVE as dependent on the skill of the technician & quality of sample

Question 16

Recommended samples for respiratory tract infections?

Answer 16

o throat swab +/- nose swab
o nasopharyngeal swab
o NP aspirate (NPA)
o bronchoalveolar lavage (BAL)
o endotracheal tube (ET) secretions

Question 17

How are the samples then run to view?

Answer 17

Multiplex PCR!

Rather than use a SINGLE test tube for each virus, use several viruses in one tube

Question 18

Recommended samples for CNS infections?

Answer 18

CSF

• PCR
• HSV, VSV, enterovirus

Stools & throat swab

• PCR
• enterovirus

Blood for serology

• PCR
• Encephalitis virus, arboviruses

Question 19

Some CNS dieases?

Answer 19

o Meningitis or encephalitis
- HSV, VZV & enterovirus

o Young child w. febrile fits
- HHV-6, parechovirus

o Immunocompromised (e.g. HIV)
 - also CMV, EBV & JC virus

o Recent travel to endemic region
- Japanese encephalitis, West Nile virus etc.

o Outbreaks e.g. mumps

Question 20

Recommended samples for D&V?

Answer 20

Stool (preferred)
Vomit - lower yield

PCR OR antigen detection assays (EIA)
e.g. norovirus, rotavirus, adenovirus, sapovirus, astrovirus

Question 21

Difference between enteroviruses vs. enteric viruses?

Answer 21

Enteroviruses:
o Genus of positive-sense single-stranded RNA viruses associated with several human and mammalian diseases
o Enteroviruses are named by their transmission-route through the intestine

Enteric viruses:
o human viruses that are primarily transmitted by the fecal-oral route, either by person-to-person contact or by ingestion of contaminated food or water

Question 22

How is PCR used?

Answer 22

To amplify specific RNA (RT-PCR) or DNA

 Carry out 30 cycles usually
 Starting block is dsDNA (so if the initial virus has RNA, use RT-PCR to form dsDNA)
 Denaturing the dsDNA into strands is achieved by heating
 Taq polymerase elongates the chain.
 Cycles of this induce exponential expansion
 “Real-time” PCR can produce a read-out of results

Question 23

3 types of sequencing applications used in virology?

Answer 23

1. Genotyping
   - sequence viral genomes
2. Antiviral resistance testing
   - vital for selecting correct antiretroviral drugs
3. Phylogenetic analysis
   - tool used to investigate outbreaks