Virology Flashcards
What can be detected in virology?
- Infectious Virus
o virus isolation & EM - Protein components
o ANTIGENS on the virus
e.g. p24 antigen in HIV - Genetic components
o DNA or RNA - Host response
o Antibody OR cell responses
What are different METHODS of diagnosing?
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)
Limitations of laboratory tests?
ALL assays give rise to FALSE NEGATIVES & FALSE POSITIVES results
Sensitivity?
Test’s ability to:
CORRECTLY identify POSITIVE samples
Specificity?
Test’s ability to:
CORRECTLY identify NEGATIVE samples
Typical samples used?
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
How is serology used to detect HIV, HepA, HBV, HCV, CMV & EBV, VZV, MMR and Parvovirus B19?
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
Difference between IgM & IgG?
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)
Antibody avidity testing?
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
Avidity?
Strength with which ANTIBODIES bind to a SPECIFIC ANTIGEN
How often is virus isolation & EM used?
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
How does HIV serology work?
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)
What happens after HIV serology?
Repeat blood sample & EDTA blood
for the HIV VIRAL LOAD - for genotyping & baseline resistance testing
Point-of-care testing?
Exists outside of lab (small equipment)
How is immunofluorescence used (IF)?
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
Recommended samples for respiratory tract infections?
o throat swab +/- nose swab o nasopharyngeal swab o NP aspirate (NPA) o bronchoalveolar lavage (BAL) o endotracheal tube (ET) secretions
How are the samples then run to view?
Multiplex PCR!
Rather than use a SINGLE test tube for each virus, use several viruses in one tube
Recommended samples for CNS infections?
CSF
- PCR
- HSV, VSV, enterovirus
Stools & throat swab
- PCR
- enterovirus
Blood for serology
- PCR
- Encephalitis virus, arboviruses
Some CNS dieases?
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
Recommended samples for D&V?
Stool (preferred)
Vomit - lower yield
PCR OR antigen detection assays (EIA)
e.g. norovirus, rotavirus, adenovirus, sapovirus, astrovirus
Difference between enteroviruses vs. enteric viruses?
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
How is PCR used?
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
3 types of sequencing applications used in virology?
- Genotyping
- sequence viral genomes - Antiviral resistance testing
- vital for selecting correct antiretroviral drugs - Phylogenetic analysis
- tool used to investigate outbreaks
