Lab 3.1 - Detection of Viruses Flashcards
what are the main purposes of virus detection? (5)
-clinical cases; need for a specific diagnosis
-certification of freedom from specific infection
-disease status prior to artificial insemination, embryo transfer, blood transfusion
-tracking zoonoses
-xenotransplantation for humans
sensitivity vs specificity
-Sensitivity: represents the likelihood that an animal with a disease will
have a positive test result
-Specificity: represents the likelihood that an animal without a disease
will have a negative test result
five requirements of an ideal diagnostic procedure
-sensitivity
-specificity
-speed
-low cost
-simplicity
what information tells you the type of specimen needs to be collected? why does it matter?
-varies based on the tissue affected and clinical signs
-important to submit the correct type of specimen in a usable condition (shipment conditions)
what type of virus detection can you do with serum samples
indirect detection of antiboies (serological tests)
what type of virus detection can you do with feces, exudate, excretion, secretion, tissue, or whole blood samples? (4)
-virus isolation
-virus visualization
-virus protein (antigen) detection
-detection of viral nucleic acid genomes
primary purpose of virus isolation (3)
- To isolate and identify viruses in clinical samples
- To do research on viral structure, replication, genetics and effects on host cell
- To prepare viruses for vaccine production
how do we isolate viruses? (3)
By inoculating the virus in:
-Cultured mammalian cells = Cytopathic effect (CPE)
-Embryonated eggs = Death of embryo (or lesions)
-Laboratory animals = Depends on the suspect virus
two methods for isolation of viruses in cell culture
primary cell line vs continuous cell line
process for primary cell line and the organs it can be used for
I. Primary cell line: prepared directly from animal tissues
-e.g. kidney, lung, spleen - no sub-cultivation
process for continuous cell line and when its used most
these type of cells are transformed for indefinite sub-cultivation. Most frequently used in diagnostic virology
what do you look for during cell culture with cytopathic effect? (4)
- Rounding and clumping of cells
- Vacuolation
- Fusion of cells
- Syncytia or giant cells
what is a syncytia
a single viral infected cell may fuse with and infect neighboring cells
examples of when you would see a cytopathic effect during virus isolation
Syncytia or giant cells of Herpes simplex virus infected cells
what is involved in the isolation/detection of viruses from embryonated eggs
-Chick embryo of 7-12 days old
-Eggs surface is disinfected and penetrated with a small sterile drill
-After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days
-After incubation, the egg is broken, and virus is isolated from tissue of egg
-Viral growth is indicated by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes
advantages of virus isolation (2)
- during new epidemics, isolated virus is useful for further characterization
- Production of vaccines and other diagnostic tests
disadvantages of virus isolation (3)
- Time consuming and technically demanding; not frequently used
- Need to have an idea of what virus you are isolating to choose the “right” cells for growth
- Some viruses cannot be propagated
two types of virus visualization
indirect and direct visualization
what is involved in indirect visualization and what are you looking for
-uses light microscope
-Cell culture: cytopathic effect
-Histology: inclusion bodies
what is involved in direct visualization
Direct visualization of the virus in transmission Electron Microscopy (EM)
advantages of virus visualization (1 plus limitation of it)
-Can provide size and shape of virion particle
-Cannot provide specific information about the identity of the virus
disadvantages of virus visualization (3)
- Expensive equipment and trained staff
- It needs high concentrations of viral particles
- Not accessible to vet clinics
what do you see in a negative stain vs thin section for virus visualization using electron microscopy
Negative stain: virus particles in a fluid matrix
Thin-section: fixed tissue sample or cell cultures
what does it mean if you detect viral protein in a sample
means the pathogen is still present in the host
tests for direct detection of viral proteins (antigens) (3)
- Immunofluorescence: Fluorescence Antibody Test (FA)
- Immunohistochemistry (IHC)
- ELISA (for antigen detection)
what do you look for in ELISAs
looks for viral proteins (DIRECT ELISA)
two options for direct detection of nucleic acids
- PCR
- Sequencing
what type of viruses can PCR detect overall
DNA or RNA viruses
what is the process of sequencing
Process of determining the nucleic acid sequence – the specific order of nucleotides
what is standard PCR used to detect
DNA
what is reverse transcriptase PCR used to detect
viral RNA
what is real time PCR used to detect
fluorescent based detection of PCR products
what is multiplex PCR used to detect
different targets in the same reaction
advantages of PCR (4)
–The most versatile, rapid, sensitive and specific test
–Can be applied for poorer quality samples
–High reproducibility
–Automation – large number of samples
disadvantages of PCR (2)
–Unlike virus isolation there will be no virus available for further characterization and
archiving
–Cannot be easily applied for a new virus since the sequence is required for PCR primer design
what do serological tests detect and what do they indicate
-Detection of antibodies (host response) against a virus infection
-Indicate the host exposure to the virus
options for indirect detection for serological tests
-ELISA
-virus neutralization
-hemagglutination-inhibition
-western immunoblotting
what does indirect vs direct ELISA tell us
-Indirect ELISA: looking for host antibody in the specimen
-Direct ELISA: looks for antigen!
what does virus neutralization detect
neutralizing antibodies
how to determine which test we should request? (6)
-Identify the problem, define the task, ask the appropriate question
-Nature of the agent to be detected
-Test availability, sensitivity, specificity
-Sample availability
-Cost
-Time
what two things can be used to do virus isolation
cell culture, egg inoculation
what two things can be used to do direct detection (visualization)
EM, histo
what three things can be used to do direct detection (antigen)
ELISA, FA, IHC
what two things can be used to do indirect detection of antibodies
ELISA, virus neutralization
what two things can be used to do direct detection (nucleic acid)
PCR, sequencing
- Which one of the following is CORRECT about diagnostic virology?
a) A rapid test is preferable than a sensitive test?
b) Virus visualization is more important than detecting its nucleic acid
c) Sequencing is employed to know the specific nucleic acid sequence of a virus?
d) Virus isolation is routinely done for diagnosis of animal infections
C
- Which one of the following is CORRECT about diagnostic virology?
a) Nasal discharge can be sampled for the diagnosis of respiratory virus infections?
b) Viruses are not alive outside of the infected tissue so storage temperature during shipment WILL
NOT affect the diagnostic result
c) There is no need to use personal protective equipment during sample collection since the type of
virus is not yet known
d) Diagnostic tests are not that important for disease control and prevention
A
- Which one of the following is CORRECT about diagnostic virology?
a) A negative diagnostic test result can always rule out the presence of a viral disease
b) No diagnostic test can be used as a standalone information in clinical diagnosis of viruses
c) Electron microscopic visualization is helpful to identify the genome of the virus
B
