L19: Lab Diagnostics of Viral Infections (Romero) Flashcards
Routine techniques to diagnose a viral infection
1) isolate virus (GOLD STANDARD*)
2) detect viral Ag
3) Detect viral NA
4) Detect virus specific Ab
5) Visualize and ID viruses by electron microscopy
Cytopathic effects (CPE)
cytopathic changes indicative of the virus involved (i.e. syncytia, intranuclear or intracytoplasmic inclusions)
viral diagnosis by viral isolation
- GOLD STANDARD
- slow
- cytopathic effects indicative of virus involved
- only assay that can achieve the “unexpected” virus isolation
- provides large amt. of virus for further characterization
- best to evaluate primary cells of fetal tissues as some cell lines can grow many viruses
viral diagnosis by EM
- based on viral morphology
- useful in detection of non-cultivable viruses
- agent independent technique
- use negative staining or thin-sectioning
viral diagnosis by immunofluorescence (IF)
- used on cryostat sections, lesion smears, tissue cultures
- not compatible with formalin
- direct and indirect (more sensitive, less specific) methods which labels serum containing specific viral Ab
- requires expensive equipment
detection of viral antigens by immunohistochemical (IHC) staining
- enzyme reacts with substrate to produce brown color
- tissue fixed in formalin
- requires only light microscope
- rxn gets stronger with longer incubation
- provides evidence of Ag localization inside cells
Negri bodies
intracytoplasmic inclusions in nerve cells infected with Rabies
Detection of viral antigens at “Point-of-care”: Immunochromatography
- migration of Ab-conjugate complexes through a filter matrix or a lateral flow matrix + controls
- results seen as colored spots or bands
Detection of viral NA
- detects non-cultivable or no-longer viable viruses
- detects latent infections w/dormant viral genome
- detects virus that has been bound or complexed with Ab
- detects virus in formalin-fixed tissues
PCR
- an in-vitro method for the enzymatic synthesis of specific DNA sequences
- amplifies DNA using a pair of nucleotide primers
- amplified products from one cycle serve as templates for the next
- 3 main steps: denaturation of dsDNA, primer annealing, extension
How is PCR performed for RNA viruses?
via Reverse Transcription Polymerase Chain Reaction (RT-PCR):
RNA transcribed to cDNA, then amplified by PCR
PCR Precautions
-false positives can result from lab contaminated with amplified DNA products
Solution: Real-time PCR or qPCR measures the accumulation of DNA product as it’s amplified
Deep sequencing
relatively low-cost sequencing and random NA amplification that can discover new viruses
Advantages of PCR
- useful to detect viruses that don’t grow in culture
- can be used on any sample which is appropriate for virus isolation
- useful when rapid dx needed
- preferred for the initial ID of dangerous viruses (Rabies, Hendra, Nipah, influenza, Ebola)**
Serology
detection and quantification of virus-specific antibodies
-defines infection status of animals (discloses past infections, determines proper responses to vax, quantifies lactogenic (maternal) immunity, determines if a specific virus linked to a clinical event)
first Ab to develop
IgM
-presence of virus specific IgM Ab in acute serum may provide a presumptive dx (which is the case for West Nile virus infection of horses)
Enzyme-linked immunosorbent assay (ELISA) for the detection of viral Ab
- rapid, cost effective
- determines qualitative and quantitative viral Ab
- does serum dilutions against Ag on microplate and looks for color change
What is the gold standard for the quantification of antibodies?**
Virus Neutralization:
- usually correlates well with protective immunity in vivo
- relies on binding of Ab to critical viral Ag preventing virus from attaching and infecting susceptible cells
- virus growth detected by the development of cytopathic changes or effects (CPE) or production of viral antigens in a susceptible cell culture
- amt. of Ab needed to completely inhibit Ag production is measured
- slow, high cost, needs infectious virus
- ideal for wildlife studies
Virus neutralization: plaque-reduction assay
-dilution of serum needed to inhibit between 30-90% PFUs is measured
Hemagglutination inhibition (HI) for Ab to hemagglutinating viruses
- HI antibodies bind to virus RBC receptors and block hemagglutination of RBCs
- wells that don’t have clotted blood contain virus, because virus has been inhibited to clot blood
- used for detection of Ab against influenza, arthropod-borne virus, parainfluenza, parvoviruses, paramyxoviruses
Agar gel immunodiffusion (AGID) for the detection of viral antibodies
- simple, inexpensive, doesn’t require infectious Ag
- gives qualitative yes or no result
- not as sensitive as ELISA and has been mostly replaced by other techniques
