Lab Dx of viral infections Flashcards
Current routine techniques to dx viral infection (5)
1) Virus isolation: Gold standard
2) Detection of viral antigens
3) Detection of viral nucleic acids
4) Detection of virus specific antibodies
5) Visualization and identification of viruses by electron microscopy
Clinical specimens appropriate for dx of virus related syndromes
- Professor prefers swabs to bxs
Virus isolation
- little influence on clinics
- cell culture is virus independant
- could culture something unexpected
- cytopathic changes (cytopathic effects or CPE) usually indicative of virus involved
- syncytia: large multinucelated cell with one huge membrane
- intranuclear or intracytoplasmic inclusions
- better than CPE
- Provides tons of virus for further work
- Primary cells best
- some animal cell lines known to be susceptible and grow many viruses

- Detection of canine distemper virus
- intracytoplasmic inclusions in conjuctival epithelia scraping
VIral dx by electron microscopy
- Virus morphology can be sufficient to characterize family
- Useful in detection of viruses that can’t be cultivated
-
Negative staining
- straight or after virus aggregation or immuno EM
- requires > or equal to 106 particles/ml
-
Thin sectioning
- most cells must contain viruses, otherwise insensitive
- expensive and takes several days

- Parvovirus in feces

- Bovine rotavirus from calf feces
- negative staining
Detection of viral antigens:
Immunofluorescence (IF)
- Used on
- cryostat sections
- lesion smears
- tissue cultures
- Not compatible with formalin fixation
- Direct IF
- uses FITC-labeled specific viral antiserum
- Indirect IF
- uses labeled anti-species antiserum after 1st antibody
- inc sensitivity but also non-specific background

- Indirect immunofluorescence detection of canine parvovirus antigen

- Immunofluorescent assay of rabies
- Gold standard for rabies testing
Detection of viral antigens:
Immunohistochemica (IHC) staining
- For tissues already fixed in formalin
- Horse radish peroxidase enzyme used
- enzyme reacts with substrate => brown color
-
Advantage of IHC over IF
- only needs light microscope
- amplifies reaction product
- color gets stronger by inc incubation time

- IHC staining: brown areas are rabies viral antigen using IHC
- Rabies infected neuronal cell
- Intracytoplasmic inclusions (Negri bodies)
- 38:53
Detection of viral antigens at ‘point-of-care’
Immunochromatography
- migration of antibody-conjugate complexes through filter matrix or lateral flow matrix
- All controles included in membrane
- Results seen as colored spots/bands
- Rapid and simple assay in clinic
- each test unit contains positive and negative control
- sensitivity varies by kit
*Iddex snap test for BVDV
Sequencing of viral genomes:
Deep sequencing
- new viruses discovered by random nucleic acid amplification
- relatively low cost sequencing
- host cell DNA must first be liminated by nuclease tx
- prior sequence knowledge not needed
- obtained sequences compared to existing ones in GenBank
Most prevalent flu types in US
- H5, H7
- Real-time RT-PCR to identify avian flue
Serology:
Detection and quantification of virus-specific antibodies
- Defines infection status of animals
- discloses past infections
- determines proper responses to vaccination
- quantifies lactogenic (maternal) immunity
-
Paired-sera
- acute and convalescent sera from same animal
- convalescent serum titer must be > equal to four-fold greater
- Virus specific IgM antibodies in acute serum
- may provide presumptive dx (e.g. west nile in horses)
Detection of virus neutralizing antibodies
test question
- Virus neutralization
- Gold standard for quantification of antibodies
- Best correlate with protective immunity against viruses
- Constant virus-variable serum dilutions
- most widely used technique
- Assay relies on binding of antibody to critical viral antigens
- prevents virus froma ttaching and infecting susceptible cells
Virus neutralization:
Plaque-reduction assay
- Uses fixed amount of virus-producing plaques (PFU) per plate
- End-point or VN titer of a serum corresponds to dilution that inhibits between 30-90% PFU
- Scored by staining cell monolayer with crystal violet
- stains live cells
- enumerates unstained plaques corresponding to dead cells
- Assay performed under an agarose overlay to slow down virus spread
- PFU with serum/PFU w/out serum X 100 =% PFU reduction
Hemagglutinating viruses of veterinary importance (5)
- 1) Orthomyxoviridae
- influenza viruses A, B, C:
- avian, canine, equine, seal, swine….
- influenza viruses A, B, C:
- 2) Paramyxoviridae
- Bovine parainfluenza virus 3 (PIV-3)
- Porcine hemagglutinating encephalomyelitis virus
- 3) Parvoviridae
- feline panleukopenia virus
- canine parvovirus
- porcine parvovirus
- 4) Adenoviridae
- Canine adenovirus 1
- Equine adenovirus 1 & 2
- Egg drop syndrome virus (EDS-76)
Agar gell immunodiffusion (AGID) for the detection of viral antibodies
- workhorse in viral diagnostics field
- Simple, inexpensive, doesn’t require infectious agent
- Crude antigens prepared from tissues can be used
- Strictly qualitative (yes/no)
- cannot be automated
- Not as sensitive as ELISA
- has been replaced in developed world