Lab Dx of viral infections

Question 1

Current routine techniques to dx viral infection (5)

Answer 1

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

Question 2

Clinical specimens appropriate for dx of virus related syndromes

Answer 2

• Professor prefers swabs to bxs

Question 3

Virus isolation

Answer 3

• 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

Question 4

Answer 4

• Detection of canine distemper virus
• intracytoplasmic inclusions in conjuctival epithelia scraping

Question 5

VIral dx by electron microscopy

Answer 5

• 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

Question 6

Answer 6

• Parvovirus in feces

Question 7

Answer 7

• Bovine rotavirus from calf feces
• negative staining

Question 8

Detection of viral antigens:

Immunofluorescence (IF)

Answer 8

• 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

Question 9

Answer 9

• Indirect immunofluorescence detection of canine parvovirus antigen

Question 10

Answer 10

• Immunofluorescent assay of rabies
• Gold standard for rabies testing

Question 11

Detection of viral antigens:

Immunohistochemica (IHC) staining

Answer 11

• 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

Question 12

Answer 12

• IHC staining: brown areas are rabies viral antigen using IHC
• Rabies infected neuronal cell
• Intracytoplasmic inclusions (Negri bodies)
• 38:53

Question 13

Detection of viral antigens at ‘point-of-care’

Immunochromatography

Answer 13

• 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

Question 14

Sequencing of viral genomes:

Deep sequencing

Answer 14

• 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

Question 15

Most prevalent flu types in US

Answer 15

• H5, H7
• Real-time RT-PCR to identify avian flue

Question 16

Serology:

Detection and quantification of virus-specific antibodies

Answer 16

• 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)

Question 17

Detection of virus neutralizing antibodies

test question

Answer 17

• 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

Question 18

Virus neutralization:

Plaque-reduction assay

Answer 18

• 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

Question 19

Hemagglutinating viruses of veterinary importance (5)

Answer 19

• 1) Orthomyxoviridae
  
  • influenza viruses A, B, C:
    
    • avian, canine, equine, seal, swine….
• 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)

Question 20

Agar gell immunodiffusion (AGID) for the detection of viral antibodies

Answer 20

• 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