Methods of viral diagnosis

• Different ways of diagnosing if a disease or the causative agent is viral
• Some viruses can only be detected using specific techniques
• It is necessary to know the various ways you can detect a virus
• Virus detection methods:
• Direct examination:

  • Antigen detection serology (immunofluorescence, ELISA etc.)
  • Electron microscopy for morphology of virus particles
  • Viral genome detection:
  • Hybridization with specific nucleic acid probes
  • Polymerase chain reaction (PCR)
• Cultivation of Viruses:
• Indirect examination:

  • Cell Culture for cytopathic effect (CPE) and hemadsorption
  • Animals for disease or death
  • Serology using Direct and indirect ELISA
  • Hemagglutination inhibition test
• Allows detection of multiple viruses that may not have been suspected initially
• Provides a viable isolate that can be used for further study of the viral agent
• Primary Cell Cultures:
• Normal cells obtained from freshly killed adult animals

  • Monkey kidney cell lines are usually used
  • Used for isolation of respiratory and enteroviruses
• Organ Cultures:

  • Small bits of organ from human or animal maintained in tissue culture media
  • Accurately model functions of an organ in various states and conditions
• Continuous Cell Cultures:
• Made up of embryonic kidney and skin fibroblasts

  • More viable for longer periods than primary cell lines
  • Example: HeLa Cells (discovered in 1951) from Henrietta Lacks
• Viral Plaque Assays:
• Many viruses can be isolated and quantified by forming visible zones (plaques) in layers of host cells

  • Plaques represent the number of infectious particles within the sample
  • Data from this assay can be used in drug and vaccine development
• Animal Inoculation:
• Embryonated Egg Culture:
• Some viral diseases can be confirmed through inoculating laboratory animals with the infective agent
• Uses embryonated eggs of 7-12 days

  • Virus can be cultured in different parts of the egg depending on the types of virus
• Direct methods for virus detection:
• Serology:

  • Detection of antigen using methods like Radioimmunoassay (RIA), Sandwich ELISA, Particle agglutination, Western Blot (WB)
  • Complement fixation tests (CFT), Immunofluorescence
• Advantages of FA assays:

  • Rapid to perform
  • Results available within a few hours
• Methods of fluorescent antigen-antibody detection (FA) include staining, Immunoperoxidase staining, and Enzyme-linked Immunosorbent Assay (ELISA)
• Fluorescent antibody staining: Labeled antibodies are incubated with suspect tissue, and if positive, antibodies will bind to the antigen, causing them to fluoresce
• Immunoperoxidase staining: Peroxidase stains cells with specific antibodies brown
• Enzyme immunoassay: Positive samples change the color of solutions in the well
• Disadvantages of FA methods:

  • Require more labor
  • Results interpretation can be challenging for untrained individuals
  • Higher occurrence of false positive and false negative results
  • High-quality specimen needed
• Complement fixation test is an example of a serology method
• Serology technique detects antibodies alone, checking for IgM antibodies against specific viruses in the body
• Hemagglutination inhibition test is another serology method
• Direct ELISA and Indirect ELISA are serology methods
• Molecular methods for viral diagnostics are based on detecting viral genome, such as Polymerase Chain Reaction (PCR)
• Advantages of PCR:

  • Extremely high sensitivity, detecting down to one viral genome per sample volume
  • Easy to set up
  • Fast turnaround time
• Disadvantages of PCR:

  • Highly susceptible to contamination
  • Requires a high degree of operator skill
  • Challenging to set up a quantitative assay
• For RNA viruses, reverse transcription PCR (RT-PCR) converts RNA into DNA before copying it
• Nucleic Acid Detection:

  • PCR analysis since 1985 allows sensitive detection of viral nucleic acids
  • PCR achieves sensitivity down to one DNA molecule in a clinical specimen
  • PCR test amplifies DNA sequences by unwinding and duplicating the original DNA sample
• Electron Microscopy:

  • Electron microscopes provide high magnification (around 50,000x)
  • Rapidly detects viruses, including those unsuitable for other tests
  • Expensive equipment maintenance and requires experienced observers
• Real-time (quantitative) PCR (qPCR) generates a fluorescent signal during PCR, monitoring DNA amplification in real-time
• Gel Electrophoresis:

  • Organic molecules respond to an electric current based on their charge
  • DNA's negative charge allows separation of fragments under an electrical current using agarose or acrylamide gel