Diagnosis 1 & 2 (lecture 4 & 5)

what are some criteria needed for the selection and collection of samples for laboratory diagnosis of viral infection?

- virus titer

- selection of specimens (based on symptoms pick best place to collect a sample)

- what stage is the disease in (is the virus being shed?)

what are some reasons for making a quick and accurate viral diagnosis?

- public health concerns (zoonotic diseases)

- economical reasons (livestock food industry, i.e. infections such as avian influenza and how that has impacted the cost of eggs)

- rapid diagnosis allows for the management and control of epidemic outbreaks

- allows for surveilance of epidemiology and pathogenicity of a virus at many levels

when is virus titer usually highest?

- at affected sites

- during the early stages of infection

viremia

presence of viruses in the blood

what techniques are used in virus isolation?

- cell culture and CPE

- egg culture

- laboratory animals (historically)

describe the technique of cell culture and CPE

Specimen (appropriately collected and filtered) --> inoculated to cultured cells as a monolayer (selection of the cell type is critical to isolate the suspected virus)--> observe for 2-10 days --> recognize characteristic cytopathic effect

what are egg cultures used for?

- Used for isolation of influenza viruses by allantoic and amniotic inoculation

- Historically, used for pox virus isolation

what technique is used for virus visualization?

Electron microscopy

- Non-specific technique: as long as virus is present, can see

what techniques are used for viral antigen detection?

o Immunohistochemistry/Immunofluorescence

o Enzyme-linked immunosorbent assay (ELISA)

o In situ Hybridization

o Hemagglutination

describe the technique of Immunohistochemistry/Immunofluorescence

Need specific antibody for virus of interest. In Immunohistochemistry the reporter enzyme turns color and can be seen with the light microscope.

- the reporter is a fluorescent dye that can only be seen with a fluorescent microscope.

what is In situ Hybridization?

a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ)

describe Hemagglutination as it applies to virus antigen detection

Some viruses (Influenza, parvo, measles/CDV) naturally bind to the surface of red blood cells and cause them to agglutinate.

Basis for detecting those viruses.

Use round-bottom assay plate, allows for rbcs to settle.

what techniques are used in characteristic gene sequencing?

o Polymerase chain reaction (PCR)

o Whole genome sequencing

o Metagenomics

what is Polymerase chain reaction (PCR)

A method to amplify a fragment of DNA.

PCR is an in vitro DNA synthesis reaction that produces many copies of a single DNA fragment.

amplifies DNA from a small amount of DNA template

what is metagenomics

applies a suite of genomic technologies and bioinformatics tools to directly access the genetic content of entire communities of organisms. The field of metagenomics has been responsible for substantial advances in microbial ecology, evolution, and diversity

Identify techniques used for detection of antibodies produced by the host in response to viral infection.

• Enzyme-linked immunosorbent assay (ELISA)

• Immunofluorescent Antibody

• Serum Virus neutralization

• Hemagglutination inhibition

• AGID

what techniques are used for virus detection?

- virus isolation

- virus visualization

- viral antigen detection

- characteristic gene sequence

describe immunofluorescent use in detection of antibodies

o Requires a microscope with fluorescence capability (light accessory and bulb)

o Used to demonstrate the presence of Antibody in serum

o Uses and anti-IgG secondary antibody labeled with a fluorescent marker (anti dog IgG).

o Can use serial dilutions of patient's serum over a number of wells to find last well that has fluorescence = 'titer'.

describe the serum virus neutralization (SN) technique used in detection of antibodies

o Serially dilute serum (1:2, 1:4, 1:8, 1:16......1:512)

o Add each dilution to separate wells of cultured cells

o Add equal amounts of virus (100 plaque forming units) to each well containing cultured cells and serum dilutions

o Last dilution that can prevent plaque formation is antibody titer

describe hemagglutination inhibition technique used in detection of antibodies

o Some viruses have the natural ability to bind to the surface of red blood cells (Influenza, parvo, measles/CDV) and cause agglutination.

o Mix serial dilutions of patient's serum with known amount of virus particles.

o Antibodies in patient's serum 'inhibit' the virus's ability to agglutinate rbcs.

o Read titer of antibodies where is stops inhibiting agglutination

describe the AGID (ouchterlony) technique used in detection of antibodies

o Our algorithmic approach to AGID testing uses multiple methodologies to identify antibodies associated with the illness.

o Diagnostic test using serum that detects antibody produced in response to infection

what are the uses of the AGID (ouchterlony) technique for antibody detection today?

Under-developed countries

Some diseases, still test of choice

Screening (EIA in horses)

Used in research and development of new diagnostic tests, such as ELISA

Identify the limitations of diagnostic techniques in terms of turn around time, cost, sensitivity & specificity, and correlation with disease

• ELISA technology is cost effective in clinical practice

• Virus isolation is expensive, but useful as vaccines can be developed from the isolates

• PCR technology is cost effective and increasingly available (e.g. Idexx), but needs a sophisticated laboratory

• Sequencing also needs a sophisticated laboratory, but increasing in use in university and state laboratories

• Metagenomics is available in only the most advanced of national diagnostic laboratories,