Diagnostic Virology Laboratory (DVL)

Isolation procedures used are specific to the host animal and suspected pathogen.  The DVL has expertise in recognizing morphological changes in cell culture and effects on embryonated avian eggs caused by viral infection.  Some of the identification techniques used include PCR, RT-PCR, serum neutralization, serology, electron microscopy, and DNA sequencing.  The DVL participates in the Select Agent Program and is a member of the USDA National Animal Health Laboratory Network (NAHLN).

Virology Laboratory Capabilities

Virus isolation and propagation:

• Perform virus isolation in tissue culture 
  • Utilize cell lines appropriate to host animal, including  Muscovy duck embryo fibroblasts (MSDEF), Madin-Darby canine kidney (MDCK), African green monkey kidney (VERO), and zebra fish (ZF4) cells 
  • Microscopically detect typical cytopathic effect in cell culture for known viruses and atypical cytopathic effect for possible novel viruses
• Perform two methods of virus isolation in embryonated chicken eggs: 
  • Allantoic inoculation [example: avian influenza virus (AIV) and avian paramyxovirus (APMV)]
  • Chorioallantoic membrane (CAM) inoculation [example: avian pox virus]

  

  Sources/Usage: Public Domain.

Identification of virus isolates:

• Prepare electron microscopy grids for viewing virus particles
• Perform virus-neutralizing tests using constant-serum varying-virus (example: duck plague) and constant-virus varying-serum (example: APMV)
• Determine structure of viral nucleic acid and presence of lipid coat
• Perform ELISA testing (example: Influenza types A and B, canine parvovirus)
• Conduct molecular testing including nucleic acid extraction, PCR, and sequencing (see molecular testing below)

Sources/Usage: Public Domain.

Characterization of novel virus isolates:

• Determine optimal cell culture necessary and number of virus particles present (TCID50)
• Describe morphology by electron microscopy
• Determine pathogenicity by mean death time and/or EID50 in embryonated chicken eggs
• Characterize viral nucleic acid as RNA or DNA and as enveloped with lipid coat or non-enveloped

Serologic tests (antibody detection):

• Conduct microtiter neutralization and plaque reduction assays to determine serum antibody titers for a variety of pathogens including;
  • Crane Herpesvirus (Inclusion Body Disease in Cranes – IBDC) 
  • AMPV subtypes 1-9
  • Novel common eider orthomyxovirus (Wellfleet Bay Virus)
  • West Nile virus (WNV)
  • St. Louis encephalitis virus (SLEV) & Japanese encephalitis virus (JEV)
  • Avian adenovirus
  • Eastern equine encephalitis virus (EEEV) and others

Nucleic acid extraction, screening, and sequencing:

• Employ multiple procedures for RNA and DNA extraction on tissue, swab, and environmental samples
• Conduct molecular screening (PCR) for known viruses, including: AIV, APMV-1, WNV, Ranavirus, Wellfleet Bay virus, Duck Viral Enteritis Virus, Buggy Creek virus, avian poxvirus, flavivirus
• Perform viral subtyping and phylogenetic analysis by Sanger, Next-generation, and whole genome sequencing methods

Virology Diagnostic Tests

The following is a reference guide to commonly requested tests.  It is not a full list of testing capabilities of the laboratories. Determination of which test will be run on any given submission will be based on case history, gross findings at necropsy, and the scientific judgment of the case pathologist.