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Virology

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Conventional Virological Assays

Antigen-capturing ELISA (AgELISA)

AgELISA is a rapid, sensitive laboratory diagnostic test for detecting virus or viral antigen(s) in a variety of clinical materials, e.g., tissues, serum, secretions, and excretions. The assay is a variant of the ELISA format in which virus-specific antibody is used to coat the surface of the plates instead of antigen. Therefore, virus or antigen in clinical specimen is captured by antibody. The use of virus-specific monoclonal antibody in AgELISAs provides high specificity. The presence of antibody-antigen complexes is subsequently visualized by a colorimetric reaction. In general, color development is proportional to the amount of viral antigen present in the specimen. One advantage of the AgELISA format is that it detects both infectious and inactivated viruses; however, expense is a consideration. At present, AgELISA is available at the VDL for the rapid detection of:

  • Rotaviruses in fecal material
  • Influenza A virus in nasal swabs and lung
  • Bovine viral diarrhea virus in sera (testing for persistently infected animals)
  • Bovine coronavirus in fecal material
  • Bovine respiratory syncytial virus in deep nasal swabs
  • Canine parvovirus in feces
  • Feline leukemia virus in whole blood

Fluorescent antibody examination of frozen tissue section (FATS)

FATS is a rapid, specific laboratory diagnostic test for detecting viral antigen in tissues and cell smears. The test utilizes virus-specific antibody labeled with a fluorochrome. When viewed with a fluorescence microscope, complexes of viral antigen and labeled antibody appear as fluorescent green areas. For best results, FATS requires very fresh clinical specimens. Freezing and thawing tissues can be detrimental to the test. If autolysis of tissues is anticipated, other antigen detection methods, such as immunohistochemistry (IHC) on formalin-fixed tissues, should be considered.

It is important to recognize that VI and EM are nonspecific tests that can detect a variety of viruses. In contrast, FATS and IHC are very specific tests that, because they are use virus-specific reagents (e.g., antibody), only detect the targeted virus. Furthermore, FATS and IHC cannot be performed on certain specimens, such as serum or feces.

Virus isolation

Virus isolation consists of two steps - the attempted recovery of virus and identification of the isolate. Isolation is attempted using in vitro cell culture. Identification of the isolate is done using immunofluorescence microscopy, electron microscopy, or molecular techniques. Although isolation of virus followed by identification is considered to be the definitive diagnosis, VI is often laborious, expensive, and time consuming. Results are not generally available for 1-2 weeks after submission. Therefore, VI should primarily be attempted under specific circumstances:

  • When other detection methods fail or when trying to isolate virus(es) from previously unrecognized diseases.
  • If there is no other detection method of similar or greater sensitivity.
  • If the virus is required for other purposes, such as differentiation (e.g., RFLP, sequencing), characterization (e.g., typing), and/or for production of autogenous vaccines.

Aseptic collection and proper handling of clinical specimens is critical for VI. Although certain viruses (e.g., parvovirus, circovirus) can withstand harsh environmental changes, this is not true for most viruses, particularly enveloped viruses. Virus isolation can be done on most clinical specimens, including biopsy and necropsy tissues, blood, secretions, and excretions. However, some clinical materials, e.g., urine, feces and semen, are difficult to work with because they are toxic to cell cultures.

The quantity of sample required for VI is usually more than that needed for rapid diagnostic assays so collect and submit the adequate amount of sample. To avoid unnecessary expense, be specific in your VI requests. Refer to specimen selection guidelines in the User's Guide or call the laboratory for advice.

 

Export Cases

Submitters should clearly indicate on the submission form that the testing is for export purposes.  Clearly indicate the specific test(s) needed, including the agent, test type, the number of passages needed if virus isolation is requested, and any special requirements imposed by the importing country.

 

 

 

VDL submission form

Best Practice Export Guide

Rabies

Submissions for Rabies Examination

Please use the VDL Rabies Examination Submission Form when submitting samples for rabies virus testing.   Provide a complete history and indicate clearly if human exposure is involved.  The absence of a complete history may delay the rabies examination until the laboratory can compile and verify the necessary information.  Information to be provided includes:

  1. Name of submitting veterinarian or physician.
  2. The species of animal submitted and any pertinent description.
  3. The name of the person making the request or the name of the person exposed.
  4. If a human exposure, the date of exposure and route of exposure.

Rabies Specimens

The entire animal, chilled (not frozen), should be delivered by private carrier.  Alternatively, submit the intact head, properly sealed to prevent leakage, and identified as a rabies suspect.

To safely prepare a rabies head:

  1. Remove the head by disarticulation of the occipito-atlantal joint.  Gloves, face, and eye protection are recommended for this procedure.
  2. Refrigerate and package in a leak-proof container with refrigerant packs.
  3. Deliver by private or commercial carrier with the package identified as "Rabies Suspect".
  4. Rabies suspects should be euthanized prior to delivery to the VDL, only in exceptional cases and with prior notice should a live animal be submitted.

Note:  According to the Centers for Disease Control and Prevention (CDC) guidelines, the cerebrum, cerebellum, and hippocampus are REQUIRED for the appropriate diagnostic evaluation of rabies virus infection.

Rabies Test Procedures

  1. A microscopic direct fluorescent antibody (DFA) test is performed routinely Monday through Friday on all rabies submissions.  Results are available the same day if received before 11 a.m.  Samples received after noon may be tested on the next business day.
  2. Emergency and weekend rabies test service is provided by the University Hygienic Laboratory (800-421-4692) in Iowa City. The UHL is a state-codified lab for rabies testing and provides 7 days/week, 24 hours/day service for rabies testing at no charge. For the State Hygenic Lab Rabies sumbission form and more specific info, see their website at http://www.shl.uiowa.edu/kitsquotesforms/rabiesslip.pdf.
  3. After-hours submissions to the ISU Vet Diagnostic Lab can now be placed directly in a refrigerator located inside the VDL submission door foyer.  Click here for complete instructions.

Rabies Results and Reporting

  1. All rabies results are reported by telephone as soon as they become available.
  2. Test results are reported as "inconclusive" when, as per CDC guidelines, any of the three parts of the brain required for the test are missing and the FA result is negative.
  3. Cases are reported as "unsuitable" when autolysis is evident and the FA test result is negative.
  4. In all cases, a written report will be provided to the submitter.

Additional tests, if requested, will not be performed on a rabies suspect case until the rabies examination is completed.  This will usually cause a one-day delay.  For this reason, it is important that the carcass and tissues be handled correctly if the differential diagnosis includes agents in addition to rabies virus.

Rabies Disease Links

For more information on rabies including prevention, control, epidemiology data, etc, please check the following websites:
 

Center for Disease Control and Prevention (CDC) 

ISU Center for Food Security and Public Health Rabies information

University of Iowa Hospitals and Clinics - Bat Rabies

IA Dept of Public Health - Rabies information

Rabies Resources Manual - For Veterinarians, Animal Control and Law Enforcement

 

 

 

VDL rabies submission form

Center for Disease Control and Prevention (CDC) 

University of Iowa Hospitals and Clinics - Rabies

IA Dept of Public Health - Rabies information