Why Perform Viral Diagnostic

Tests?
 Can aid the physician with further therapeutic
measures and interventions
 Results have prognostic value
 Availability of rapid methods and antiviral
chemotherapeutic agents
 Early and specific viral diagnosis may permit
earlier institution of preventive public health
measures
 Approaches to Viral Diagnosis
 Cytologic studies
 Intracellular viral inclusions, giant cells, syncytia

 Direct examination by electron microscopy

 Especially helpful for non-cultivable agents of human
viral gastroenteritis
 Isolation of viruses in tissue culture, avian host
systems, or animals
 Demonstration of viral components directly in clinical
specimens or in host-cell systems by immunologic or
molecular methods
“Owl’s Eye” Inclusion of Cytomegalovirus
(CMV) in Lung Tissue
Electron Micrograph of Herpes
Simplex Virus type 1 (HSV-1)
General Guidelines for Viral
Specimen Collection
 Best chance for isolation of viruses exists
when the specimen is collected as early in
the clinical course as possible
 Respiratory viruses shed for 5-7 days, with
highest titers during the prodromal period
 HSV and VZV may not be recoverable as
soon as 5 days after onset of symptoms
 Collect 7-10 ml of clotted blood (“red top
tube”) during the acute phase of the illness
Time Course of Viral Infection
Swabs for Collection of Virology
Specimens
 Swab material should be made of sterile Rayon, or
Dacron
 DO NOT USE CALCIUM ALGINATE SWABS FOR
VIROLOGY SPECIMENS!
 Toxic to enveloped viruses (e.g., HSV)
 False-positive direct fluorescent antibody tests
 DO NOT USE SWABS WITH WOODEN SHAFTS!
 Formaldehyde in wood may inhibit recovery of
viruses
Viral Transport Medium (VTM)
 Buffered saline or other isotonic solution with a
protein stabilizer
 Stabilizers
 Gelatin, whole serum, bovine serum albumin,
bacteriologic broths (e.g., brain-heart infusion),
tissue culture medium (e.g., HBBS) with serum
 Antibiotics
 Penicillin or vancomycin
 Streptomycin or gentamicin
 Amphotericin B
 pH of 7.2-7.4
Identification of Viruses in Culture
 Type of cell line, time to detection and nature of the
cytopathic effect (CPE)
 Confirmatory identification
 Neutralization
 Hemagglutination/hemadsorption inhibition
 Direct and indirect fluorescent antibody tests
 Immunoperoxidase testing
 Enzyme immunoassay
 In situ hybridization
Identification by Neutralization
Identification by Direct Fluorescent
Antibody (DFA) Staining
 Employs monoclonal antibodies conjugated to fluoro
isothiocyanate (FITC, fluorescein)
 Rapid
 Can be used to identify CMV, HSV, and other viruses
in shell vials after 24-48 hours
Characteristic CPE of RSV Observed in A549
Cell Line (l), Cells Removed from Monolayer (r)
Patient with Herpetic Vesicles on Lower Lip (l) &
Direct Scraping of Cells from Unroofed Lesion
Placed on Slide (r)
Other Methods for Direct Detection of
Viruses in Clinical Specimens
 Enzyme immunoassay
 RSV and influenza A in nasopharyngeal aspirates
 Rotavirus and adenovirus types 40/41 in stool
specimens
 Electron microscopy/immune electron microscopy
 Non-cultivable agents of gastroenteritis (e.g.,
Norwalk agent, caliciviruses, astroviruses)
 Nucleic acid amplification techniques
 PCR, in situ hybridization
EIA for Detection of Rotavirus
Antigen in Stool Specimens
Immunoelectron Micrograph of
Norovirus (i.e.Norwalk-like Viruses)
 Also called “small, round-
structured viruses” about 27
nm in diameter
 Cause nausea, diarrhea,
comiting, and cramping and
constitutional symptoms
 Transmitted by close contact
with infected people, fomites
contaminated with the virus,
and eating contaminated
food or drinking
contaminated liquids
 Cause of several outbreaks
of gastroenteritis on cruise
ships in recent years
Rapid Tests for Influenza
 Several available
 Performed on
nasopharyngeal
aspirate specimens
 Vary significantly in
sensitivity/specificity
In Situ Hybridization Assay
Direct Detection by Polymerase
Chain Reaction
Methods for Viral Antibody
Detection
 Complement fixation
 Hemagglutination inhibition
 Enzyme immunoassay
 Indirect fluorescent antibody assay
 Latex agglutination assay
 Western immunoblot
 IgM capture assay
Complement Fixation Test
Hemagglutination Inhibition (HAI)
Hemagglutination Inhibition Assay for Detection
of Abs in Acute and Convalescent Serum

 Highest dilution of serum where hemagglutination of RBC’s by

the added virus occurs = endpoint
 Acute phase = serum dilution of 1:10 (titer = 10)
 Convalescent phase = serum dilution of 1:160 (titer = 160)
 Greater than or equal to a 4-fold increase in titer is serum
dilution of >= 1:80
Hemagglutination Inhibition (HAI) Assay for
Detection of Anti-Influenza A Abs in Acute and
Convalescent Sera
 Highest dilution of acute
serum specimen
producing
hemagglutination
inhibition is 1:10 (titer =
10)
 Highest dilution of
convalescent serum
producing
hemagglutination-
inhibition is 1:320 (titer
= 320)
Enzyme Immunoassay for Detection of
Specific Viral Antibodies
Indirect Fluorescent Antibody (ITA) Test
for Detection of Specific Viral Antibodies
Latex Agglutination Tests for Viral
Antibodies
Western Immunoblot Procedure
Structure of Human Immunodeficiency
Virus type 1 (HIV-1)
Western Immunoblot for HIV-1
Antibodies
 Blots shows antibodies directed against
 gp160/120
 p66
 p51
 gp41
 p31
 p24
 P17
 Positive blot criteria
 1. gp160/120 and p24
 2. gp41 and p24
 3. gp160/120 and gp41
 No bands = Negative
 Any other combination =
indeterminant
IgM Capture Assay for Detection of
Specific IgM Antibodies