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DR SONNIE P. TALAVERA
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¬ Viruses are the most frequent cause of
human infectious diseases
± range from trivial colds to fatal
immunoimpairment and opportunistic infections.
¬ Virus isolation in cell culture and viral antigen
identification
± community hospital laboratories to diagnose
infection
± molecular methods.
¬ Accurate viral diagnosis is fundamental for
optimal patient management
± reasonable use of antiviral drugs,
± reduction of unnecessary lab testing
± inappropriate antimicrobial therapy,
± application of infection control procedures and
for public health control of community outbreaks.
¬ any common viruses grow in vitro in cell
culture
± primary, diploid or heteroploid cell lines;
¬ cytopathic changes that develop in the cell culture
monolayer
± detection of viral antigen in the cells
¬ fluorescence-labeled specific antibodies.
± propagate in newborn mice or embryonated egg
tissue,
¬ .
¬ Shell vial culture technique uses the cell
monolayer
± short incubation (1-3 days), the monolayer is
stained with fluorescein-labeled specific viral
antibody.
± ID of herpes simplex virus (HSV),
cytomegalovirus (C V), varicella zoster virus
(VZV), respiratory viruses, and enteroviruses.
¬ Hybrid culture systems employ two cell lines
± recovery of the common respiratory viruses
(influenza, parainfluenza, respiratory syncytial
virus (RSV), and adenovirus), the enteroviruses,
and HSV/VZV.
¬ Nucleic acid hybridization and nucleic acid
amplification tests (NAAT)
± DNA or RNA viral genome.
± human papillomavirus (HPV), C V, HSV, and
parvovirus B19 in tissue;
¬ NAATs such as polymerase chain reaction,
strand displacement amplification, and
nucleic acid sequence-based amplification
± molecular diagnosis beyond culture or antigen detection
methods.
¬ Direct visualization of viral particles by
electron microscopy
± ID morphologic features of agents of
gastroenteritis
¬ rotavirus, enteric adenovirus, norovirus, astrovirus and
calicivirus;
± none of these viruses replicates in standard cell
culture lines.
¬ Viral serology has two major applications ±
diagnosis of recent infection and
determination of immunity.
± Ig -specific antibody during acute illness or 4-fold rise
in IgG antibody between acute and convalescent sera
designates 
 .
± Persistent specific IgG is marker of 



for
several viruses such as measles, mumps, VZV, hepatitis
A and B.
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¬ community hospital with adult and pediatric
primary care and high risk obstetrics,
neonatology, hematology±oncology, and HIV
services.
¬ Average detection time for most viruses is
often 2 days or less
¬ reflects the use of rapid antigen assays,
¬ shell vial cultures with short incubation
schedules,
¬ nucleic acid amplified tests.
¬ Overall recovery rates for viruses are high±
16-23%± several times greater than for
routine bacterial blood or stool cultures,
¬ any common viruses exhibit seasonal
variations
¬ Influenza, respiratory syncytial virus, and
parainfluenza virus 1 and 2 circulate every
winter.
¬ Adenovirus, parainfluenza virus 3,
cytomegalovirus, and herpes simplex
infections occur year-round.
¬ Enterovirus disease clusters in late summer
and early autumn.
Y
 

¬ clinically significant viruses replicate in vitro,

¬ virus recovery correlates well with acute
disease.
¬ culture
± labor-intensive,
± requires technical proficiency,
± means at least one-day turnaround for positive
results.
Y
 
¬ Replication in newborn mice or embryonated eggs
± essential for some fastidious viruses
¬ Tissue culture cells are divided into three
categories:
± primary cell cultures,
± diploid cell lines,
± heteroploid cell lines.
Y
 

¬ Primary cell cultures

± prepared directly from the parent organ (e.g.,
monkey or rabbit kidney)
± by mincing and trypsinization
± mixture is transferred to a tube or shell vial
where the cells attach
± form a confluent monolayer on the glass surface.
Y
 

¬ Primary cell cultures

± Overlay medium with buffered isotonic inorganic
salts, glucose, amino acids, vitamins, and
cofactors at a physiologic pH
± Eagle minimum essential medium in Earl or
Hanks balanced salt solution ±
¬ to maintain a healthy monolayer but not promote cell
proliferation.
Y
 

¬ Diploid cell lines

± usually human diploid fibroblasts (HDF) derived
from lung or newborn foreskin,
± majority of cells have a normal diploid
chromosome number.
± propagated in serum-rich growth medium
¬ examples include RC-5, WI-38, and foreskin
fibroblasts.
± serially subcultured 20-50 times;
Y
 

¬ Heteroploid cell lines

± derived from malignant tumors and can be
passaged indefinitely;
± they are aneuploid and divide rapidly.
± Heteroploid lines include HEp-2 (laryngeal
carcinoma), HeLa (cervical carcinoma), and
A549 (laryngeal carcinoma).
¬ Primary cultures must be checked for
contamination with endogenous virus.
Y
 

¬ Diploid and heteroploid cell lines should be

tested periodically
± for m
 contamination
± continued sensitivity to viral infection.
¬
Hybrid¶ cells are a new concept; the
monolayer is a mixture of two cell types that
together support growth of a wide range of
viruses.
Y
 

¬ R- ix contains A549 and mink lung cells ±

± respiratory agents±influenzas, parainfluenzas,
respiratory syncytial virus (RSV), and
adenovirus.
¬ E- ix contains Buffalo green monkey kidney
and A549 cells -- enteroviruses.
Y
 

¬ Tube monolayers are versatile:

± Viral replication in tube monolayers -- inverted
phase-contrast microscope for cytopathic
changes
± a single cell line may support growth of several
viruses,
± each producing distinctive cytopathic effect
(CPE)
Y
 

¬ Tube monolayers are versatile:

± other identifying features (hemagglutination,
hemadsorption, interference).
± cytopathic effect (CPE)
¬ 1-3 days (herpes simplex virus [HSV])
¬ may not be recognized for 5-20 days (RSV, varicella
zoster virus [VZV], C V);
¬ extended incubation for 2 weeks or more.
Y
 

¬ shell vial technique

± cell sheet rests on a round coverslip, monolayer
side up, in a shell vial.
± specimen is inoculated into the vial, which is
then centrifuged to enhance viral attachment to
the cell surface.
± 1-3 days' incubation, the monolayer is tested for
specific viral antigen, usually with a 


 

± ideal for identification of a single virus (HSV) or
limited number of viruses (RSV and influenza),
Y
 

¬ shell vial technique

± faster detection of slow-growing agents such as
C V and VZV.
¬ HSV vesicular genital lesions are excellent candidates
for shell vial culture; sensitivity is almost 100%
turnaround for negative specimens is decreased from
1 week to 1 day.
¬ comparable or superior to tube methods for recovery
of C V from urine,
± backup tube culture is recommended to maximize C V
recovery from peripheral blood, body fluids and tissue
biopsies.