MycVir | Chapter 5

Lester Joseph M. Ballad, RMT

 Learning objectives

• Define and discuss the different parts of a

virus
• Identify the different types of viruses
• Discuss viral replication
• Discuss the pathogenesis of viruses
• Discuss viral transport and specimen
collection
• Discuss and identify the different viral
detection methods
 VIROLOGY

• Virology is the field of science that deals with the

study of viruses and virus-like agents, including, but
not limited to, their taxonomy, disease-producing
properties, cultivation, and genetics

• Viruses are filterable agents; size ___________________

• __________________ is a protein coat which surrounds

the nucleic acid genome of the virus

• Genomes can either be DNA or RNA

• ___________________ are protein subunits of capsids,

which assemble into an icosahedral or irregularly-
shaped or helical in form
 VIRAL REPLICATION (Infectious Cycle)

• Viruses are obligate intracellular parasites

1. ____________________ - process by which the virus must

be recognized and bind to a suitable host cell
2. ____________________ - process by which viruses enter
the host cell
3. ____________________ - necessary process to release
the viral genome before the viral DNA or RNA is
delivered to its intracellular site of replication in
the nucleus or cytoplasm
4. ____________________ - production of nucleic acid and
protein polymers
5. ____________________ - structural CHON, genomes and,
in some cases, viral enzymes are assembled into
virions
6. ____________________ - occurs following cell lysis or
by budding from cytoplasmic membrane
 VIRAL PATHOGENESIS

• Mode of transmission
• Respiratory
• Fecal-oral route
• Sexual contact
• Trauma/injection with contaminated objects or needles
• Tissue transplants (blood transfusion)
• Arthropod or animal bite
• Vertical transmission
• Local infection can lead to viremia, which inoculates
secondary target tissue from the primary site and
releases mediators of human cell functions
• Tissue is damaged by:
• Lysis of virus-infected cells
• Immunologic mechanism directed against the virus
• __________________________ - cross-reaction of virus with
human tissue leading to tissue destruction
• Reactivation may occur accompanying immune suppression
• _________________________ - viruses that stimulate
uncontrolled growth of host cells
 SPECIMEN COLLECTION

• Viruses are in highest concentration during the first

several days following onset of symptoms
• Sample should generally come from the infected site

THROAT, NASOPHARYNGEAL SWAB OR ASPIRATE

• Nasopharyngeal aspirates are superior
• Swabs are considerably more convenient
• Rhinovirus detection requires a nasal specimen
• Throat specimens are collected by rubbing inflamed,
vesiculated or purulent areas of the posterior pharynx
with dry sterile swab
• Nasopharyngeal secretion specimens are collected by
inserting a swab with a flexible shaft through the
nostril in the nasopharynx or by using a bulb syringe
with 3 to 7 mL of buffered saline
BRONCHIAL AND BRONCHEOALVEOLAR LAVAGE
• Wash and lavage fluid collected during bronchoscopy are
excellent specimen for the detection of viruses
infecting lower respiratory tract

RECTAL SWABS AND STOOL SPECIMEN

• Stool and rectal swabs of fecal specimens are used to
detect rotavirus and enteroviruses
• Rectal swabs are collected by inserting a swab 3 to 5
cm into the rectum
• 5-10 mL of freshly passed diarrheal stool or stool
collected in a diaper from young infants is sufficient
for rotavirus and enteric adenoviruses

URINE
• ____________________________ can be detected
• Virus can shed intermittently or in low numbers
• At least ______________________________________
SKIN AND MUCOUS MEMBRANE LESIONS
• Enteroviruses, HSV, VZV and rarely CMV or poxviruses
• Detection of virus is difficult once the vesicle has
ulcerated or crusted
• _____________________ is a smear of cells from the base
of the vesicle stained by Giemsa or Papanicolau and
detects typical uninucleated giant cells and inclusions
BLOOD
• Used primarily to detect CMV
• HSV, VZV, enteroviruses and adenoviruses may
occasionally be encountered
• 5 to 10 mL of anticoagulated blood collected in a
vacutainer tube
• Heparin, citrate and EDTA may be used for CMV
detection; citrated blood for other viruses

BONE MARROW
• Detection of ___________________________

SERUM FOR ANTIBODY TESTING

• Acute and convalescent serum specimen
• Acute specimen should be collected as soon as possible
after the appearance of symptoms
• Convalescent specimen is collected a minimum of 2-3
weeks after acute specimen
• 3-5 mL
 SPECIMEN TRANSPORT & STORAGE

• Specimen for viral isolation should not

be allowed to sit at room temperature or
higher
• Should be placed on iced and transported
to the laboratory. If a delay is
unavoidable, refrigerate the specimen
• __________________________ inactivates
HSV
• Specimen on swabs should be emulsified in
viral transport medium before transport
• VTM contains buffered saline, protein
stabilizers, and antimicrobial that
inhibits bacteria and fungi
• Samples for viral culture can be
refrigerated in VTM for about 48 hours.
But should never be frozen. Samples can
be stores at 70OC, however, infectivity
will be diminished
 SPECIMEN PROCESSING

• Specimens should be processed immediately

• Processing should be done in BSC level II
• Any specimen that might be contaminated with bacteria
or fungi, or any swab specimens should be added to a
VTM
• Fluid specimens that are normally sterile can be
inoculated directly to cell culture
• Each viral cell culture tube is inoculated with 0.25 mL
of specimen
 VIRUS DETECTION METHOD

CYTOLOGY AND HISTOLOGY

• Detection of viral inclusion or syncytia in Giemsa or
Pap-stained cytologic smears
• Cytology is most frequently used to detect VZV or HSV
(Tzanck smear)
• Tissue stained with H&E or Pap is used to detect
inclusion of CMV, adenovirus, parvovirus, papilloma
viruses and molluscipoxvirus
ELECTRON MICROSCOPY
• _________________________________ – negative staining
(most common staining for rapid EM)
• Most helpful for the detection of viruses that do not
frow readily in cell culture
• Detection of gastroenteritis viruses that cannot be
detected by other methods (noroviruses, coronaviruses,
and astroviruses) and virus causing encephalitis that
are not detected with cell culture (HSV, measles virus
and JCV)

IMMUNODIAGNOSIS (ANTIGEN DETECTION)

• Fluorescent antibody, enzyme immunoassays,
radioimmunoassays, latex agglutination and
immunoperoxidases
• Direct and indirect immunofluorescent methods
• Nuclear and cytoplasmic staining patterns are typical
for influenza virus, adenoviruses and herpes viruses
• Cytoplasmic staining is typical for RSV, parainfluenza,
mumps viruses
CELL CULTURE

1. CONVENTIONAL CELL CULTURE

• Cell culture are inspected microscopically for the
presence of virus indicated by areas of dead and
decaying cells called ___________________________
• Two kinds of media
• Growth media – serum rich (10% fetal, newborn or
agammaglobulinemic calf serum) nutrient medium designated
to support rapid cell growth
• Maintenance media - similar to growth media but contain
less serum (0-2%) ang used to keep cells in a steady
state of metabolism
• Cell culture becomes a cell line once it has been
passed or subcultured in vitro. Cell lines can be
classified as:
1. Primary cell line
2. Low passage cell line
3. Continuous cell lines
2. SHELL VIAL CELL CULTURE
• Rapid modification of conventional cell culture
• Virus is detected more quickly because the infected
monolayer is stained for viral antigens produced soon
after infection, before the development of CPE
• Specimen are inoculated onto shell vial monolayer by
low speed centrifugation to increase viral infectivity
• Disadvantage – only one type of virus can be detected
End of Chapter 5