Cyrelle D.

Solijon
BMLS 3B
Mycology and Virology (3:00-5:00PM)

CHAPTER 65
Overview of the Methods and Strategies in Virology

GENERAL CHARACTERISTICS
VIRAL STRUCTURE
Virions these are virus particles.
Consist of two or three parts:
An inner nucleic acid core, consisting of either ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA)
A protein coat the surrounds and protects the nucleic acid which is the capsid
A lipid containing envelope that surrounds the virus in some of the larger
viruses
Naked Viruses viruses that do not have an envelope and are very resistant to
environmental factors and transmitted by the fecal-oral route.
Nucleocapsid used to describe the nucleic acid genome surrounded by a symmetric
protein coat; enclosed in a lipid envelope.
Responsible for viral entry into the host cell.
The function of the nucleic acid genome is to encode the proteins required for viral
penetration, transmission, and replication.

Viral capsid protects the viral genome and is responsible for the tropism to specific
cell types in naked viruses.
Capsomeres associate to form the capsid and a characteristic symmetric structure.

Most common capsid

structures

Helical irregularly shaped capsids

Icosahedral cubical and have 20 flat sides

Viral Proteins:

Hemmagglutinin (HA)
Neuraminidase
Glycoprotein spikes assist in stabilization of attachment for the lipid
envelope and for attachment to the host cell to facilitate viral entry.

VIRAL REPLICATION
The six steps of virus replication, called infectious cycle:
1. Attachment also referred as adsorption, first step. Involves recognition of a
suitable host cell and specific binding between viral capsid proteins and
carbohydrate receptor if the host cell.
2. Penetration a process which viruses enter the host cell.
Syncytia used to determine the presence of virus in cell cultures or stained
smears of clinical specimens.
3. Uncoating a process by which the capsid is removed; this may be degredation
of viral enzymes or host enzyme or by simple dissociation. Release the viral
genome before the viral DNA or RNA is delivered to its intracellular site of
replication in the nucleus or cytoplasm.
4. Macromolecular Synthesis production of nucleic acid and protein polymers.
5. Viral Assembly process by which structural proteins, granules, and in some
cases viral enzymes are assembled into virus particles.
6. Release this occurs after cell lysis or by budding from cytoplasmic membranes.

EPIDEMIOLOGY
Viruses are transmitted from person to person by:

Respiratory
Fecal oral
Sexual contact routes
Trauma or injection with contaminated objects or needles
Tissue transplants
Arthropod or animal bites
During gestation (Transplacental transmission)

THREE CHARACTERISTICS OF VIRAL INFECTION:

Acute viral infection displaying evident signs and symptoms
Latent infection no visible signs and symptoms, but the virus is still present.
Chronic or persistend infection low level virus are detectable and the degree
of visible signs and symptoms varies.
Viremia after local viral infection this occurs, which is a viruses present in the patients
blood.
Secondary viremia occur in variety of tissues, such as skin, salivary glands, kidneys,
and brain tissues.
Auto-immune pathogenesis pathogenic viruses stimulate an immune reaction that
cross-reacts with related human tissue, resulting in damage to host function. This
occurs after the acute viral infecton has resolved.
Oncogenic viruses with the ability to stimulate uncontrolled growth of host cells.

LABORATORY TEST FOR IDENTIFICATION

CELL CULTURE
Host cells, referred to cell cultures (referred to by some as tissue cultures),
originate as a few cells and grow into a monolayer (single confluent layer) on the sides
of glass or plastic test tubes.
The cells are kept moist and supplied with nutrients by keeping them
continuously immersed in a cell culture medium.

Cell cultures are routinely incubated in a roller drum that holds cell culture test
tubes tilted to 5 to 7 degrees while slowly resolve at 35 to 37C.
Bicarbonate buffering system used in the culture medium to keep the cells at
physiologic pH (7.2).
Phenol red a pH indicator that is red at physiologic pH, yellow at acidic pH, purple at
alkaline pH, is added to monitor adverse pH changes.
Once inoculated with specimen, cell cultures are incubated for 1 to 4 weeks, depending
on the viruses suspected.
Cells are inspected microscopically with an inverted light microscope for the presence of
the virus, indicated by areas of dead or dying cells, called cytopathic effect.
QUANTITATIO
N
Negative
Equivocal

INTERPRETATION

Uninfected monolayer
Atypical alteration of monolayer involving
few cells
1% to 25% of monolayer exhibits
cytopathic effects
25% to 50% of monolayer exhibits CPE
50% to 75% of monolayer exhibits CPE
76% to 100% of monolayer exhibits CPE
Quatitation of Cell Culture Cytopathic Effects

1+
2+
3+
4+

Virus-induced CPE also presents two other important considerations

Rate at which CPE progresses

The type of cell culture in which the virus grows may be used for presumptive
identification
Viruses are most often detected in cell culture by the recognition of CPE.
Viruses have distinct CPEs

Flourescent-labeled antisera available for most viruses, used for confirmation.

Acid lability used to differentiate enteroviruses from rhinoviruses.
Neutralization used to identify viruses with many serotypes for which fluorescentlabeled antisera are not available.
Viruses that produce little or no CPE:

Influenza
Parainfluenza
Mumps viruses
Poliovirus and echovirus produce similar CPE in primary rhesus monkey kidney
(RMK) cells, but echovirus does not induce CPE in continuous cell lines, whereas
poliovirus does.

Two kinds of Media:

1. Growth medium is a serum-rich nutrient medium (10%) designed to support
rapid cell growth. This is used to initiate the growth of cells in a tube when cell
cultures are prepared in-house or to feed tubes of purchased cell cultures that
have incomplete cell monolayers.
Feeding refers to the removal of old medium, followed by the addition of fresh
culture medium.
2. Maintenance medium similar to growth medium but contain less serum (0% to
2%) and is used to keep cells in a steady state of metabolism.

Both are prepared with:

Eagles minimum essential medium (EMEM)

Hanks or Earles balanced salt solution (HBSS or EBSS).

HBSS has a better buffering capacity with carbon dioxide.

EBSS has a better buffering capacity in ambient air.

Inuculated cell cultures should be incubated immediately at 35C. Incubation should be

continued for 5 to 28 days, depending on the suspected agent.

Blind passage refers to passing cells and fluid to a second cell culture tube. This is
also used to detect viruses that may not produce CPE in the initial culture tube but
produce CPE when the beefed-up inoculum is passed to a second tube.

VIRAL SEROLOGY

Determine immune status

Confirm diagnosis of infection when the virus cannot be cultivated in cell culture
or detected readily by immunoassay or molecular assays.

Serology was the primary means of laboratory diagnosis of viral infections until
the mid-1970s.

IgM is undetectable 1 to 4 months after the acute infection resolves.

A positive result with a sensitive, virus-specific IgG test indicates past infection.

TWO APPROACHES TO DIAGNOSE ACTIVE DISEASE

Detection of virus-specific IgM in an acute phase specimen
Detection of a fourfold antibody titer
Serologic methods used to detect antiviral antibody

Complement fixation (CF)

ELISA
Indirect immunoassay fluorescence
Anticomplement immunofluorescence
Western immunoblotting used for viral antibody detection

SPECIMEN COLLECTION
Specimen selection depends on the specific disease syndrome, viral etiologies
suspected and time of year. Selection of appropriate type of specimen is one of the keys
to correct test result. Specimens should be collected as early as possible, virus may no
longer be present as early as 2 days after the appearance of symptoms.

Throat, Nasopharyngeal Swab or

Aspirate

Throat swabs are acceptable for the

recovery of enteroviruses, adenoviruses,
and HSV.
This are collected with a dry, sterile swab
by passing the swab over the inflamed,
vesiculated or purulent areas on the
posterior pharynx. The swab should not
be touched to the tounge, buccal
mucosa, teeth or gums.

Bronchial and Bronchoalveolar Washes

Rectal Swabs and Stool Specimen

Urine

Skin and Mucous Membrane Lesions

Sterile Body Fluids Other Than Blood

Bone Marrow

Tissue

Genital Specimens

Serum for Antibody Testing

Nasopharyngeal swab or aspirate

specimen are preferred for the detection
of RSV and influenza and parainfluenza
viruses.
This is collected by inserting a swab with
a flexible shaft through the nostril into the
nasopharynx or by washing and
collecting the secretions by rinsing with a
bulb syringe and 3 to 7 mL of buffered
saline.
Washing and lavage fluid collected during
bronchoscopy excellent for lower
respiratory tract infection.
Rectal Swab is inserted 3 to 5cm into the
rectum and rotated against the mucosa to
obtain feces, this is for detecting
enteroviruses, such as aseptic meningitis.
Stool specimen must collected atleast 5
to 10mL of freshly passed diarrheal stool
and preferred for rotavirus and enteric
adenovirus detection.
Collect 10mL from a clean-catch firstmorning urine.
CMV; mumps, rubella, and measles
viruses; polyomaviruses; and
adenoviruses can be detected.
Collection of specimens from cutaneous
vesicles for detection of HSV or VZV may
require a Tzanck smear if PCR testing is
not available.
This may contain enteroviruses, HSV,
VZV, influenza viruses, or CMV.
These specimens are collected
aseptically by the physician and sent to
the laboratory for processing.
Used to detect CMV.
5 to 10 mL of anticoagulated blood
collected in a whole blood tube is
needed.
Heparinized, citrated, or
ethylenediaminetetraacetic acid (EDTA)
anticoagulated blood is acceptable for
CMV detection.
Specimens are collected during surgical
procedures. Fresh tissue is preferred for
nucleic acid assays, but formalin-fixed
and paraffin-embedded tissue may be
used after removal of the paraffin and
extraction.
Genital swabs should be used for
ulcerations and placed in appropriate viral
transport media. Cervical specimens may
be collected using a swab or brush and
placed in viral transport media.
Acute specimen must be collected as
soon as possible after the appearance of
symptoms. The covalescent specimen is
collected a minimum of 2 to 3 weeks after

acute specimen. In both cases, an

appropriate specimen is 3 to 5 mL of
serum collected by venipuncture.

SPECIMEN PROCESSING
Laboratory Processing of Viral Specimens
SOURCE
Blood
Cerebrospinal fluid (CSF)
Stool or rectal swab

Genital, skin
Miscellaneous
Respiratory Tract
Tissue

Urine

PROCESSING
Separate leukocytes
Inoculate directly
Place 2 mL of viral trasnsport medium
vortex. Centrifuge at 1000x g for 15
minutes and use supernatant fluid for
inoculum
Emulsify in viral transport medium
Emulsift in viral transport medium
Fluid, inoculate directly
Dilute with viral transport medium
Mince with sterile scalpel and scissors
and gently grind. Prepare 20%
suspension in viral transport medium.
Centrifuge at 1000x g for 15 minutes and
use supernatant fluid for inoculum.
Clear: Inoculate directly.
Turbid: Centrifuge at 1000x g for 15
minutes and use supernatant fluid for
inocula.

PREVENTION AND CONTROL

Immunizations are available for some viruses capable of causing disease in
humans. However, for viruses for which there are no available vaccines, the most
effective means of preventing viral infection involves regular, thorough hand washing
and avoiding contact with others during episodes of evident signs and symptoms, such
as fever, cough, diarrhea, and respiratory infections.