This document provides an introduction to medical virology. It defines key terms like clinical virology and virion. It describes how viruses are classified based on their nucleic acid and describes traditional laboratory methods for viral diagnosis like electron microscopy, viral isolation, and serology. It discusses virus growth in tissue culture and preparing samples for scanning electron microscopy. Key equipment for a virology laboratory including laminar flow hoods and cell culture materials are also outlined. The process of inoculating a patient sample onto a cell monolayer and observing for cytopathic effect is described.

1. Medical Microbiology Laboratory
Introduction to Medical Virology
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. TERMINOLOGY
 Clinical or medical virology is a branch of medicine
which consists in isolating and/or in characterizing one or
several viruses responsible for some human pathologies
by various direct or indirect techniques.
 A virus is a small infectious agent that replicates only
inside the living cells of other organisms. Viruses consist
of genetic material (DNA or RNA), capsid and in some
there are an envelop surrounds the capsid.
 Virion, an entire virus particle, consisting of an outer
protein shell called a capsid and an inner core of nucleic
acid (either RNA or DNA).

3. VIRAL CLASSIFICATION
 Viruses could be classified according to the type of nucleic acid
they use as genetic material into:
1. DNA viruses: double-stranded DNA (ds-DNA) viruses, and
single-stranded DNA (ss-DNA) viruses.
2. RNA viruses: positive-sense single-stranded RNA [(+) ss-RNA]
viruses, negative-sense single-stranded RNA [(-) ss-RNA]
viruses, and the much less common double-stranded RNA (ds-
RNA) viruses.
3. Reverse transcribing viruses: double-stranded reverse-
transcribing DNA (ds-RT DNA) viruses and single-stranded
reverse-transcribing RNA (ss-RT RNA) viruses including
retroviruses.

5. The traditional approaches to
laboratory diagnosis of viral infections
have been:
a. Direct Examination-EM
b. Isolation of the virus in tissue
culture
c. Detection and measurement of
antibody in the patient's serum
(serology)

6.  Viruses need a “host” system.
 Viruses can be grown in:
◦ Animals
◦ Embryonated eggs
◦ Tissue (cell) cultures (preferred
method)

7. Scanning Electron
Microscope
Transmission Electron
Microscope

9. The Scanning Electron Microscope, or
SEM, is an incredible tool for seeing the
unseen worlds of microspace.

10. Conventional light microscopes use a series
of glass lenses to bend light waves and
create a magnified image.

11. The Scanning Electron Microscope
creates the magnified images by using
electrons instead of light waves

12. The SEM shows very detailed 3-
dimensional images at much higher
magnifications than is possible with a
light microscope. The images created
without light waves are rendered black
and white

16. Samples have to be prepared
carefully to withstand the vacuum
inside the microscope.

17. Now the prepared specimen is ready.

18. The sample is placed inside the microscope's vacuum
column through an air-tight door

19. After the air is pumped out of the column, an
electron gun [at the top] emits a beam of high
energy electrons. This beam travels downward
through a series of magnetic lenses designed to
focus the electrons to a very fine spot

20. Near the bottom, a set of scanning
coils moves the focused beam back
and forth across the specimen, row
by row.

21. As the electron beam hits each spot on the
sample, secondary electrons are knocked loose
from its surface. A detector counts these electrons
and sends the signals to an amplifier.

22. The final image is built up from the number
of electrons emitted from each spot on the
sample

23. The Scanning Electron Microscope is revealing
new levels of detail and complexity in the
amazing world of micro-organisms and
miniature structures

27.  Virologist’s facility
 Laminar vertical flow
hoods
◦ Contains HEPA filter
Removes 99.97% of particles
of 0.3μM or higher
Figure 5-3: Vertical laminar flow hood.

35.  Tissue culture flasks.
 Cell lines: Vero, MDCK, A549, HeLa, McCoy.
 Cell culture medium: Minimum Essential Medium
(MEM).
 10% MEM: Growth Medium
 2% MEM: Maintenance Medium
 Foetal Calf Serum (FCS)

36.  2 Sucrose-Phosphate Transport Medium
(2SP)
 Trypsin
 Double Strength R.Q. Water
 Phosphate Buffered Saline (PBS)
 Note: 2 causes of contaminating tissue
culture:
 1. Aspergillus sp. and 2. Mycoplasma sp.

37.  A throat swab is taken from patient, and put
into 2SP transport medium and sent to the
laboratory.
 On arrival at laboratory, it is vortexed
vigorously to release virus from swab.
 The cells were grown and now are in
maintenance medium for a single monolayer

38.  Remove the maintenance medium and add
2ml of growth medium.
 Inoculate 0,2-0,5ml of the specimen
containing virus.
 Place in a 37˚C incubator with 5-10%CO2,
leaving the lid slightly open.
 Examine flasks daily for seven days for
presence of CPE using Inverted Microscope.

42. MONOLAYER OF CELLS

43. Near-confluent monolayer of
precapillary endothelial cells

44. Uptake of fluorescently-labelled
acetylated low-density lipoprotein by
precapillary endothelial cells

45. CYTOPATHIC EFFECT ( CPE )

49. Figure 5-19a
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual,
Third Edition . ASM Press, 2000.
Figure 5-19b

53. © Hank Morgan/Science Photo Library/Photo Researchers, Inc.
Figure 5.20: HIV ELISA
test.