2. John Franklin
Enders
• Known for polio viruses
• Classified viruses on the
basis of morphologic effects
on cells.
"The Father of Modern Vaccines."
Nobel laureate
3. Virus
NOT considered as cells
• Small, obligate intracellular parasites
• Strucure – DNA/RNA and protein coat
• Mobile genetic elements
• Virion - Function
5. Cytopathic effect(CPE)
• Morphological changes in cells caused by viral
infections; the responsible virus is said to be
cytopathogenic
• Degree of CPE depends on
o Virus and host type
o Multiplicity of infection (MOI)
o Some viruses do not cause CPE – Interference/ hemadsorption
• Application- Virus identification, unknown virus
6. How virus infects
• Live inside cells- avoid immune response
• Attachment receptors- DOCKING
• PENETRATION- cytoplasm
• BIOSYNTHESIS of viral components
• MATURATION
• RELEASE
7. Sampling
• What types of specimens
are
collected to diagnose?
• Respiratory tract
infections:
Nasal
and bronchial
washings, throat
and nasal swabs, sputum
• Eye infections: throat and
• conjunctival
swab/scraping
• Gastrointestinal tract
infections:
stool and rectal swabs
• Vesicular rash: vesicle
fluid, skin
Scrapings
• Maculopapular rash:
throat, stool,
and rectal swabs
• CNS (encephalitis and
meningitis
cases): stool, tissue,
saliva, brain biopsy,
cerebrospinal fluid
• Genital infections:
vesicle fluid or
Swab
• Urinary tract infections:
urine
• Bloodborne infections:
blood
8. Techniques of virus study
• Direct
1. Electron microscopy/ Immuno-electroon microscopy
2. Light microscopy
3. Ag detection, molecular methods etc..
• Indirect
1. Cell culture
2. Egg pocks on chorio-allantoic membrane
3. Animal disease/death
9. • Find suitable host – primary, semi-continuous,
continuous cells
• Host grown as MONOLAYER on a solid surface
• Infect cell with virus of interest- ONE VIRION/cell
• Circle of death- easily counted and quantified
13. • Requires experience to use CPE as diagnostic tool
• Virus may not conform to norm for its family
• CPE best viewed by daily observation of cultures
admixed with low MOI (<0.1)
• Normal cell ageing to be distinguished
• Rate of CPE appearance- characteristic
• General rule : Slow virus- 4-5 days
• Rapid – 1-2 days for CPE to appear
14. Types
1) Total destruction
o Of all monolayer-
most severe
o Cells shrink rapidly,
become pyknotic
o Detach from glass
within 72 hrs
o Typical of
ENTEROVIRUSES
20x objective
within 24 hrs
Bovine fetal spleen cells
15. 2) Subtotal
destruction
• Some cells dead/detached
• Togavirus
• Some picornavirus
• Paramyxovirus
20x objective
Bovine fetal
spleen cells 2
days
postinfection
Rhabdovirus
16. 3) Focal
degeneration
• Herpesvirus, Poxvirus
• Localised foci of infection
• D/t cell to cell virus trasnfer
rather than extracellularly
• Enlarged, rounded, refractile
cells
• Strangling of cytoplasm
• Cell fusion
Bovine fetal spleen cells 2 days postinfection, Herpesvirus
Black arrows - cell rounding in a focal pattern.
Blue arrows -cytoplasmic stranding.
20x objective . IMP: view at a low MOI
17. 4) Swelling and
clumping
• Adenovirus
• Cells greatly enlarge and
clump together in ‘grape-
like’ clusters
Bovine fetal spleen cells 4 days postinfection with a high MOI
of the bovine adenovirus, an Adenovirus, showing cell
rounding and small amounts of clumping.
10X objective
18. 5) Foamy
degeneratioon
• d/t large/numerous
cytoplasmic vacuoles
• Retrovirus
• Paramyxovirus
• Flavivirus
• Difficult to see without
staining
Giemsa-stained bovine fetal spleen cells 1 day
Flavivirus, showing vacuoles (arrow).
40X objective
19. 6) Cell fusion
• Syncytium/polykaryon
• Plasma membranes of 4 or
more cells fuse
• Enlarged cell with 4 or more
nuclei
• Easily seen when stained
• To be distinguished from cell
clumping/clustering
• Paramyxovirus
• Herpesvirus
Giemsa-stained bovine fetal
spleen cells 4 days
postinfection with the
bovine respiratory syncytial
virus, a Paramyxovirus,
showing syncytia (arrows)
and faint basophilic
cytoplasmic inclusion
bodies (dashed arrows).
20x objective
20. 7) Inclusion bodies • Areas of altered cell staining
• CANNOT be seen in live cell
cultures
• Single or multiple- depends on
virus
• Large/small
• Round/irregular
• Intranuclear/intracytoplasmic
• Eosinophilic/basophilic
• Chromatin margination
• Indicate areas of viral protein
synthesis or nucleic acid synthesis
or virion assembly
• Some cases no virus is present
Viral scarring
Herpesvirus, 40x.
22. Poxvirus, showing pink eosinophilic cytoplasmic
inclusion bodies (arrows) and cell swelling near the
top of the field, 20x.
Feline nasal turbinate primary cell culture infected
with feline herpes virus-1, a Herpesvirus, showing
chromatin margination (dark ring around the edge of
the nucleus).
23. CPE of Common Viruses
• MEASLES
o dilated skin vessels, edema
o Lymph nodes, lungs
o Warthin-Finkeldey cells
24. • MUMPS
o Parotitis
o Orchitis
o Pancreas
o Rarely encephalitis
Mumps parotitis
Enlarged glands, C/S reddish brown,
Glistening.
Macrophage, lymphocyte infiltration.
Oedematous
25. • HERPES SIMPLEX
o Fever blisters
o Gingivostomatitis
o Genital herpes
o Herpes epithelial keratitis
o Herpes stromal keratitis
o Cowdry type A inclusion bodies
• Pink to purple intranuclear
inclusions
Glassy intranuclear herpes simplex
inclusion bodies
26. • Varicella-Zoster Virus
o Chickenpox
• Intranuclear inclusions
o Shingles/ Herpes zoster
• Reactivates in dorsal nerve root ganglia
• Infects keratinocytes
• Vesicular lesions
27. • Cytomegalo virus (CMV)
o Intranuclear basophilic inclusions
o Clear halo
o Enlarged cells (40 microns)
o “Owl eyes”
o Cellular and nuclear
pleomorphism
28. • Epstein-Barr virus (EBV)
o causes infectious
mononucleosis
o Absolute lymphocytosis (T cells)
o 5-80% are atypical, large
o Multiple clear vaculations in
cytoplasm
o Express CD8
o RS like cell may be seen
29. • HPV- LSIL
o Multinucleation
o Perinuclear halos
o Nuclear enlargement
o Hyperchromasia
(KOILOCYTES)
31. • RABIES
o Negri bodies
o Eosinophilic , sharply outlined
o 2-10 microns diameter
o Cytoplasmic
o RIBONUCLEAR PROTEINS of
Rabies virus.
o Found in
• Pyramidal cells of Ammon’s
horn
• Purkunje cells of cerebellum
32. References
1. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical
Branch at Galveston; 1996. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7627/
2. http://www.microbelibrary.org/component/resource/laboratory-test/2875-cytopathic-effects-of-
viruses-protocols
3. http://classes.midlandstech.edu/carterp/Courses/bio225/chap15/lecture4.htm
4. http://en.wikipedia.org/wiki/John_Franklin_Enders
5. Thomas C. Wright, Jr.; Pathology of HPV infection at the cytologic and histologic levels: Basis
for a 2-tiered morphologic classification system; International Journal of Gynecology and
Obstetrics (2006) 94 (Supplement 1), S22---S31
6. Robbins & Cotran; Pathologic Basis of Disease; 8th edition
