INTRODUCTION TO

VIROLOGY
Department of Microbiology
SRMC & RI
Chennai
 General property of viruses
• Do not posses a cellular organization
• Contain only one type of nucleic acid and never both
• Obligate intracellular parasites
• Lacks enzymes necessary for protein and nucleic acid
synthesis and dependent on host synthetic machinery
• Multiply by complex mechanism and not by binary fission
• Unaffected by antibacterial antibiotics

• ‘Virus’ name came from the Latin word for 'poison'

 History
• Perhaps the first written
record of a virus
infection consists of a
heiroglyph from
Memphis, the capital of
ancient Egypt, drawn in
approximately 3700 BC,
which depicts a temple
priest called Ruma
showing typical clinical
signs of paralytic
poliomyelitis.
Pharoh Ramses V, who died in 1196 BC, is believed to
have succumbed to smallpox - compare the pustular
lesions on the face of the mummy & those of more
recent patients.
 Morphology
• The extra cellular infectious virus particle is called
“virion”

• Smaller than bacteria,its filterability through bacterial

filters led to their recognition

• Cannot seen under light microscope

(ultramicroscopic),except few (small pox v)

• Size varies from to 20 to 300 nm

 Estimation of size
• Passing the virus particle through colloidon membrane filters of
graded porosity – old method

• Can be determined from the rate of sedimentation of virus

particle by ultracentrifuge

• Directly we can measure the size of virus by electron

microscopy, both size and shape of virus can be determined
Structure and shape
• Virion consist of a nucleic acid
surrounded by protein coat,
CAPSID
• Capsid with enclosed nucleic acid is
called NUCLEOCAPSID
• Capsid is composed of large number
of CAPSOMERS
• Capsid adsorbs readily into host
cells and introduces the viral
genome
• Shape of the capsid is either
HELICAL (ROD shaped) or
ICOSAHEDRAL (CUBICAL
shape), Pox v is complex shaped
 Structure and shape…..
• Virions may be NON ENVELOPED or ENVELOPED (outer
covering),derived from host cell membrane
• Envelop is lipoprotein in nature, protein subunits (virus coded)
seen as projecting spikes, called PEPLOMERS
• A virus may have more than one type of peplomers
(Hemagglutinin and Nuraminidase in Influenza virus)
• Enveloped has antigenic and biological properties and they are
susceptible to lipid solvents like soap, ether, chloroform and
bile salts
 Resistance
• Heat labile, inactivated within seconds at 57ºC, minutes at 37ºC
and days at 4ºC
• Long term storage is by lyophilisation (except polio v)
• Some are resistant to acidity (entero v) but all are susceptible to
alkaline PH
• Generally inactivated by sunlight, UV and ionizing radiation
• Phenolic disinfectants are only weakly virucidal
• Formaldehyde, beta propriolactone and chlorination are actively
virucidal
 VIRAL PROTEINS AND ENZYMES
• Structural proteins are essential for the formation of viral
particles.

• Enzymes are essential for the formation of viral replicator

cycle.

• DNA polymerase, DNA dependent RNA polymerase,

RNA dependent RNA polymerase, protease, endonuclease.
 VIRAL REPLICATION

• Viruses utilize the biochemical machinery of the host

cell, stages of replications are:
• Adsorption
• Penetration
• Uncoating
• Biosynthesis
• Maturation and Release
 Nucleic Acid
• DNA or RNA
• Single stranded or double stranded
• Segmented or non segmented genome
• Depending upon the mRNA transcription, RNA
viruses may be POSITIVE sense or NEGATIVE
sense
• In positive strand or sense RNA viruses, the viral
RNA itself acts as the mRNA
 Cultivation of virus
• As they are obligate intracellular parasites they
cannot grow in inanimate culture medium
• Three methods are employed for cultivation:

i. animal inoculation
ii.chick embryo (egg) inoculation
iii. tissue culture
 Animal inoculation
• The earliest culture medium for cultivation of viruses causing
human disease were MAN
• Then Monkeys are employed esp., for Polio virus, not routine
because of cost and risk involved
• Theiler (1903) introduced white mice, which extended scope of
animal inoculation
• Mice (suckling mice for Coxsackie and Arbo V) are still the
most widely used animal in virology
• Mice can be inoculated intracerebral, subcutaneous,
intraperitoneal and intra nasal
• Other animals are Pigs, Rabbits, Ferrets etc.,
 Embryonated eggs
• 8-11 days old hens or ducks egg used, incubation is for 2-9 days
• Offers several sites for the cultivation
• Inoculation in chorioallantoic membrane(CAM) produces
visible lesions (Pocks)
• Pock morphology differs with virus and counting of pocks used
for assay
• Inoculation in allantoic cavity results in rich yield of Ortho and
paramyxo V
• Yolk sac can be used for cultivating JE,Toga V later cause death
of embryo
 Tissue culture
• First application of tissue culture in vitro was to maintain
Vaccinia V in fragments of rabbit cornea
• Bacterial contamination was major obstacle before Abs available
• Three types of tissue cultures are used:

I. Organ culture- small bits of organs used

II.Explant culture- fragments of minced tissue embedded in
plasma clots
III.cell culture- routinely used for cultivation of viruses
 Cell culture

• Tissues are dissociated into component cell with Trypsin and

suspended in growth medium and fetal calf serum
• Dispensed in bottles, tubes and petri dishes as monolayer
• Cell cultures are classified into 3, based on origin, chromosomal
characters and number of generations they can maintain:
1. Primary
2. Diploid
3.Continuous
Cell culture
 Cell culture…..

• Primary cell culture- normal cells used for primary

isolation of virus, preparation for vaccine, capable of
limited growth-eg., Monkey kidney cell and Human
amnion cell lines, chick embryo cell lines
• Diploid cell culture- has original diploid
chromosomes,can be sub cultured for about 50 times,
eg., Human fibroblasts are used for production of
vaccines
 Cell culture….
Continuous cell lines- cells are derived from cancer cells,
-capable of serial cultivation indefinitely
- cell lines derived from human cancers,
HeLa, Hep-2 and KB used throught the
world
- used for years when stored at -70ºC
-generally not used for vaccine production
except Vero cell lines
 IDENTIFICATION OF VIRUS GROWTH

A. Morphological examination- EM

B. Cytopatic effect (CPE)

C. Neutralization of CPE

D. Haemadsorption / Haemadsorption inhibition test

E..Haemagglutination

F. Direct immunofluorescence
G. Immuno electron Microscopy
 Cytopathic effect

• Morphological changes produced by growing viruses

• Readily observed by microscopy
• Characteristic of each viruses and helps in presumptive
identification
• Rounding, syncytium formation, focal degeneration, grape like
clusters formation and Vacuolization are some of the CPE seen
in cell lines
 Inclusion bodies
• Most characteristic feature of viral infected cell seen under light
microscope
• Has distinct size, shape, location,& staining properties
• May be situated in cytoplasm (Pox V), nucleus (herpes V) or both
(Measles)
• Generally acidophilic and seen as pink structure when stained
with Giemsa or eosin-methylene
• Demonstration helps in presumptive identification (Negri bodies)
• Guarnieri bodies (Vaccinia), Bollinger bodies (Fowl pox),
Molluscum bodies (Molluscum contagiosum, Cowdry type A &B
Negri bodies (Rabies)
• May be crystalline aggregates of virions
 CLASSIFICATION
BASED ON
• Nucleic acid content-DNA or RNA virus
• Morphology
• Presence of envelope
• Nucleic acid symmetry
 DNA VIRUSES

• Herpeviridae
• Poxviridae
• Adenoviridae
• Hepadnaviridae
• Papovaviridae
• Parvoviridae
 RNA VIRUSES
• Orthomyxoviridae
• Paramyxoviridae
• Coronaviridae
• Arenaviridae
• Rhabdoviridae
• Togaviridae
• Flaviviridae
• Picornoviridae
• Retroviridae
 Laboratory diagnosis
• Microscopy:

Electron microscopy-virus
Immuno electron Microscopy
Microscopic demonstration of IB
IFT
 Serology
• Haemagglutination inhibition test
• Complement fixation test
• Immunofluorescence test
• Neutralization test
• Enzyme immuno assay- antibody/antigen
• Western Blot (Immuno blot test )
 DETECTION OF VIRAL GENETIC
MATERIAL
• Nucleic acid probe
• Polymerase chain reaction
• Reverse transcriptase PCR
• Viral assay – Total particle count
Infectious virions assay
• Plaque assay
• Pock assay
 Viriods

• Protein free infectious agent

• Genome much smaller than those of known virus

• Usually has single stranded RNA

• Shown to produces disease in plant

 Prion
• Unconventional, virus like agent
• Proteinaceous infectious particle without any
detectable nucleic acid
• Highly resistant to heat, UV rays and nucleases
• Sensitive to proteases
• Responsible for Scrapie, Kuru, Cruetzfeldt-Jacob
disease and some chronic viral degenerative
diseases