Virology (1,2, 3)

Dr. Tarek Mahbub Khan
MBBS, M.Phil
Senior Lecturer
MD-UKM-AUCMS
1
Copy right: Dr.Tarek Mahbub Khan/11.12.2013
Corespondence: 0111-4429185

LEARNING OBJECTIVES
 At the end of session , students will be able to:
 Appreciate the history in the research of virology
 Describe important characteristics of virus
 Differentiate virus from other microorganism
 Describe the effect of physical and chemical
agents on virus
 Outline viral growth medium
 Describe atypical virus like substances
 Explain the difference between virus and prions
2

History of research on viruses
 1884: Charles Chamberland a colleague of Louis
Pasteur design a porcelain filter to sterilize drinking
water
 1892: Iwanowski a Russian scientist showed that
such a filter allowed passage of infective agent
 1898-1899: Beijernick observed that the agent
multiplied only in dividing cells and it withstood
desiccation but inactivated by boiling.
3

 1898: Loeffler and Frosch, both associates of Robert
Koch, reported passage of the agent of foot and mouth
disease and ruled out the idea of an inert toxin,
concluded that-
The agent must be able to replicate
It must be smaller than the smallest bacterium that
was beyond the resolving power of the microscope
at that time
4

 1887: Buist visualized one of the largest vaccinia
virus
 1906-1907: Harrison devised the first tissue
culture not for propagating virus but for studying
the growth of frog nervous tissue in clotted lymph.
 1913: Steinhardt, Israeli and lambert exploited
Harrison technique to grow vaccinia in fragments
of guinea pig cornea embedded in clotted plasma
5

 1928: Maitland and Maitland propagated vaccinia in
suspensions of minced hen’s kidneys
 1931: Woodruff and Goodpasture found that fowlpox
virus inoculated on to chorioallantoic membrane of
10-15 days embryos produce discrete lesion (pocks)
6

 1946: Beveridge and burnet adopt the method of
inoculation through allantoic and amniotic routes to
grow a range of viruses and used to grow yellow
fever and influenza virus in large quantities for
vaccine production
And later, flask cultures of trypsinized fragments of
monkey kidney were used to grow polio virus on a
large scale for vaccine production
7

The discovery of animal and plant viruses were
soon followed by finding of others that affected
insects and bacterias
1908-1911: Oncogenic viruses were discovered by
Ellerman, Bang and by Rous who respectably
showed that a leukemia and a sarcoma of fowls
could be transmitted by filterable agents
8

 1915-1917: Twort and d’Herelle proved bacterial
viruses and their importance in the study of viral
replication and bacterial genetics
 1929: Elford describe a method of making series of
collodion membrane filters of known average pore
diameter (APD)
 Thus by 1940 the sizes of about 25 animal viruses
ranging from enterovirus (20nm) to pox viruses
(250nm) had been measured
9

 1939: Kausche, Pfannkuch, Ruska visualizes a
virus with newly invented electron microscope
 After second world war several techniques were
adopted to visualize virus structure in details-
Shadow-casting technique- viral morphology
Negative staining- demonstrated that the outer
surfaces of virus particles are composed of
subunits
10

 1935: Stanley suggested that viruses are
composed of proteins
 1937: Bawden and Pirie claimed that viruses are
not compose of pure protein only but also contain
nucleic acid.
 1956 : Crick and Watson suggested that viral
nucleic acid is protected by a shell of identical
protein subunits
11

 1960-1962: Klug and Caspar mention that viral
nucleic acid being with few exceptions, double
stranded DNA or single stranded RNA
 1962: Following terminology were proposed-
Capsid: Protein shell
Nucleocapsid: Complex of protein shell and
nucleic acid
Virion: The mature nucleocapsid, surrounded in
some viruses by an outer envelope.
12

13

Evolution of viruses
 TWO THEORIES
 Virus derived from DNA and RNA nucleic acid
components of host cell and evolve independently
 Virus may be degenerate form of intracellular
parasite
14

 Virus: Virus are the smallest infectious
agents ranging from 20nm to about 300
nm in diameter and contain only one
kind of nucleic acid either DNA or RNA
as their genome
15

 Virion: A complete virus particle
In some instances this includes
nucleocapsid plus a surrounding envelope
This structure, the virion, serves to transfer the
viral nucleic acid from one cell to another.
16

Important characteristics of virus
 Obligate intracellular parasite
 Viruses are acellular
 Extremely small in size usually beyond the
resolution of light microscope (20-300 nm)
 Have distinctive life cycle inside a living cell
 Posses either DNA or RNA as their
genome
17

Differences between Bacteria, Rickettsiae, Climydiae,
Mycoplasma and virus
Traits Bacterria Rickettsiae Chlamydiae Mycoplasma Virus
-----------------------------------------------------------------------------------------------------------------------
Size 1-3 x 0.4 – 0.8µm 0.25 - 2 µm 200 – 300nm 50 – 300nm 20 – 500nm
Both
DNA & + + + + Ether of two
RNA
Growth
On artificial + - - + -
Medium
Intracellular
Replication - + + - +
18

Differences between Bacteria, Rickettsiae,
Climydiae, Mycoplasma and virus
Traits Bacterria Rickettsiae Chlamydiae Mycoplasma Virus
------------------------------------------------------------------------------------------------------------
Multiplication
By binary + + + + -
Fission
Muramic acid
In cell wall + + + + -
Sensitive to
Anti-bacterial
Agent + + + + -
19

Reaction to physical and chemical agents
 HEAT AND COLD
 Envelop viruses are more susceptible to heat
 Most of the viruses are killed at 500-600 C for 30
minutes
 Well preserved at subfreezing temperature
 STABILIZATION
Can be stabilized with MgCl2
Useful for vaccine production
20

 PH
 Stable between 5.0-9.0
 Viruses are destroyed by alkaline PH :
 RADIATION
 ETHER SUSCEPTIBILITY
 DETERGENTS
 Nonionic: Triton 100
 Ionic: Sodium dodecyl sulphate
 FORMALDEHYDE
 PHOTODYNAMIC INACTIVATION
Reaction to physical and chemical agents
21

Methods of inactivating virus
 STERILIZATION UNDER PRESSURE
 DRY HEAT
 ETHYLENE OXIDE
 GAMMA-RADIATION
 SURFACE DISINFECTANTS:
 Hypochloride
 Gluteraldehyde
 Formaldehyde
 SKIN DISINFECTANTS:
 Chlorohexidine, 70% ethanol
 VACCINE PRODUCTION:
 Formaldehyde, β-propiolactone
22

VIRAL GROWTH MEDIUM
 Grows in cell culture and fertile eggs
 TYPE OF CELL CULTURE:
 Primary cell culture:
 Secondary cell culture :
 Diploid cell lines
 50 passage
 Chromosomes are not altered
 Continuous cell culture:
 Growth in diploid cell or malignant cells
 Indefinite passage
 Altered chromosome
23

Inoculation in fertile eggs
24

Cell culture
25

Difference between virus and prions
Virus Prions
Protein particle contain
nucleic acid
Protein encoded by viral
gene
Inactivated by UV radiation
or heat
Icosahedral or helical
symmetry under EM
Infection induces
protective antibody
Protein particle does not
contain nucleic acid
Protein encoded by cellular
gene
Not inactivated by UV
radiation or heat
Filamentous or rod shape
under EM
Does not induces
protective antibody
26

 Defective virus: Composed of viral
nucleic acid and protein but can not
replicate with out the help of helper virus
 Pseudovirions: Contain host cell DNA
instead of viral DNA within capsid
 Viroids: Contain single molecule of
circular RNA with out protein coat or
envelope
 Prions: Infectious protein particle
27

28

MBBS, M.Phil
Senior Lecturer
MD-UKM-AUCMS
29

LEARNING OBJECTIVES
 At the end of the session, students will be
able to:
 Describe the basic structure of virus
 Conceptualize viral symmetry
 Explain the clinical significance of envelop and non-
envelop virus
 Describe structure and functions of various viral
proteins, both structural and functional protein
 Correlate viral structure in their classification
 Appreciate the concept of positive sense and negative
sense virus
30

Influenza virus
31

Structure of virion
 Naked virus
Composed of nucleocapsid as infectious viral
particle.
Nucleic acid is either of RNA or DNA
Capsid is composed of viral structural protein
encoded by viral gene.
Naked viruses are released by disintegration or
destruction of host cell
32

Structure of virion
Capsid has the following function
1. Protection of fragile nucleic acid
2. Antigenicity
3. Adsorption of viral particles on the
specific sites of host cells
 Capsomeres: These are spherical morphological
units of capsid consists of polypeptide responsible
for different geometric symmetry of virus
33

Structure of virion
34

Structure of virion
 Envelope virus:
Viral nucleocapsid is surrounded by a
lipoprotein membrane
Lipoprotein membrane derived either from
nuclear membrane or cytoplasmic membrane
of host cell
So envelope viruses are released by the
process of budding
Peplomers: Are virus encoded glycoprotein
spikes present on the viral envelope.
35

Structure of virion
36

Clinical importance of envelope virus
 Fusion of virus with host cell membrane- Some
antiviral fusion blocker drugs are effective in
preventing viral infection to occur
 The envelope viruses are more sensitive to heat,
detergent and lipid solvents such as alcohol and
ether
 Produce neutralizing antibody-Important as a
diagnostic tool for detection of disease and
immune status
37

Morphological structure of virus
Size: In virology the unit of measurement is taken as
nanometer (nm)
1 nm = 10-9 m =10-3 µm
Resolution power of-
Electron microscope: 0.3 nm – 200 nm
Light microscope: > 200 nm
Example
Picorna virus: 20- 40 nm
Staphylococcus: 1000nm
38

 Viral symmetry
Arrangement of capsomere gives two types of viral
Symmetry and a complex structure-
1. Icosahedron symmetry: The capsomeres are
arranged in 20 equilateral triangles with 12
vertices that form a cubic symmetric figure
( Icosahedron)
Example: Adenovirus, Herpes virus
2. Helical symmetry: Capsomeres are arranged
in a hollow coil that appears rod shaped
Example: Paramyxovirus, Rhabdovirus
3. Complex structures: Pox viruses
39

40

41

Viral Proteins
Viral proteins are of two types-
1. Structural proteins: Capsid proteins and matrix
Proteins
2. Functional proteins: These are the viral
enzymes helps in the process of viral
replication.
Example: RNA polymerase, Reverse transcriptase
42

Viral Proteins
 Function of viral proteins:
1. Protect the viral genome
2. Participate the viral attachment to susceptible
host cell
3. Provide structural symmetry of the virus particle
4. Facilitate transfer of viral nucleic acid from one
host cell to another
5. Determines antigenic characteristics of virus
6. Helps in viral replication
43

Viral glycoprotein
 Viral envelope contains glycoproteins
 It is virus encoded
Functions:
1. Attaches the virus particle to target cell by interacting
with a cellular receptor
2. Involve in membrane fusion steps in viral infection
3. Acts as surface antigen and involve in viral
neutralization with neutralizing antibody
Example: Hemaglutinin, neuraminidase glycoproteins of
influenza virus
44

Glycoproteins of HIV
45

Viral
nucleic acid
Viral DNA
ds DNA ss DNA
Viral RNA
ds RNA ss RNA
+ss RNA
- ss RNA
Un segmented
Segmented
Viral nucleic acid
46

Viral nucleic acid(DNA)
47

Viral nucleic acid
 Positive sense RNA: Viral RNA that acts as mRNA
Is infectious RNA
Example: Picornavirus, Retrovirus
 Negative sense RNA: Viral RNA is complimentary to mRNA
Viral nucleic acid is not infectious
Virions contain RNA polymerase
Example: Rhabdoviruses, Paramyxoviruses,
 Ambesense : Some genes have both + and - polarity
48

5'CGCTATAGCGTTTCAT 3'
DNA antisense strand
(template/noncoding), Watson strand
Used as a template for transcription.
3'GCGATATCGCAAAGTA 5'
DNA sense strand
(nontemplate/coding), Crick strand
Complementary to the template
strand.
3'GCGAUAUCGCAAAGUA 5' mRNA Sense transcript
RNA strand that is transcribed from the
noncoding (template/antisense)
strand. Note1: Except for the fact that
all thymines are now uracils (T-->U), it
is complementary to the noncoding
(template/antisense) DNA strand
(identical to the coding
(nontemplate/sense) DNA strand).
Note2 There is an AUG start codon at
the 5' end (although written backwards
here).
5'CGCUAUAGCGUUUCAU 3' mRNA Antisense transcript
RNA strand that is transcribed from the
coding (nontemplate/sense) strand.
Note: Except for the fact that all
thymines are now uracils (T-->U), it is
complementary to the coding
(nontemplate/sense) DNA strand
(identical to the noncoding
(template/antisense) DNA strand.
49

50

MBBS, M.Phil
Senior Lecturer
MD-UKM-AUCMS
51

LEARNING OBJECTIVES
 At the end of the session, students will be
able to:
 Describe the steps of viral replication
 Explain the mode of replication of different RNA and
DNA viruses
 Explain the formation and use of different viral proteins
in replication
 Appreciate the strategy of viral genome replication
 Describe viral growth curve
 Explain eclipse period and latent period with the
clinical importance
52

Steps of viral replication
53

A. Attachment
 Viral attachment: Interaction of virions
with specific receptor site on cell surface
Example: HIV binds to CD4 receptor on
cells of immune system
54

B. Penetration
 Penetration: Process of taking up of
viral particle inside the host cell
 Can occur in several mechanism-
1. Receptor-mediated endocytosis
2. Direct penetration
3. Fusion of viral envelope with cell
membrane
55

C. Uncoating
 Uncoating: It is the physical separation
of viral nucleic acid from outer structural
component of virion
Favor by low PH of endosome
56

D. Early transcription, translation
genome replication
Viral nucleic acid Viral mRNA
1
2
Functional proteins
(Enzymes)
3
Viral genome replication
57
1 Early Transcription
2 Early Translation
3 Viral genome replication

Formation of viral mRNA
DNA genome mRNA
Cellular DNA-depended RNA polymerase
+ Sense RNA mRNA
Act as
- Sense RNA mRNA
Viral RNA depended RNA polymerase
58

Transcription of messenger RNA
59

Some important points in genome replication
 DNA viruses replicate in the nucleus
Exception: Pox viruses
 Most RNA viruses replicate in the host cell
cytoplasm
Exception: Retroviruses, Influenza viruses
60

E. Late transcription, translation
1
Viral gene expression Late mRNA
2
Late protein
(Structural: eg. Capsid)
61
1 Late transcription
2 Late translation

F. Morphogenesis and release
 Progeny viruses: Newly synthesized
viral genomes and capsid polypeptides
assemble together to form progeny
viruses
 Released by-
1. Budding: Envelope viruses
2. Cell lyses: Naked viruses
62

Steps of viral replication
63

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Virology (1,2, 3)

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