General Virology Obligate intracellular parasites

General Virology Obligate intracellular parasites

2. Viruses
Acellular infectious agents
Obligate intracellular parasites
Containing only one kind of nucleic acid as genome and
protein coat designed to protect the genome.
Other way even simplest microorganisms are cells enclosed
within cell wall, containing both nucleic acid (DNA and
RNA), synthesising their own macromolecular constituent
and multiply by binary fission

3. •Viruses are twilight zone that separates the living from
the non-living
•Viruses could be crystallised like chemicals
•Infectious nucleic acid form a virus that could infect the
host cells and yield complete virus progeny made it
appear that viruses were only “living chemicals”

4. Viruses
vs. cellular organisms
Viruses
•Simple organization
•DNA or RNA but not both
•Lack enzymes for protein &
nucleic acid synthesis & so
unable to reproduce outside
living cells
•Obligate intracellular
parasites
Cellular Organisms
•Complex organization
•Both DNA and RNA
•Carry out cell division
•Some are obligate
intracellular parasites

5. D
EFINITIONS:
•Virus – viruses are the smallest obligate intracellular
infectious agents containing only one type of nucleic acid
either DNA or RNA
•Virion - Physical particle of the virus
•Core - Nucleic acid and tightly associated proteins within the
virion
•Capsid - Protein shell around NA or core
•Capsomere - Protein subunit making up the capsid
•Nucleocapsid - Core and capsid

6. •Envelope - Lipid membrane found on some viruses, often
derived by budding from infected cells.
•Peplomer - ("spike”)- Morphological unit projecting
from the envelope or surface of a naked virion
D
EFINITIONS:

7. Morphology
SIZE AND SHAPE
•Size of viruses ranges from 20 to 400nm in diameter.
•Largest being smallpox virus (400nm) as large as
smallest bacteria mycoplasma.
•Smallest virus is parvovirus(20nm).
•They too small and cannot be seen by light microscope
•They ultramicroscopic.

8. SIZE AND SHAPE
Size demonstration by
•Passing through colloidon membrane of graded porosity
•Ultracentrifuge: Rate of sedimentation
•Electron microscope: Direct method

9. SIZE AND SHAPE
Shape of the viruses varies in different groups
•Animal viruses roughly spherical
•Pox virus is rectangular or brick shaped
•Rabies virus is bullet shaped
•Tobacco mosaic virus is rod shaped
•Some are irregular and pleomosphic shape.

10. STRUCTURE
Simplest naturally occurring infective virion consists of a
nucleic acid genome of either RNA or DNA but not both,
packaged into a protein coat (capsid)

11. GENOME
•It contains either DNA or RNA
•Nucleic acid exist
– Single strand as in RNA and double strand as in DNA
–Single segment or several segments( segmented genome as in
some RNA)
•Both ssRNA and dsDNA genomes are seen in viruses of clinical
significance.
•Genome can be linear or circular ( most of viruses possess linear
genome except papova virus contain supercoiled circular genome.

12. There are two major structures of viruses called the naked
nucleocapsid virus and the enveloped virus.

13. CAPSID
•Protein shell or coat
•That encloses the nucleic acid genome
•Viral capsides composed of varying numbers of protein
subunits – capsomeres
•Each capsomeres made up of one or more polypeptide chains –
protomers ( structural unit)
•Protomers may be monomeric (made of single repeating
polypeptide chain) or polymeric (composed of six different
polypeptide chain)

14. C
APSID
C
APSID
Capsid symmetry
Arranged symmetryically
around the NA core
Two types of capsids
Helical
Icosahedral

15. Icosahedral (cubical) :
•It is a polygon with 12 vertices or corners and 20 facets or sides
•Each facets is in shape of an equilateral triangle.
•Two types of capsomeres
–Pentagonal capsomers at the vertices (pentons)
–Hexagonal capsomers making up facets (hexons)
•They are always 12 pentons but number of hexons varies with
the virus group.

16. Helical (rod shaped or coiled)
•Capsomers and nucleic acid are wound together to form
a helical or spiral tube.
•Tube is rigid in Tobacco mosaic virus.

17. C
omplex viruses
•Structure is more intricate than helical and icosahedral viruses
•Pox virus
–Several layers of lipoproteins
•Bacteriophage
–Polyhedral head
–Helical tail
–Fibers for attachment

18. Func
tions of the capsid
Protect nucleic acid from the host’s acid and protein-
digesting enzymes
Assist in binding and penetrating host cell
Stimulate the host’s immune system

19. E
nvelope
•Virions may be enveloped or non-enveloped (naked)
•Envelope or outer covering is derived from the host cell
membrane.
• It is made up of Lipid and proteins (lipoprotein)
•During release of animal viruses, a part of the host membrane is
taken
•Proteins from viral
•Lipids from host

20. •Envelope spikes made of proteins (peplomers) eg:- influenza
virus (hemagglutinin & neuraminidase)
•Enable pleomorphic shape of the virus
- Spherical, Filamentous
•Envelopes confer chemical, antigenic and biochemical
properties on viruses.
•Enveloped viruses are susceptible to lipid solvents (ether,
chloroform & bile salts)
•Biological properties such as attachment to host cell surface or
hemagglutination depend on the envelope

21. Specific viral neutralization by Ab to surface antigens
Attachment to host cell surface
Hemagglutination
Func
tionS of the envelope

22. Chemical properties
Nucleic acid
•Viruses contain only one type of nucleic acid, either single or
double-stranded DNA or RNA
Proteins
•Viruses also contain proteins which make up capsid
•Viral protein protecting the nucleic acid also determine the
antigenic specificity of the virus.
Lipid - Enveloped viruses contain lipids derived from the host
cell membrane.

24. Resistance
•Heat sensitivity
–Protein denaturation (enveloped viruses more sensitive), 56
o
C
in seconds & 37
o
C in mins & at 4
o
C in days. Can be stored
for long time at -70
o
C & freeze drying/ lyophilisation/
cryodesiccation.
•pH sensitivity
–Viruses vary greatly in their resistance to acidity.
–All viruses are disrupted under alkaline condition.

25. •Lipid solvents ( ether, chloroform and bile salts)
–Enveloped viruses generally more sensitive
–Naked viruses resistance
•Chemicals
–React with amino acids of proteins, but some
inactivate DNA or RNA. Eg;- H2O2, hypochlorites &
KMnO4.
•Humidity
–Different viruses respond differently

26. Viral
Classification :
26
Based upon:
Tropism / affinity to different systems or organs of the body
Genome (RNA, DNA {SS, DS} etc)
Morphology of virion, envelope
Replication strategy
Serological relationships (Serotypes)
International Committee onInternational Committee on
Taxonomy of VirusesTaxonomy of Viruses

27. Tropism / affinity to different systems or organs of the body
•Dermotropic:
–Affinity to skin
–Smallpox, chickenpox, measles
•Neurotropic:
–Affinity to CNS
–Poliomyelitis, rabies
•Pneumotropic:
–Affinity to Respiratory system
–Influenza, common cold
•Viscerotropic:
–Affinity to viscera
–Yellow fever, hepatitis

28. Viral
Genomes
28

29. Examples of medically important DNA viruses.

30. Examples of medically important RNA viruses.

31. REPLICATION OF VIRUSES
•Viruses multiply only on living cells
•Due to lack of biosynthetic enzymes
•Depend on the synthetic machinery of the host cell for
replication.

32. Viral multiplication cycle can be divided into six sequential
phases.
–Adsorption
–Penetration
–Uncoating
–Biosynthesis
–Maturation
–Release

33. R
eproduction of Viruses

34. Viral li
fe cycles

• Virulent – These viruses lyse (kill) their host cell after
infection.
• Temperate – These viruses can replicate their genome
along with the host cell genome without killing the host cell.
These viruses are also capable of lysing the host cell

35. 1.
Adsorption of Virions
•Virion come in contact with cell by random collision
•Viral surface proteins and/or enzymes mediate attachment to
specific host receptors
•Infectious nucleic acid can infect without adsorption by injection
into the cell.

36. 2. Pene
tration
•Bacteria posses rigid cell walls
•So only nucleic acid enters host cell
•Animal cells do not have rigid cell walls and whole virus
can enter into host cell
•One of two mechanisms used by most viruses
–fusion of envelope with host membrane release
nucleocapsid into the cytoplasm.
–Endocytosis (viropexis)( virus particles engulfed)

37. 3.
Uncoating
•It is a process of stripping the virus of its outer layers and capsid
so that nucleic acid is released into the cell.
•Most steps of uncoating take place within the cell
•Uncoating with the help of host lysozomal enzymes.
•But some complex viruses required 2 steps . Ex : Poxviruses
•1
st
step – outer coat is removed by lysosomal enzymes in the
phagocytic vacuole.
•2
nd
step – inner core effected by viral incoating enzyme and
liberate internal protein and NA into the cytoplasma.

38. Penetration of animal viruses occur by endocytosis or
fusion between the viral envelope and the host cell
membrane.
Two principal means by which animal viruses penetrate.

39. 4. BIOSYNTHESIS
•Synthesis of viral nucleic acid and capsid protein.
•Along with enzymes necessary in the various stages of viral
synthesis, assembly and release.
•Regulatory proteins – to shut down normal cell metabolism
•Viral DNA replication - usually occurs in nucleus except
pox virus
•Viral RNA replication - usually occurs in cytoplasm except
myxovirus & retrovirus

40. Biosynthesis consists of the following steps;
Transcription of mRNA from viral nucleic acid
Translation of mRNA into early proteins
Replication of viral nucleic acid
Synthesis of structural proteins which are the
components of daughter virion capsids

41. Critical step is the transscription of mRNA from the viral
nucleic acid.
Once its achieved the host cell resources can be utilised
for translating m RNA into viral components.
Viruses have be categorised into six classes Baltimore
based on their replication.

42. mRNA
ds DNA
ss DNA
ds DNA
ds RNA
+ sense ss RNA
Acts as
- sense ss RNA
ss RNA
RNA:DNA
hybrid
ds DNA
Bal
timore classification
Bal
timore classification
Pathway of transcription of viral mRNA
Reverse transcriptase
CLASS 1
(Adeno, Herpes, Papovaviruses)
CLASS 2
(Parvovirus)
CLASS 3
(Reoviruses )
CLASS 4
(Picorna, Togaviruses )
CLASS 5
(Rhabdo,
Othomyxo,
Paramyxoviridae)
CLASS 6
Retroviridae
Viral polymerase

43. 5.
Maturation
•Assembly of daughter virions fallows the synthesis of viral
NA and protein.
•Capsid proteins - encoded by late genes
•Assembly of viruses
–Empty procapsids formed then nucleic acid inserted
–Assembly take place in host cell nucleus and cytoplasma.
•Site of morphogenesis varies
- Nucleus: DNA viruses except Pox viruses
- Cytoplasm: RNA viruses except myxo & retro viruses

44. 6. Virion
Release
In bacterial viruses progeny release by lysis of host cell
In animal viruses release usually occurs with out cell lysis.
Myxoviruses are released by a process of budding from the cell
membrane (host cell unaffected and may divide, doughter cells
continuing to release virions in sourrounding medium and may
infect other cells)
•In varicella , transmission occurs directly by cell to cell
•In polio virus cause profound damage to the host cell and may
release by cell lysis.

45. Eclipse phase
•From the stage of penetration till the appearance of mature
daughter virions the virus can not be demonstrated inside the
host cell this period during which the virus seems to be
disappear is known as eclipse phase.
•Time taken for a single cycle of replication is about
–15-30 min for Bacteriophages
–15-30 hours for animal viruses.

47. A
bnormal replicative cycles
•Incomplete viruses: Defective assembly (Influenza virus)
von Magnus phenomenon: Virus yield with high
hemagglutinin titre but low infectivity.
•Pseudoviruses: Capsid enclosing host cell nucleic acid
•Abortive infection: Infection of non permissive cells
•Defective viruses: Genetically defective. Yield of progeny
virions occurs only if the cells are simultaneously infected with a
helper virus, which can suppliment the genetic deficiency. Eg:-
Rous sarcome virus & avian leukosis virus, HDV & HBV, Adeno
associated satellite virus & adeno virus.

48. VIRAL PATHOGENESIS

49. •Pathogenicity – (“patho”-pain) : ability to cause disease.
•Pathogen : organism able to cause disease
•Pathogenesis : means by which organism produces disease in
host
•A result of :
–Injury to discrete populations of cells.
–In particular target organs.
–Producing signs and symptoms of disease in a given host.
•Virulence : capacity to produce disease.

50. Pathogenesis of viral infection
•The ways in which viruses produce disease
•Involves virus –host interactions:
–Initiation and spread of virus infections in the body.
–Host immune response
–Effects of viruses at cellular level in tissues and
organs of infected host.

51. Stages in pathogenesis
•Viral entry and replication
•Viral spread and tropism
•Cell injury and clinical illness.
•Host immune response.
•Recovery.

52.    MODES OF TRANSMISSIONS
Horizontal transmission:
Viruses enter the body through one of the body surface
Transmission between person of any age after birth, usually
excluding via the maternal milk.
Exception are viruses transmitted by blood transfusion – no
portal of entry – viruses spread directly to the organ or tissue.

53. 1)
   Horizontal transmission

54. Vertical transmission:
Spread of infection occurs at any stage, from the
development of the ovum up to birth from parent to the
young via the placenta or genital tract.
Transmission via the maternal milk is sometime included.

55. Zoonotic transmission
Viral diseases shared by animals & humans.
(Arboviruses)

56.  

TYPES OF VIRAL INFECTIONS
1)   Inapparent / Subclinical infections
2)   Apparent infections
A.   Acute infection
B.   Persistent infection
a) Chronical infection: HBV
b)  Latent infection: HSV
Varicella--zoster virus

57. TYPES OF VIRAL INFECTIONS
C) Slow virus infection （HIV, Kuru in humans &
Scrapie in sheep)
D) Delayed complication after acute viral infection.
SSPE

58. SPREAD OF VIRUS IN THE BODY
•Infection established at the site of entry - most
viruses spread locally by cell-to-cell transmission
•Some remain localized whereas others spread widely
•Dissemination determined by: pattern of viral release
from polarized epithelial cells.
–released at apical surface remain localized,
–released at basal surface tend to disseminate

59. Spread - dissemination
•First step is transport of virus via efferent lymphatics to
the regional lymph nodes (free virions or virus-infected
phagocytes)
•Then route of dissemination is blood circulation -
potentially carry viruses to any site in the body.
“VIRAEMIA”

60. Spread - dissemination
•Viraemia - sources of viraemia.
–efferent lymphatic flow
–shed from infected endothelial cells
–circulating mononuclear leucocytes.
•Viruses can circulate free in plasma - 'plasma' viraemia
- enteroviruses, togaviruses
•Viruses may be associated with cells ('cell-associated‘ viraemia)
- measles, CMV

61. Neural spread
•Some viruses can disseminate by spreading along peripheral
nerves. Eg. rabies and herpesviruses
–entry mechanisms similar to those into other cell types
•nucleocapsid along axons or dendrites, axoplasmic flow.
•After transport, virus may replicate in the perikaryon,
–this is a slower process and not required for neural
spread.

62. •Virus –host interactions

63. Virus-host interaction
1)   Cell level
Lytic infection (Cytocidal infection): Cytopathic effect
( CPE ) Alternation in the Microscopic appearance of cell in
culture following infection with a virus.
Steady state infection: Fusion of cells, new antigen.
Apoptosis
Cell transformation: Alternation in cell morphology and /
or behaviour, …….
Inclusion body: An area of abnormal staining in a virus-
infected cell.
Viral effect on cell

64. Inclusion bodies
Structures with distinct size, shape, location & staining
properties that can be demonstrated in virus infected
cells under the light microscope.
These may be crystalline aggregates of virions or made
up of viral antigens
Some represent degenerative changes produced by viral
infection
Cytoplasm: Poxviruses
Nucleus: Herpesviruses
Both: Measles virus
Helps in diagnosis of viral infection

65. Negri bodies Rabies
Guarnieri bodies Vaccinia
Bollinger bodies Fowlpox
Molluscum bodies Molluscum contasiosum
Cowdry type A Herpes & Yellow fever virus
Cowdry type B Adeno & Polio virus

66. Cell injury
•Destruction of infected cells of the host tissue
•The resistance to injury is variable for different tissues.
•Can lead to physiological alterations and not cell death
•Clinical illness is due to variety of factors – general
symptoms of viral infections (malaise, anorexia) are
primarily due to host response like production of IFN
and other cytokines

67. Recovery
•Recovery from infection is host mediated
•Due to host immunity – humoral and CMI

68. 2)
   Host immune response
A.
   Inflammatory reaction
Bacteria : Inflitration of polymophonuclear
leukocyte
Viruses : Inflitration of mononuclear cell and
lymphocytes.
B. Immune pathology
a. Cell-mediated immunity : Measles virus , HIV
Normal immunity in agammaglobulinemics
b. Antibody-mediated immunity : IgG, IgM &
IgA classes depending upon site of infection

69. Viral
pathogenicity and
I
mmunity

II. Immunity for viral infection
A) Non-specific immune response
Interference phenomenon
Definition: Prevention of the replication of one virus
by another.
Important mediator: Interferons
Can also be produced by destruction of cell receptors
eg:-Influenza virus

70. Phagocytosis – macrophages phagocytose viruses and
are important in clearing viruses from the bloodstream.
Body temperature – fever may act as a natural defense
mechanism against viral infections as most viruses are
inhibited by temperature above 390C
Hormones – corticosteriod administration enhances
most viral infection.
B)
  
Interferon (IFN)

71. •Malnutrition – some viral infections such as measles
produce a much higher incidence of complications
and a higher case fatality rate in malnourished
children.
•Age – most viral infections are commoner and more
dangterous at the two extremes of age.

72. I
NTERFERONS

73. L
aboratory Diagnosis of Viral
Disease
Introduction
Object
Procedure
I.
   Specimens
1.
   Kinds of specimen
2.
   Time

74. O
bjectives of diagnosis in virology
•To identify an etiological agent as early as possible
•To provide a rational basis for treatment and
prognosis
•To provide quick epidemiological information
during outbreaks.

75. 1.Throat
First presentation with fever (measles, mumps,
rubella, also viral meningitis caused by enteroviruses and
neonatal HSV). Vigorous swab, because you need cells.
2. Nasopharyngeal swab or wash
RSV, Rhino-, CMV (if lots of virus)
3. Rectal
Entero- and adenoviruses, rotavirus
4. Urine
Adenovirus (hemorrhagic cystitis)
MMR, after cleared from throat or sometimes concomitant
CMV and HSV (rare)
Wha
t specimen to collect?
When?

76. 5. CSF
PCR for HSV, VZV, CMV,
adeno or flu
Rarely can grow coxsackie or
echo
6. Lesion
VZV, CMV, measles (scrape
for cells)
HSV, Tzanck smear
7. Genital
HSV, vulvar swab (not
endocervical) in last
month of pregnancy
8. Bronchial and BAL wash
RSV, Flu, Adeno-, CMV etc
9. Other
Biopsy, autopsy specimens

77. 1.Since we still depend on viral growth for diagnosis,
rapid transport to lab is essential
2.Specimen on ice
3.Refrigerate if delay inevitable, DO NOT FREEZE
4.If need to store for more than 6 days, freeze at -70°C
5.Transport and store in viral transport medium (VTM)
6.Enteroviruses more stable and will tolerate some delay
7.Hand delivery encouraged (also for better
communication: viruses suspected, source of material)
Trans
port to lab

78. Diagnosis o
f Viral Infection
1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology

79. Direc
t Examination
1. Antigen Detection Immunofluorescence, ELISA
etc.
2. Electron Microscopy Morphology of virus particles
Immune electron microscopy
3. Light Microscopy Histological appearance
Inclusion bodies
4. Molecular Methods Hybridization with specific
Nucleic acid probes
Polymerase chain reaction
(PCR)

80. R
apid Diagnosis Based on the
De
tection of Viral Antigens
Nasopharyngeal Aspirate RSV
Influenza A and B
Parainfluenza
Adenovirus
Faeces Rotaviruses
Adenoviruses
Astrovirus
Skin HSV
VZV
Blood CMV (pp65 antigenaemia test)

81. I
mmunofluorescense
HSV-infected epithelial cells
from skin lesion. (Source:
Virology Laboratory, Yale-New
Haven Hospital)
Positive immunofluorescence test for
rabies virus antigen. (Source: CDC)

82. E
lectron Microscopy
10
6
virus particles per ml required for visualization. Viruses may be
detected in the following specimens.
Faeces Rotavirus, Adenovirus
Norwalk viruses
Astrovirus, Calicivirus
Vesicle Fluid HSV
VZV
Skin scrapings Papilloma virus,
Molluscum contagiosum

83. E
lectronmicrographs
Rotavirus
Adenovirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)

84. M
olecular Methods
•Methods based on the detection of viral
genome are also commonly known as
molecular methods.
•It is often said that molecular methods is the
future direction of viral diagnosis, and it is
certain that the role of molecular methods will
increase rapidly in the near future.

85. I
ndirect Examination
1.Cell Culture Cytopathic effect (CPE)
Haemabsorption
Immunofluorescence
Haemagglutination
Haemagglutination inhibition test
Immunoelectron microscopy
2. Eggs Pocks on CAM
Haemagglutination
Inclusion bodies
3. Animals Disease or death

86. 1
st
by Steinhardt & colleagues (1913) for vaccinia virus.
Enders, Weller & Robbins – turning point in cultivation
of viruses.
Cell Cultures are most widely used for virus isolation,
there are 3 types of cell cultures:
1.Organ culture (Tracheal organ ring culture for Corona
virus)
2.Explant culture (Adenoid tissue explant culture for
adenovirus)
3.Cell culture
C
ell Culture Methods

87. Routinely employed.
Tissue dissociation Washing Count &
suspend in a growth medium.
Essential constituents:
Amino acids & vitamins
Salts, glucose
Bicarbonate buffer
5% co2
5% calf or fetal calf serum
Antibiotics
C
ell Culture Methods

88. Virus
Isolation
Based on their origin, chromosomal characters & number of generations of subculture
1. Primary cells - e.g., Monkey Kidney cell line, Human amnion, Chick embryo
2. Semi-continuous cells - Skin fibroblasts, Human embryonic lung
3. Continuous cells - HeLa, Vero, Hep2.
Primary cell culture are widely acknowledged as the best cell culture systems available
since they support the widest range of viruses. However, they are very expensive and it
is often difficult to obtain a reliable supply. Continuous cells are the most easy to
handle but the range of viruses supported is often limited.
Detection of virus-infected cells can be done by;
a. CPE
b. Haemadsorption
c. Interference
d. PCR
e. Immunofluorescence
f. Transformation
g. Metabolic inhibition

89. Pro
blems with cell
cul
ture
•Long period (up to 4 weeks) required for result.
•Often very poor sensitivity, sensitivity depends on
a large extent on the condition of the specimen.
•Susceptible to bacterial contamination.
•Susceptible to toxic substances which may be
present in the specimen.
•Many viruses will not grow in cell culture e.g.
Hepatitis B, Diarrhoeal viruses, parvovirus,
papillomavirus.

90. S
yncytia

91. E
mbryonated egg

92. S
erological Methods

93. E
LISA for HIV antibody
Microplate ELISA for HIV antibody: colored wells indicate reactivity

94. Wes
tern Blot
HIV-1 Western Blot
•Lane1: Positive Control
•Lane 2: Negative Control
•Sample A: Negative
•Sample B: Indeterminate
•Sample C: Positive

95. Bal
timore classification
Bal
timore classification