Isolation of viruses. - INAYA Medical Science College

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22 févr. 2014 (il y a 3 années et 5 mois)

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Virus are the smallest infectious agent known. They can infect man,
animals, insects, plants and bacteria

1.
They are very small in size

2.
They contain one kind of nucleic acid (RNA or
DNA ) as their genome

3.
They are metabolically inert, as they do not
possess
ribosomes

or protein synthesizing
apparatus.

4.
They are obligate intracellular parasites i.e. can
only replicate inside living cells

5.
They cannot be grown on artificial culture media
and are grown in tissue culture,
embryonated

eggs or living animals.



They

can

pass

through

bacterial

filters
.


They

require

high

speeds

(ultracentrifugation

)

for

their

sedimentation

10
.
000

-
30
.
000

rpm

(bacteria

requier

1
.
000



3
.
000

rpm)
.


They

are

only

seen

by

electron

micro
-

scope(EM)

No

Property

Viruses

Bacteria

1

Size


20
-
300 nm

1000 nm

2

Genome

(type of nucleic acid)

DNA or RNA but

not both

DNA and RNA

3

Cell wall

Envelope present

in some viruses

Cell wall

4

Ribosomes

No
ribosomes

Ribosomes

5

Sensitivity to antibiotics

_

+

6

Growth in culture media

Grow only in

living host cell

+



Protein coat
(Capsid)

A



Nucleic acid core

B

Each virus particle or virion is composed of :

It is composed of small protein subunits called
capsomers
.


It protects the viral genome (DNA or RNA )against
inactivation by nucleases.


It is responsible for the structural symmetry of virions i.e.
icosahedral or helical


It participates in attachment of virion to susceptible cells.


Capsid proteins are important antigen that induce antibodies
that neutralize virus infectivity and activate cytotoxic T cells
to kill virus infected cell


Variation in capsid proteins is responsible for the different
viral serotypes in non
-

enveloped viruses.


Viruses contain either DNA or RNA but not both.


The nucleic acid may be linear or circular.


The molecular weight and type of nucleic acid are specific for
each virus group


All viruses have one copy of their genome (haploid) except
retraviruses

which have two copies(diploid)


It is the infectious part of the virus


I
t

carries

the

genetic

information

for

virus

replication

,

virulence

or

ability

to

parasitize

cells

and

antigenic

specificity

of

the

protein

coat`




Many

viruses

are

surrounded

by

lipid

or

lipoprotein

envelope,

which

may

be

covered

by

glycoprotein

spike

which

attach

to

host

cell

receptor

during

the

entry

of

the

virus

into

the

cell


The

surface

proteins
;

whether

the

virus

capside

proteins

or

the

envelope

glycoproteins,

are

the

principal

antigens

against

which

the

host

mounts

its

immune

response


They are also the determinants of type
specificity.


Some

viruses

carry

enzymes

e
.
g
.
RNA

polymerase,

which

is

present

in

negative

sense

RNA

viruses

to

copy

their

mRNA

and

the

reverse

transcriptase

enzyme

(RT)

present

in

retroviruses

to

make

a

c

DNA

copy

of

the

viral

RNA
.

The

arrangement

of

the

capsomers,

in

the

capsid,

gives

the

virus

its

geometric

symmetry
.


Viruses have three type of symmetry:


These

viruses

resemble

a

crystal

and

are

called

icosahedral

viruses

e
.
g
.

herpes

viruses

Cubical symmetry:


The

capsomers

are

arranged

in

a

ribbon

which

is

wound

in

the

form

of

a

helix

or

spiral

around

the

spiral

nucleic

acid

e
.
g
.

influenza

virus
.

Helical symmetry


In

which

the

viruses

are

complicated

in

structure

e
.
g
.

poxviruses

Complex symmetry


:

Are composed of viral nucleic acid and proteins but cannot
replicate without a helper virus, which provides the missing
function. These usually have a mutation or a deletion of part of
their
genetic

material.

Contain host cell DNA instead of viral DNA within the capsid.
They are formed during infection with certain viruses when the
host cell DNA is fragmented and pieces are incorporated within
the
capsid.Pseudovirions

can infect cell, but they don’t replicate.

Are infectious particles that are composed solely of protein. They
contain no detectable nucleic acid. They cause slow diseases.


Viruses

have

no

metabolic

activity

of

their

own
.

Therefore,

they

depend

on

living

cells

for

providing

energy

and

synthetic

machinery

for

synthesis

of

:


1
-

Viral nucleic acid (genome)


2
-

Viral proteins

The virus growth cycle within a cell can be divided into different stages

6
-
Relase

5
-

Assemply


4
-
Intracellular viral
synthesis

3
-
Uncoating.

2
-
Penetration

1
-
Attachment

1
-
Attachment

Virus

and

cell

are

brought

into

contact

by

random

collision

but

attachment

is

specific

and

occurs

if

the

cell

membrane

contains

specific

receptors

for

the

virus

,e
.
g
.

human

immunodeficiency

virus

(HIV)

binds

to

CD
4

receptors

on

immune

cells


2
-
Penetration

Following

attachment

the

virus

must

penetrate

the

external

plasma

membrane

of

the

cell

rapidly

and

release

its

genome

into

the

cell

for

subsequent

replication
.

are

taken

into

animal

cells

by

endocytosis

where

they

are

uncoated

by

lysosomal

enzymes
.

penetrate

the

membrane

by

fusion

between

the

virus

envelope

and

the

cell

membrane

releasing

the

nucleocapsid

into

the

cell
.

4
-
Intracellular viral synthesis


It

includes
:


(
1
)

synthesis

of

viral

nucleic

acid

(transcription)



(
2
)

Synthesis

of

proteins(

translation)
.


3
-
Uncoating.

In

this

phase

the

protein

coat

of

the

virus

is

removed,

this

is

mainly

carried

out

by

host

cell

enzymes

contained

in

lysosomes
.

Virus

nucleic

acid

is

then

released

and

made

accessible

for

the

production

of

mRNA


The

viral

nucleic

acid

(genome)

replicates

by

using

a

strand

of

the

parental

nucleic

acid

as

a

template

for

the

production

of

progeny

DNA

or

RNA

molecules
.


The

essential

step

in

protein

synthesis

is

transcription

of

mRNA

from

viral

nucleic

acid
.


The

mRNA

is

translated

by

the

host

cell

ribosomes

into

viral

proteins
.


+
ve

stranded

RNA

viruses

:

the

+
ve

stranded

parenteral

viral

RNA

used

directly

as

mRNA


-
ve

stranded

RNA

viruses
:

In

case

of


ve

stranded

RNA

viruses,

a

virus

associated

RNA

polymerase

(transcriptase)

must

first

make

mirror

image

copies

of

the

original


ve

strand

viral

RNA

and

these

copies

which

are

now

+
ve

stranded

then

function

as

mRNA
.

Virus

mRNA

attaches

to

ribosomes

and

direct

the

synthesis

of

virus

specified

proteins

.
These

are

of

two

main

types
:

(1)
Structural

proteins

:

which

made

up

the

virus

particle

or

virion

(Capsid

protein)

(
2
)

Non

structural

proteins
:

Which

are

not

incorporated

into

new

part
.

Many

of

these

are

enzymes

required

for

the

processes

of

viral

replication

especially

the

synthesis

of

virus

nucleic

acid
.

DNA

viruses

produce

viral

mRNA

transcripts

by

a

host

cell

enzyme

DNA

dependent

RNA

polymerase
.

5
-

Assembly

Assembly

of

viral

nucleic

acid

and

protein

to

form

mature

virus

particles
.

6
-
Relase

The new
virus

particles are released from the cells by budding
through cell membrane or by rupture of cells.

Since viruses are obligate intracellular parasites, they have to be
grown in living cells. There are three systems for their cultivation:


Cell culture

(
1
)


Embryonated

eggs

(
2
)


Laboratory animals

(
3
)


Viruses

should

reach

a

susceptible

cell

before

they

can

produce

disease

therefore

,

they

should

have
:

1.
Portal

of

entry
;

mainly

the

respiratory

tract,

gastrointestinal

tract(GIT)

,skin,

urogenital

tract

.

2.
A

pathway

through

the

body
;

namely

the

blood,

lymphatic

or

nerves
.

3.
A

target

organ

which

may

be

CNS,

skin

,glands,

liver


.
etc


Many viral infections are subclinical.


The same virus may produce a variety of diseases.


The same disease may be produced by a variety of viruses.


The outcome of virus infections is determined by the
interaction of the virus and the host and is influenced by
the genetics of each


Which

occur

at

the

portal

of

entry

with

no

viremia
.

Local

infection

are

characterized

by

short

incubation

period

e
.
g
.

viral

influenza

in

respiratory

tract

and

Rota

virus

infection

in

GIT
.

Local infection


After

primary

replication

at

the

site

of

entry,

the

virus

travels

through

the

blood

or

lymphatic

causing

viraemia
,

or

through

the

nerves

to

reach

a

distant

target

organ

that

has

specific

receptor

for

the

virus

e
.
g
.

poliovirus

measles

systemic infections


Sometimes

viruses

persist

for

a

long

time

in

the

host

in

one

of

the

following

form
:



(A)

Chronic

infections
:

In

which

the

virus

can

be

continuously

detected

with

no

or

mild

symptoms

e
.
g
.

hepatitis

B

and

C

chronic

carriers
.


(B)

Latent

infections

:
In

which

the

virus

persists

hidden

most

of

the

time

with

periodic

reactivation

and

development

of

clinical

lesions

containing

the

virus

e
.
g
.

Herpes

virus

and

HIV

infections
.

persist infections

Viruses

may

produce

local

or

systemic

infections
.

Some

viruses

persist
.

Some

DNA

viruses

that

synthesize

DNA

during

their

replication

are

capable

to

induce

malignant

transformation

of

cells
.





Increase rate of multiplication and metabolism.




Cells show morphological and chromosomal abnormalities




Can be propagated indefinitely in the laboratory.




Possess new tumor antigens on their cell surface.

Virus

Tumor

DNA Viruses:



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灡灩汯浡

癩牵獥猨vP嘩




Epstein Barr virus (EBV)




䡥灡瑩瑩猠䈠癩牵猠⡈B嘩



䍡r捩湯浡c潦⁳歩o



Nasopharyngeal carcinoma



Hepatocellular

carcinoma

RNA viruses:


Human T
-
cell
Lymphotropic

Retrovirus (HTLV
-
1 )




䡥灡瑩瑩猠⁃ 癩牵猠⡈䍖C


Adult T
-
cell
leukaemia

lymphoma


Liver cancer

Since

viruses

are

obligate

intracellular

parasites,

it

is

difficult

to

find

an

antiviral

drug

that

selectively

inhibits

the

virus

without

affecting

the

cell

(selective

toxicity)
.
Few

antiviral

drugs

are

in

clinical

use
.

They

act

by

stopping

virus

attachment,

penetration,

uncoating

or

intracellular

synthesis
.


Interference

with

virus

adsorption

or

attachment

to

the

receptor
-

binding

site

on

the

cell
.


Inhibition

of

viral

uncoating


Inhibition

of

viral

nucleic

acid,

transcription

and

replication


Prevention

of

virus

budding

or

interference

with

virus


maturation


Interferon

Interferon

are

proteins

made

and

released

by

host

cell

in

response

to

the

presence

of

host

cell

such

as

viruses,

bacteria,

parasites

and

tumor

cells
.


After
a cell has come into contact with a virus or some
other
interferon
inducer, infected cell produces interferon
molecules, which diffuse out from the
cell


Interferon molecules attach to receptors on the nearby cells
and induce the formation of proteins, which interfere with
the translation of viral mRNA and inhibit viral protein
synthesis and viral
replication

Therapeutic uses of interferon:

With recombinant DNA
technology, interferon was now
commercially produced.
Interferon(INF
-

) treatment
may be helpful in certain severe
viral infections(herpes), in
chronic virus infection (hepatitis
Band C viruses).

The laboratory procedures used in diagnosis of viral diseases include:


Direct detection of viruses

(A)


Isolation of viruses.

(B)


Serologic detection of
antiviral antibodies

(C)

Dire
ct detection of viruses can be Achieved by different techniques:



1.
Light microscopy:

This can be used to visualize some
large viruses e.g. poxviruses

2. Electron microscopy:

Is

used

to

demonstrate

virus

particles

in

vesicular

fluid

or

tissue

extracts

treated

with

special

stain
.

Its

only

successful

if

large

numbers

of

particles

are

present
.

3
. Immunofluorescence microscopy:

Detection

of

virus

in

smears

from

lesions

using

fluorescein

labeled

specific

antisera


4
. Nucleic acid hybridization :

Using

DNA

probes,

it

is

possible

to

detect

virus

nucleic

acid

in

pathologic

specimen

or

in

tissue

samples
.

The

probe

which

is

a

single

strand

of

the

nucleic

acid

of

the

virus

in

question

will

hybridize

with

its

complementary

strand

in

the

specimen
.

Probes

are

labeled

and

can

be

easily

detected
.

5
. Polymerase chain reaction:

This

technique

involves

amplification

of

a

short

sequence

of

a

target

DNA

or

RNA

(which

may

be

in

low

concentration

e
.
g
.

one

copy)

leading

to

accumulation

of

large

amounts

of

that

sequence,

so

it

can

be

easily

detected
.

PCR

can

be

used

to

determine

the

quantity

of

viruses

in

patients

blood

i
.
e
.

virus

load

e
.
g
.

in

HIV

patients
.

This

is

used

to

monitor

the

course

of

the

disease

and

to

evaluate

treatment

and

prognosis
.


Isolation of virus from clinical specimens by inoculation on cell
culture or laboratory animals

Serologic diagnosis used in virology:

1
. Enzyme linked
immunosorbent

assay(ELISA):

Enzyme linked
immunosorbent

assay (ELISA) can be used for
detection of viral antigens in different clinical specimens e.g.
detection of HBV ,HIV, HCV and Rota virus.

2
. Radioimmunoassay(RIA):

The principle of the test is the same as ELISA but radiolabeled
anti
-
immunoglobulin are used instead of enzyme labeled anti
-
immunoglobulin.

3. Immunofluorescence assay :

In this test patient serum is added to the antigen preparation
(intracellular antigen or virus infected cells fixed on glass slides).
Detection of attached antibody is performed by the addition of
fluorescein

labeled
antiglobulin

which fluoresces green when
stimulated by ultra violet light.

4
.
Immunodiffusion

:

Antibodies and soluble antigens when brought into contact with
a gel slowly diffuse into the gel and insoluble antigen
-
antibody
complexes are formed that precipitate and become visible as
white line