virology - prac. microbiology

GENERAL VIROLOGYGENERAL VIROLOGY

Viruses

The smallest infectious agents (20-300 nm) only seen under

Electron Microscope (except Poxviruses)

Electron Microscope- The source of illumination is a beam of electrons

- Electromagnetic lenses

- Magnification is 100.000 or more

Electron Microscopy

Bacterial fimbriae

Structure of Viruses

Virus particle (virion)

Structure of viruses

7

8

Viral Symmetry

1. Icosahedral symmetry: e.g., all DNA viruses, except poxviruses & some RNA viruses

2. Helical symmetry: e.g., many RNA viruses (e.g. rabies virus)

3. Complex symmetry: e.g., poxviruses (brick-shaped)

Ico

Icosahedral Symmetry

Rotavirus Calicivirus Astrovirus

Icosahedral Symmetry

Helical Symmetry

Complex symmetry

Differences between viruses & bacteria

1. They are obligatory intracellular parasites They can not be cultivated on artificial culture media. can only replicate inside living cells.

2. Viruses contain only one type of nucleic acid (DNA or RNA), never both.

3. They can not be cultivated on artificial

culture media.

4. They are not susceptible to antibacterial agents.

Diagnosis of viral infectionsDiagnosis of viral infections

A- Direct methodsA- Direct methods B- Indirect methodsB- Indirect methods

I. Direct detection of viruses and / or their components

I. Direct detection of viruses and / or their components

II. Isolation of viruses

II. Isolation of viruses

I. SerologyI. Serology II. Skin testsII. Skin tests

I. Direct detection of viruses & / or their components:

1. Light microscopy: Examination of large viruses as PoxvirusesDetection of giant cells in Herpes infectionDetection of inclusion bodies e.g. Negri bodies in nerve cells in rabies

A- Direct methodsA- Direct methods

2. Electron microscopy (EM): Large number of viruses in the sample. Size and shape of viruses.

3. Immunoelectron microscopy (IEM):

Sample (unknown virus) + known specific antibody

aggregation of unknown virus particles e.g. hepatitis A virus in stools

4. Fluorescent microscopy:

Direct immunofluorescent antibody technique (IF) e.g. diagnosis of rabies in brain smears.

5. Immunoassays :

For detection of the virus antigens by ELISA / RIA e.g. hepatitis B antigens in blood

ELISA

6. Nucleic acid hybridization:

A highly sensitive and specific method.

Viral nucleic acid in sample + Specific labeled probe

hybridization

fluorescence

7. Polymerase chain reaction (PCR) Amplification of a specific sequence of nucleic acid

Detection e.g. by gel electrophoresis.

A- Direct methodsA- Direct methodsII. Isolation of viruses:

• Obligatory intracellular parasites • Replicate only in living susceptible

cells

a. Laboratory animals

b. Embryonated eggs

c. Cell culture (tissue culture)

Used in:

• Isolation of coxsackie viruses by inoculation in

white suckling mice.

• Rabies virus inoculation in mice.

• For research.

a. Laboratory animals:

Cultivation of viruses

b. Embryonated eggs

Cultivation of viruses

c. Cell culture (tissue culture)

Cultivation of viruses

The most widely used method

Tissues + trypsin → separate cells

Cells + growth media in

flat-sided bottles

monolayer culture

Virus inoculation → incubation at 37ºC (CO2 incubators)

Primary cell lines

Diploid cell

linesContinuous cell

lines

Prepared from

Organ fragments

Human fibroblasts derived from

embryonic tissuesTumor cells

ExamplesMonkey kidneyHuman embryo lung

tissue HeLa cells from

carcinoma of cervix

Number of Passages

(subcultures)5-1050-100Unlimited

Types of cell lines

Detection of Virus Replication in Tissue Culture:

1- Cytopathic Effect (CPE): i- Cell death or lysis

ii- Syncytial formation (multinucleated giant cells ):

2- Transformation: Viral nucleic acid + cellular DNA → cell transformation → foci of malignant cells.

3- Inclusion bodies

Site of virus assembly or degenerative changes Their location and appearance are diagnostic for a

particular virus.

a- Intracytoplasmic: e.g. Rabies (Negri bodies)

b- Intranuclear: e.g .Herpes viruses

c- Both: e.g. Measles virus and CMV

4- Plaque formation:

Infected monolayer + vital dye → unstained areas (plaques)

5- Haemadsorption:

Monolayer + hemagglutinating virus + RBCs → hemadsorption (RBCs clumping) to infected cells

6. Interference phenomenon: Monolayer + rubella virus → no change for

weeks

Add CPE-producing virus → NO CPE

(due to interference)

7- Detection of viral antigens

8- Direct fluorescent antibody staining of infected cells (DFA):

9 -Neutralization test:Monolayer + unknown virus + known specific Ab

NO CPE (due to neutralization)

Diagnosis of viral infectionsDiagnosis of viral infections

A- Direct methodsA- Direct methods B- Indirect methodsB- Indirect methods

I. Direct detection of viruses and / or their components

I. Direct detection of viruses and / or their components

II. Isolation of viruses

II. Isolation of viruses

I. SerologyI. Serology II. Skin testsII. Skin tests

I. Serological diagnosis:

Detection of antiviral antibodies

2 serum samples acute phase & 2-3 weeks later, to demonstrate a rising titer (4 fold increase or more is diagnostic).

Only one sample may be used in the acute stage to detect IgM

B- Indirect methodsB- Indirect methods

I. Serological diagnosis:

Serological methods include:

Neutralization test Complement fixation test Haemagglutination inhibition test Indirect IF ELISA RIA

II. Skin tests

Used as an indication of cell-mediated immunity (CMI) in some viral infections, e.g. mumps.

MCQs

1) Viruses differ from bacteria in all of the following EXCEPT:

a) Viruses are very small in size.b) Viruses are obligatory intracellular parasites.c) Viruses contain both DNA and RNA.d) Viruses cannot be cultivated on artificial

culture media.e) Viruses are not susceptible to antibiotics.

2) Sites of viral assembly in tissue culture are called:

a)Plaquesb)Inclusion bodiesc) Viral capsidd)CPEe)Areas of transformation

3) Regarding continuous cell lines, all of the following are true EXCEPT:

a)They allow unlimited number of passages.b)They are prepared from tumor cells.c) After viral inoculation, they should be

sterilized by autoclaving.d)They are used for isolation of viruses.e)HeLa cell line is an example of continuous cell

lines.

4) PCR may be used to diagnose viral infections by detecting:

a)Antiviral IgM antibodiesb)Viral antigensc) Rising titer of antiviral antibodiesd)Viral nucleic acide)CPE produced by the virus in tissue culture

5) Direct detection of viruses and/or their components can be done by all of the following EXCEPT:

a)Fluorescent microscopeb)Electron microscopec) Light microscoped)PCRe)Biochemical reactions

6) Cultivation of viruses can be done on:a)Blood agarb)Tissue culturec) MacConkey’s mediumd)Nutrient brothe)Anaerobic media

THANK YOU

1) Viruses differ from bacteria in all the following EXCEPT:

a) Viruses are very small in size.b) Viruses are obligatory intracellular parasites.c) Viruses contain both DNA and RNA.d) Viruses cannot be cultivated on artificial

culture media.e) Viruses are not susceptible to antibiotics.

2) Sites of viral assembly in tissue culture are called:

a)Plaquesb)Inclusion bodiesc) Viral capsidd)CPEe)Areas of transformation

3) Regarding continuous cell lines, all of the following are true EXCEPT:

a)They allow unlimited number of passages.b)They are prepared from tumor cells.c) After viral inoculation, they should be

sterilized by autoclaving.d)They are used for isolation of viruses.e)HeLa cell line is an example of continuous cell

lines.

4) PCR may be used to diagnose viral infections

by detecting:a)Antiviral IgM antibodiesb)Viral antigensc) Rising titer of antiviral antibodiesd)Viral nucleic acide)CPE produced by the virus in tissue culture

5) Direct detection of viruses and/or their components can be done by all of the following EXCEPT:

a)Fluorescent microscopeb)Electron microscopec) Light microscoped)PCRe)Biochemical reactions

6) Cultivation of viruses can be done on:a)Blood agarb)Tissue culturec) MacConkey’s mediumd)Nutrient brothe)Anaerobic media