BY:

Amr Shalaby

Definition:

Viruses are the smallest known infectious agents, they can infect man, animals, plants, insects & even bacteria ( Bacteriophage ) .

NB: The word ( Virus ) is a latin word meaning ( Poison )

Properties:1- Their size is very small (20-300nm) so they

only can be seen by EM except ( poxvirus ).

2- They are obligate intracellular parasites ,which can replicate only inside the host cells as they lack the required machinery for replication.

3- They can’t be grown on artificial culture media , but only on living cells.

4- Their genome is either DNA or RNA ( but never both )

Structure:The virion ( the complete infectious virus

particle ) is constructed of two components ..

1- The nucleic acid core .

2- The capsid ( a protein coat protect the genome ).

3- Other components : enzymes & matrix protein ( may be present )

NB: They are together called the nucleocapsid

NB: Some viruses are enclosed in an envelope.

1-viral nucleic acid genome:- Either DNA or RNA.

- Single or Double stranded.

- Linear or Non-linear.

- Segmented or Non segmented.

- All virus genomes are haploid ( 1 copy )

except : Retrovirus which are 2 copies of linear RNA ( diploid )

2- Viral Capsid :- protein coat of the virus

- makes most of the virion mass

- composition small protein subunits called capsomers ,this arrangement determines the virus symmetry

functions of capsid :

1) Protection of genome from destruction 2) Attachment to specific receptors on cells 3) Antigenic : induce Ab & activate Tc4) Arrangement of the capsomers

Viral Envelope:Structure :

surrounded by lipid or lipoprotein envelope & covered by glycoprotein spike like projections

Source : partially or totally derived from host cell

membrane during release of virus by Budding Importance :

1- Sensitive to heat , detergent & ether 2- Loss of envelope causing loss of infectivity

Envelope proteins : A- matrix protein : link nucleocapsid to

envelope

B- spike glycoprotein attached to cell receptors C- antigenic structure

D- determine type specificity of the virus

Viral enzymes:Carry enzyms important for their replication.

Atypical virus like agents :A- Defective viruses : viruses can't replicate

without a helper providing the missing function

B- Pseudovirions : contain host cell DNA instead of viral during viral replication parts of host DNA incorporated in the viral capsid infectious but not replicative

C- Viroids : infectious agents composed only of Single Stranded circular RNA with some double stranded regions causing plant diseases

D- Prions : infectious particles composed of solely of proteins i.e. no nucleic acid & cause slow diseases e.g mad cow disease

Definition : It is the intracellular phase of

virus life cycle that starts by the contact of a virion depending completely on the cell's metabolic function .

Stages :1-Adsorption 2-Penetration

3-Uncoating 4-Transcription

5-Translation 6-Synthesis of nucleic acid

7-Assembley of new virions

8-Release of new virions

1-Adsorption of virus to host cell:Binding between receptor binding site (on

viral surface) & specific receptor on host cell .

The exact fitting between them is the basis of

viral tropism ( the specificity of the virus to a particular host tissue ) .

2-Penetration:

-Enveloped viruses: by fusing to plasma membrane

-Non enveloped: by endocytosis: by endocytosis (then liberation inside cell)

3-Uncoating:- proteolytic enzymes of the host attack virus

destructing its capsid (liberation of genome)

NB: Uncoating mark the beginning of eclipse phase ( virus cannot be detected but genome is detected by molecular test )

5-Transcription:

-1- In double strand DNA viruses :

-mRNA is transcribed from the negative strand of

DNA by host cell DNA dependent RNA polymerase .

2- In ssRNA viruses of +ve sense:

ssRNA itself act as mRNA for translation into protein .

3- In ssRNA viruses of –ve sense:

transcribed by RNA dependent RNA polymerase into

complementary mRNA

6-Translation :Viruses use the cellular ribosomes to translate their viral mRNA into:

-non structural ptns (early ptns): replication enzymes

-structural ptns (late ptns): coat ,core, cote ptns

7-Genome synthesis:By using single strand as template .

8-Assembly:Assembly of genome & coats to form mature

viruses e.g herpes virus

9-Release:- Non enveloped: lysis of cells

-Enveloped: budding (taking part from cell membrane) with no cell lysis

Defenetion: It's the process by which a viral infection

leads to disease .

NB: Infection Vs disease

�Infection : entry of the virus into the body produces no symptoms or transient symptoms due to local infection .

�Disease : virus at target organ produces signs and symptoms associated with disease .

NB: The majority of viral infections are subclinical

Outcome of viral infection :Acute infection:

Recovery , death , progression to chronic

Chronic infection:

- Silent subclinical infection for life .

- Long silent period before disease .

- Reactivation to cause acute disease .

- Chronic disease with relapses and exacerbations .

- Cancers .

Factors in viral pathogenesis :1- Cellular pathogenesis :

cells can respond to viral infections in 3 ways :

�No apparent change ,Death ,Transformation .

2- Viral entry :Skin, Conjunctiva and other mucous membranes,

Respiratory tract, GIT and Genitourinary tract .

3- Cell tropism :

viral affinity to specific tissue determined by :

�cell receptors for virus

�cell transcription factors that recognize viral promoters and enhancer sequences .

�ability of the cell to support virus replication .

4- Course of viral infection :

Primary replication :

The virus replicates after gaining initial entry into the host . This determines whether the infection will be localized at the site of entry r spread to become a systemic infection .

Systemic spread :

Apart from direct cell-to cell contact, the virus may spread via the blood or lymphatic causing viraemia or through the nerves to reach the target organ .

Secondary replication :

Takes place at susceptible organs/tissues following systemic spread .

5- Cell damage :

- Viruses may replicate throughout the body without any symptoms if they don't cause significant cell damage or death .

- Retroviruses don't generally cause cell death, being released from the cell by budding rather than cell lysis, and cause persistent infections .

- Picornaviruses cause lysis and death of the cell in which they replicate .

6-Immune response :

It has the greatest impact in the outcome of infection . Cellular immunity plays the major role in clearing the viral infection whereas humoral immunity protects againre reinfection .

7- Viral clearance or persistence :

The majority are cleared but some viruses may cause persistent infections .

Mechanism of viral persistence 1- Antigenic variation

2- Immune tolerance, causing a reduced response to an antigen, may be due to genetic factors or prenatal infection .

3-Infection of immuneprivileged sites within the body e.g HSV in sensory ganglia in the CNS .

4-Direct infection of the cells of the immune system itself e.g Retrovirus (HIV) often resulting in immunosuppression .

1-Direct detection in clinical specimens:1- light microscopy : can be used for large viruses e.g. Poxviruses in skin lesions and for inclusions bodies e.g. Negribodies of Rabies viruse in nerve cells .

2- Electron microscopy : used to demonstrate large number of virus particles in vesicular fluid or tissue extract treated with special stains.

3- Immuoelectron microscopy (IEM) : by addditionof specific antisera to clinical samples leading to aggregation of virus particles which can be see easier than separate virus . e.g. Hepatites A virus ad rotavirus detectio in stools.

4- Immunofluorescence microscopy : fluorescein labeled specific antisera are added to smears from lesions and visualized by fluorescense microscope.e.g. diagnosis of rabies in brain smears.

5- Solid-phase immunoassays : Radioimmunoassay (RIA) and Enzyme linked immunosorbent assay (ELISA) are used to detect virl antigens in clinical specimens.e.g. detection of p24 antigen of HIV in blood .

6- Nucleic acid hybridization : labeled DNA probes are used to detect virus nucleic acid by hybridization with its

complementary part in the sample.

7- Polymerase chain reaction : by amplification of a short sequence of the target nucleic acid which maybe of a small amount in the sample to facilitate its detection .

2- Isolation of viruses :A- cell cultures :

These are pieces of animal or human tissue to which trypsin is added to separate the cells .Cells are grown with a growth medium containing serum , on glass or plastic tubes , bottles or plates with a flat side . A monolayerofcells is formed on the flat side into which the viruses is

inoculated there are 3 types of cell cultures :

1. primary cell lines : prepared from organ fragments

2. Human diplod cell lines : which are fibroblasts from human embryo tissue , the grow rapidly.

3. Continuous (heteroploid) cell lines : from tumor cell , they can divide identically.

Detection of viruses in cell cultures:1.Cytopathic effects (CPE) : change :

a.Cell death & detachment from the glass surface (poliovirus)

b.Rounding & cluster formation (adenovirus)

c.Syncytium or multinucleated giant cell formation (measles)

d.Transformation (tumor viruses)

2. Plaque formation : virally infected areas in cell cultures that can be seen by naked eye as unstained areas when stained with crystal violet .

3.Inclusion bodies : structures seen by LM :

-Intranuclear (Herpesvirus)

-Inracytoplasmic (pox & rabies virus *Negri bodies*)

4.Haemagglutination : cell culturebfluid + RBCs >> clumping of RBCs on areas infected with virus.

5.Interference : growth of some viruses can be detected by their interference with growth of CPE producing viruses .

6. Flouresence ab staining : florescein labeled ab + infected cells >> fluorescence by fluorescent microscope.

7.Neutralization tests : viruses with CPE + ab + cells >> NO more CPE .

8.Serology for ag : ELISA & complement fixation .

9. Decrease acid production : in dying cells & detected

by color change using a PH indicator .

B- Embryonated eggs : Viruses can be grown in the amniotic or allantoic cavity on

the choriallantoic membrane or in the embryo itself .

The virus is detected by lesions (pocks) on the chorioallantoicmembrane or by the hemagglutination in the fluid from cavities

C- Laboratory animals :

Inoculation of the virus into laboratory animals was mainly used in the past before using tissue culture . However it still used to study viral pathogensis.oncogensis and immune response against viruses .white suckling mice are the most widely used for virus inoculation

4- Serelogical methods to detect anti viral antibodies :

By detecting arising antibody titer throughtobtaining two samples

The first one in the acute phase and the second after 10-14 days .

A four-fold rise in antibody titer indicates infection Detection of IgM antibodies in a single serum

sample indicates infection . Also the presence of IgMto any virus in a serum of newborn indicates infection in utero , the used test include neutralization ,ElIsa, RIA,IF,complement fixation or haemagglutination inhibition

4- Skin test:Are used to detect cell mediated immunity against

some viral infections e.g. mumps

1- chemotheraputic antiviral .

2- Cytokines therapy .

3- Antiviral immunoglobulins .

1- chemotheraputic antiviral :Chemotherapy aims at the use of a chemical

that inhibit virus replication steps .

1 . Nucleoside analogues :a-azidothymidine :

-Synthetic thymidine analogue.

- Inhibit replication of HIV by inhibiting viral reverse transcriptase and block proviral DNA synthesis .

- Dideoxyinosine (DDI) : same action , less toxic

b-Deoxythiacytidine and stavudine :

-used if resistance to treatment with AZT or DDI develop .

c-Acyclovir :

-Guanisine analogue inhibting Herpes simplex virus .

-Inhibits virus specific DNA polymerase .

2 . Nucleotide analogues :They differ from the nucleoside analogs in having

an attached phosphate group . their ability to persist in cells for long periods of time increases their potency e.g. cidofovir

3 . Non nucleoside reverse transcriptase inhibitors :

They act by binding directly to reverse transcriptase and disrupt the enzyme catalytic site e.g. nevirapine.

4 . Protrease inhibitors :

Such drugs inhibit the viral protease that is required at the late stage of replication cycle . e.g. saquinavir used for treatment of HIV infection

5 . Fusion inhibitors :Fuzeon is a large peptide that blocks the

virus and cellular membrane fusion step involved in entry of HIV-1 into cells.

6. Other types of antiviral agents :- Ribavirin : Synthetic nucleotide effective against

many DNA & RNA viruses .-Amantadine & rimantadine : Synthetic amines

inhibiting uncoating of influenza A but not B virus .-Zanamivir : Inhibits the release of influenza virus

from infected cells .-Foscarnet : A viral polymerase inhibitor of herpes

viruses . It also inhibits the reverse transcriptase of HIV

-Methisazone : Is of historical interest as an inhibitor of poxviruses .

Combination chemotherapy:These are strategies to use more than one drug

for therapy of viruses especially those that undergo mutation inside the body , Examples:

1-HAART(highly active antiretroviral therapy)used to treat HIV with nucleoside analogue AZT ,a

nonnucleoside nalogue Lamivudine ,and protease ihibitor eg, Saqwinavir,Ritonavir.

2-Pegylated interferon alpha , ribavirinandprotease inhibitor to treat HCV

3- Pegylated inter feron alpha with Lamivudin to treat HBV

2-Cytokines Therapy:Examples:

1-Antiviral cytokines

Type 1 interferon :inhibits viral replication .therapeutic targets are HBV,HCV,herpeszoster,papilloma,HIV.

side effects of therapy are fever,malaise,fatigue,musclepain and depression

Type 2 interferon :up regulates expression of class2 MHC,enhance activity of CTL and macrophageandinhibit viral replication . Therapeutic targets are viral encephalitis ,slow viruses,prions. side effects are rare.include kidney,liver, heart,and bone marrow toxicit

2-Regulators of lymphocyte functions

Interleukin-2 (IL-2) induces proliferation of B ,T cells and CTL and stimulates NK cells.

Therapeutic targets is local treatment of viral skin and mucous membranes lesions .

Side effects of therapy are : depression, ascites,renal failure, hepatic failure and mental changes

3- Antiviral immunoglobulins :These are used for passive immunization and immunotherapy .The

injection of human hyperimmune globulin provides an immediate partial or complete

protection last for 2-4 weeks Examples :

1- Vaccine induced anti HBs human immuneoglobulin (HBIG)

- Co-administered with r-HBs vaccine to newborns of mothers with

active HBV- infection .

- Persons with needle stick injuries with first dose of accelerated

vaccination with r-HBs

2- Vaccine induced human anti rabies immunoglobulin (HRIG) :to

provide rapid protection after exposure to rabies virus until vaccine

immunity develops by active vaccination .injected IM around the bite site .

SourceBasics in medical microbiology and

immunology

part IV VIROLOGY

ByStaff members of medical microbiology and

immunology department

Faculty of medicine ,Zagazig university

Thank you

ASh