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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
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