Virology 2nd Year

VIROLOGY The smallest infectious agents and the smallest form of life. Viruses can be viewed only by Electron microscope.

General charachteristics of viruses: 1- The viral nucleic acid become active only when it enters to the host cells. 2- Viruses are a live when they multiply in the host.

Differ from other infectious agents:  They are very small.  They need host cells for multiplication.  Contain a single type of nucleic acid, DNA or RNA.  Contain protein coat.



 



Multiply inside living cells using the synthetic machinary of the cell. Can transfer the viral nucleic acid to other cell. No ATP-generating system which is very important for design of antiviral drugs as the drug will be toxic for the virus and the host cell. The virus contains lipid layer so may be the virus become sensetive to disinfectant or emulsifing agents e.g. Bile salt and detergents.

HOST RANGE: 2) Animal viruses 3) Plant viruses 4) Bacterial viruses (bacteriophage). •

The specification of the virus to infect the host cells depend on the requirment for the viral multiplication found in the host cells.

•

The virus interact with specific receptors site on the surface of the cells-----hydrogen bonds----by pilli or flagella (Bacteriophage)--------Receptor sites are plasma membrane of the host cells (animal viruses).

CHEMICAL COMPOSITION OF THE VIRUSES: 1) Nucleic acid: In a centeral core of protein include polymerase enzyme Viruses are unique contain RNA or DNA, single or double standed, Surrounded by capsid. 2) Protein Coat (Capsid): Units that make up the capsid are called capsomers. Protect the inside nucleic acid core. Complete infective viral particles called virion which responsible for infection. 3) Lipoprotein: The nucleocapsid (nucleic acid + capsid) is covered by an outer membrane called envelope (animal viruses). Viruses without envelope called naked viruses.

SIZE: 20-300 nm, size can be determined by:  Ultrafilter  High speed centrifuge  Electronic microscope Viruses are obligate intracellular parasite because: 1- Defficiency in ATP generating system. 2- Absence of ribosomes which is essential for protein synthesis. Viruses envelope may or may not be covered by spikes which are carbohydrate- protein complexes that project from the surface of the envelope e.g: Influenza virus.

GENERAL MORPHOLOGY (Shape): 1) Helical virus: Long rods may be rigid or flexible, their capsid is a hollow cylinder with helical structure surrounded nucleic acid e.g. Tobaco mosaic virus. 2) Polyhederal virus: The capsid is an icosahederal form an equilteral triangle e.g. Adenovirus, poliovirus. 3) Enveloped virus: Roughly, spherical, but highly pleomorphic as the envelope is not rigid. E.g. Enveloped helical ---------------Influenza Enveloped polhederal-----------HSV 4) Complex virus: Bacterial viruses have very complicated structure e.g. poxvirus, bacteriophage

CLASSIFICATION OF VIRUSES: Old classification according to hot and organ: Animal- Bacterial – Plant Modern Classification: Morphological classification Type of nucleic acid Size of capsid Number of capsomers Method of transmission DNA----envelope: Herpes, poxvirus Non envelope: adenoviruses RNA-----Envelope: Retrovirus (HIV) Non envelope : Polio, Retero virus

isolation, cultivation and identification of virus: -The requirment of living host cells for multiplication complicate their detection. -Cultivation on living cells, plant or animals. -Bacteriophage growing in bacteria culture. Gwrwoth of bacteriophage: -Can be grown on solid bacterial culture or suspension. -Use solid medium may plaque which use for detection and counting viruses: Bacteriophae + host bacteria + melted agar-----poured on a peteray plate containing a hardened layer of agar----each virus infect bacterium, multiplies, infect all bacteria cells----destroyed---plaques on surface agar--uninfected bacteria---turbid background PFU---------------Plaque forming unit.

BACTERIOPHAGE CLASSIFICATION: 1) Virulent phage: -Host cells-----lytic cell -The phage inject DNA inside host cell leaving the protein material ouside. -DNA enter host cell and control the genetic machine of cell stimulating it to transcript the DNA of the virus then capsid formed around DNA and capsid -----rupture of bacteria--viruses---mature phage. 2) Temperate phage: -Host cells-----lysogenic as no. 1 but no rupture of the bacteria due to DNA of virus integrated with bacterial chromosome.

REQUIREMENTS FOR VIRAL GROWTH  All viruses are obligate intracellular parasites but they can survive in certain conditions as non-replicating particles 

Temperature Heat - there is great variability in the heat stability of different viruses but icosahedral viruses tend to be relatively stable, enveloped viruses are heat labile and most pathogenic viruses are inactivated at 55-60oC because their capsid protein is destroyed (an important exception is the hepatitis virus. Cold - most viruses can be preserved at sub-freezing temperatures, some can withstand lyophilisation and can be stored in the dry state at 4oC or even at room temperature while enveloped viruses tend to lose infectivity after prolonged storage at –90oC.





pH Most viruses are stable in the pH range 5-9. Enteroviruses that have to pass through the stomach can withstand low pHs. All viruses are destroyed by alkaline conditions. Radiation UV produce damaging results on double-stranded DNA that can cause inactivation of the virus. If conditions are right the DNA can repair itself. x-ray, gamma rays and beta particles inactivate viruses.. Stabilisation by Salts magnesium chloride stabilises polioviruses, magnesium sulphate stabilises influenza viruses and sodium sulphate stabilises herpes virus. Important in the preparation of vaccines e.g. non-stabilised polio vaccine must be stored at <0oC whereas stabilised vaccine remains potent for weeks at ambient temperature which is an advantage when immunising in rural areas



Ether Susceptibility and Lipid Solubility

Enveloped viruses are inactivated by ether whereas non-enveloped ones are not (simple efficient test for the presence of envelopes). Other organic solvents and sodium deoxycholate also destroy the envelope. 

Detergents

Non-ionic detergents solubilise lipid constituents but do not denature the proteins of the capsid. Anionic detergents solubilise the lipid constituents and disrupt the capsids into separated polypeptides 

50% Glycerol

Many viruses remain alive in 50% glycerol for many years. Vaccinia virus is preserved in 50% glycerol for many years while bacteria are killed.



Formaldehyde Destroys viral infectivity but has minimal adverse affect on viral antigenicity and is used in the productions of inactivated viral vaccines



Photodynamic Inactivation

Heterocyclic dyes like neutral red, proflavine and toluidine blue intercalate between the bases of replicating viral nucleic acids and on exposure to light, they become susceptible to inactivation 

Antibacterial Agents Antibacterial antibiotics and sulphonamides have no effect on viruses although some antiviral drugs have been developed. Oxidising agents are the most effect disinfectants e.g. hydrogen peroxide, hypochlorite.

Growth of animal viruses in the lab: 1) Living animal: e.g. mice, rabbit, guinea pig Styding immunoresponse, a clinical specimen with virus inoculated the animal ----obseved signgs of disease or killing animal and examine the tissue. 2) Embryonated eggs: Conveniant, inexpensive, the viral suspension injected into the fluid of the egg, viral growth is detected by death of the embryo, or lession on the membrane of the egg. It is the most common method for viral growth and isolation , and used for preperation of viral vaccine.

3) Cell culture: The recent method for cultivation and grwoing of virus. It is homogenous collection of cells. More convneient than animal or egg. Prepared by treated the animal tissue with enzyme ---separate the individual cells.Suspended in solution with nutrient and grwoth factor required by the cell---grow– adhere to glass or plastic container----monolayer----by virus infection---deterioration of cells (cytopathic effect CPE)---plaques counted. Cells isolated from human embryo, can live for hundared generation e.g Hela cells.

Cell culture problems: -

Must be kept from microbial contamination. Need antibiotics for preventing contamination. Need experience technical worker. Identification of viral isolates is not easy so immunological method mainly used as depend on antibodies.

Multiplication of animal viruses:

Virus---attachement to the cell---penetration--uncoating---replication---assembelly---release— viruses.

Effect of animal viral infection on host cells: 

CPE: inhibition of DNA or protein synthesis---Damage of the cell.

Inclusion bodies: 









One type of CPE, a result of accumulation of viruses in the nucleous or cytoplasm of the host cells or both. Other arise at sites of earlier viral synthesis. Important: usefual for identification of the particular virus causing an infection. e.g. Rabies----Negri bodies in cytoplasm of nerve cells and presented in the brain tissue----(Diagnostic test for rabies, also for measles, vaccinia, small pox, herpes. Polykeryocytes: infection cell accumulation from myxoviruses.

Viruses and cancer: Tumor: cell multipication in an uncontrolled way. Malignant cell multiply in an uncontrolled way. Most patients die from metastatic ( spread cancer to other parts of the body) than primary tumor. Leukomia: blood cnacer, increase white cells in blood. Virus-cancer relation discover 1908, chicken leukomia transfer from sick chiken to healthy. In 1963: virus induced adenocarcinoma (cancer of glanular tissue) in mice was discovered. Cancer: The viral particles infect the cells without cancer Cancer may not developed untill long infection Cancer do not seem to be contagious.



Transformation of the normal cell to tumor cell: Alter the genetic material of the cell----cancerous cell e.g. chemical, high-energy radiation, viruses (oncogenic)

Reseptor gene®--------- Oncogen 1,2,3 chromosome Reprosser protein oncogen blocked Viral gene (V) Reprosor gene oncogen The viral protein interfere with the reprossor protein so the reprosor gene blocked and the oncogen will be function

Interferons: -Protect neighboring cells from viral infection. -It is antimicrobial cytokines which interfer with viral replication. Inter. α,β interact with specific receptor or, produced within 42 hour infection. Inter. γ with oher receptor, a T-cell product, later infection. Protein kinase, 2’,5’ oligoadenylate synthatase----inhibit the viral RNA translation----inhibit protein synthesis. Used in chronic hepatitis B and AIDS treatment.

Mechanism of Action of Interferon: Interferon itself is not the anti-viral agent. It induces an anti-viral state by prompting the synthesis of other proteins that inhibit viral replication. The action is as follows: 1) The viral nucleic acid stimulates the cell nucleus to synthesise interferon which can through the cell membrane into the tissue fluid. Interferon molecules bind to the cell surface receptors which induce a signal that is transmitted to the cell nucleus. 2) The binding triggers or the synthesis of several enzymes responsible for the development of the antiviral state. 3) These enzymes block viral reproduction by inhibiting the translation of viral mRNA while cellular DNA remains unaffected. 4) Interferon molecules interact with components of the immune system by modulating humoral (B lymphocytes) and cellular (T lymphocytes) immunity increasing antibody secretion and cytotoxicity and enhancing phagocytic macrophage activity. Interferon has broad cell-growth regulatory activities 