Subject: Microbiology Topic: Introduction to Virology Lecturer: Dr.

Barzaga Date of Lecture: June 20, 2011 Transcriptionist: Micro Group 24 Pages: 7

I. GENERAL CHARACTERISTICS AND STRUCTURE OF VIRUSES Distinctive Characteristics of Viruses Smallest infectious agent, (20-300nm) in diameter Contains only one kind of nuclear genome (May be RNA or DNA but never both) Inactive: in extracellular environment Active: biologically active during intracellular replication Replicates only in living cells so it needs a host to replicate Obligate intracellular organisms: since they have no metabolism of their own they are obliged to invade cells and take over the sub cellular machinery and using it to their advantage Structure of Virus and Functions Capsid –symmetric protein shell or coat enclosing the nucleic acid genome. o Functions of Capsid  facilitate transfer of viral nucleic acid from one host cell to another  protects the enclosed nucleic acid genome  important in adsorption: A process that enables the virus to attach to susceptible animal cells  Acts as an antigen to stimulate formation of antibodies Nucleocapsid o Structure consisting of the nucleic acid protected by a protein capsid o Represents the packaged form of viral genome o Capsid + enclosed nucleic acid Capsomeres o Morphologic units that comprises the capsid o Seen in EM on surface of iscosahedral (cubic) viral particle o Represents clusters of polypeptides

Virion o The complete or mature infectious viral particle o May be a nucleocapsid or nucleocapsid + envelop  Not all viruses have envelop  Envelop is derived from host cell membrane Defective Virus o A viral particle that is functionally deficient in some aspect of replication Viroid o Smallest known agent of infectious disease o Consist of nucleic acid only: low molecular weight RNA o Does not possess a protective capsid coat Chemical Composition Protein o major component of the virus which determines its antigenic characteristics  ex. Capsid Nucleic Acid o either DNA or RNA o encodes the genetic information necessary for replication of the virus o may be single stranded or double stranded, circular or linear, segmented or non segmented Lipids o Acquired when the viral nucleocapsid buds through a cellular membrane in the course of maturation o If present then virus may be sensitive to treatment with ether and other organic solvents Glycoproteins o Attaches the viral particle to a target cell by interacting with a cellular receptor o Serve as viral antigen

VIRAL ENVELOPE – membrane like outer coat

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Composition o Lipids  Derived from host cell membrane  Neutral lipids, phospholipids, glycolipids o Virus – Specific envelop proteins  Glycoprotein – found as a surface structure Spike proteins: viral coded envelope glycoprotein that have specialized functions such as hemagglutination or neuraminidase activity.  Matrix Protein- hydrophobic protein located on the inner surface of the envelop o Enzymes  Neuraminidase  RNA polymerase  DNA polymerase Function o Responsible for infectivity o Responsible for hemmaglutinating and adsorption property o Responsible for initiating replicative cycle o Capable of producing an immune response.

Specific viral cell and tissue tropism due to attachment of a virus to a specific cellular receptor 8. Pathology o Inclusion body formation (cytopathic effects) 9. Symptomatology o Oldest and easiest way of classification o Has not been satisfactory in developing an international classification scheme because the same virus may be found in several disease groups and structurally dissimilar viruses may cause similar symptoms. Two Major Components of the Virus Used in Classification nucleic acid - its molecular weight and structure capsid – size, symmetry and whether it is enveloped or not Criteria for Virus Classification (3 level system) virus strains that are recognizably different in more than one gene: SPECIES Species that exhibit readily apparent genetic similarity are grouped together: GENERA Genera grouped together based on morphology, physical and chemical nature of viral proteins and on molecular strategies: FAMILY Effects of Physical and Chemical Agents on Viruses Heat: Most are inactivated at 56 o C for 30 mins. or at 100 o C for a few seconds.

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II. CLASSIFICATION OF VIRUSES Basis for Classification of Viruses 1. Nucleic acid type o Viruses are either DNA type (nearly all double stranded) or RNA type (predominantly single stranded) 2. Size a. Morphology: i. Presence or absence of envelop b. Type of Symmetry: i. Helical symmetry, cubic symmetry or complex symmetry. c. Number of capsomeres d. Presence or absence of membranes 3. Presence of Specific Enzymes 4. Susceptibility to Physical and Chemical Agents o Sensitivity to viral inactivation by ether is generally demonstrated in viral families that possesses envelopes 5. Immunologic Properties o Antigenic determinants 6. Natural Modes of Transmission

Cold: Stable at low temperatures Can be stored at -40 o C to -70 o C Partially activated by thawing and freezing

Drying: Variable o Some will survive o Others rapidly inactivated

7. Host, Tissue and Cell Tropism

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Ultraviolet Irradiation: Inactivates viruses Chloroform and Ether Inactivates viruses with envelopes, those without envelopes are resistant Oxidizing and Reducing Agents Inactivates viruses Ex. Formaldehyde, chlorine, iodine, hydrogen peroxide Phenols Most viruses are relatively resistant Antibiotics Doesn’t have any effect on viruses. III. VIRAL REPLICATION Steps in Viral Replication: 1. ADSORPTION or ATTACHMENT property of a solid substance to attract and hold to its surface virus adsorb to specific receptors on cell plasma membrane o eg. - Influenza attaches to the sialic acid receptor on Upper Resp.tract/lungs o Rabies attaches to the ACH Receptor adsorption is best at 37 C and slow at 4 C enhanced by divalent cations: Mg or Ca++

3. UNCOATING removal of the capsid and core Proteins liberating viral nucleic acid, the viral uncoating process generally leads to a loss in infectivity and marks the true beginning of the viral eclipse period. Protein coat of the virus is removed by host cell enzymes viral nucleic acid is then released for the production of virus mRNA 4. TRANSCRIPTION production of virus mRNA is the key to the successful infection of the cell (virus takes over the cellular machinery of the cell) utilizing the virus genome or nucleic acid information coding for virus protein is passed to the ribosomes

5. TRANSLATION mRNA attaches to the ribosomes and directs the synthesis of virus-specific proteins Structural proteins o capsid proteins o envelope protein non structural proteins o Enzymes required for virus replications (especially the synthesis of virus nucleic acid) 6. ASSEMBLY newly synthesized nucleic acid molecules and structural proteins come together to form the new virus progeny assembly can take place in the: o nucleus o cytoplasm o call plasma membrane

2. ENTRY or PENETRATION process by which a virus enters a cell the entire virus enters the cell temperature dependent ; takes place at 37 C, not at 4 C may enter the host in 3 ways: 1. fusion with cell surface o important mechanism utilized by enveloped viruses entails a fusion of the viral envelope with the cellular membrane resulting in the release of the nucleocapsid into the cytoplasm 2. endocytosis Naked capsid viruses generally enter a cell by phagocytosis and are initially found in vacuoles 1. receptor mediated o

7. RELEASE release from the host cell takes place by: o a gradual process of extrusion (Budding) through the cell membrane  Enveloped virus o rupture of the cell  non-enveloped virus

Modes of Transmission of Viruses 1. Direct Contact Physical contact with someone who is a symptomatic or an asymptomatic carrier of the virus droplet or aerosol –respiratory viruses fecal-oral –enteroviruses  e.g. poliomyelitis

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sexual  e.g. herpes simplex I hand-mouth hand-eye mouth-mouth  e.g. kissing disease

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replicating virus can continuously be detected at low levels

2. latent infections o persists in a hidden form 3. slow virus infections o years before infection can manifest o long incubation period (can take years)

2. Transmission from animal to animal humans are accidental hosts E.g. rabies infection 3. Transmission by means of an arthropod vector e.g. dengue virus PATHOGENESIS Ability of viruses to cause disease can be viewed on 2 levels: 1. The changes that occur within individual cells. o 4 main effects:  death of cell  fusion of cells to form  multinucleated cells  malignant transformation  no apparent morphologic or  functional change 2. The process that takes place in an infected patient o Pathogenesis in the infected patient involves:  transmission of the virus and its entry to the host  replication of the virus and damage to the cells  spread of the virus to other cells and organs  the immune response, both as a host defense and as a contributing cause of certain diseases  persistence of the virus in some instances  PERSISTENT VIRAL INFECTIONS Mechanisms: 1. Integration of a DNA provirus into host cell DNA (ex. Retroviruses) 2. Immune tolerance (no neutralizing antibodies formed) 3. Formation of virus-antibody complexes which remain infectious 4. location within an immunologically sheltered sanctuary (ex. Brain) 5. rapid antigenic variation 6. Intracellular spread ; virus is not exposed to antibody 7. Immunosuppressions (ex. AIDS) 3 Types: 1. chronic carrier infections

IV. HOST RESPONSE TO VIRUS INFECTION immunity encompasses all of the mechanisms by which a host may either specifically or nonspecifically recognize foreign viral substances and ultimately respond directly or indirectly to eliminate these foreign invaders Non-Specific Defense Mechanism Interferon o A complex of protein regulatory molecules which is released from virus infected cells o when taken by uninfected cells, these are rendered resistant to virus infection  note: interferons do not affect viral replication directly but act by binding to specific cell surface receptors, which in turn induces antiviral proteins Phagocytosis o mononuclear cells and lymphocytes  neutrophils  macrophages Respiratory Tract o due to constant upward movement action of the ciliated epithelium, and effect of mucus secretory IgA Stomach Acid o inactivates acid-labile viruses (rendering them non-infectious) o acidity destroys the virus Skin o forms an impermeable barrier; resists viral penetration Specific Defense Mechanism can be elicited by both B lymphocytes (bursal dependent) and T lymphocytes (thymus dependent) 1. Humoral Immunity (B lymphocytes) due to antibodies immunoglobulins IgM, IgG and IgA are associated with antiviral activity

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produced in response to both natural infection and active immunization antibodies can neutralize a virus or render it noninfectious 2. Cell-mediated Immunity (T lymphocytes) delayed hypersensitivity important in recovery from certain viral infections *note: both are important not only in recovery but also in prevention of repeated infections Other Factors Which Influence Virus Infection 1. Age viral infections are generally acquired in childhood and are followed by long-lasting immunity most of the time, severe viral infections are encountered in childhood 2. Immune Deficiency 3. Pregnancy V.LABORATORY DIAGNOSIS OF VIRUS INFECTIONS Effects of Viruses on Cell Cell death - the infection is lethal and kills the cell causing cythopathic effect (CPE) o When viruses grow in culture they often produce changes in the infected cells or CPE Cell transformation - the cell is not killed but is changed from normal cell to one with properties of a malignant or cancerous cells Latent infection - the virus remains within the cell in a potentially active state but no obvious effects on the cell function ISOLATION OF VIRUSES (3 Main Systems) 1. Tissue Culture (1st system) Medium - balanced and buffered salt solution with amino acids and vitamins Atmosphere - often grown in closed vessels Temperature - optimum growth is 37 degrees Celsius Types of tissue culture 1. Primary Culture  derived directly from animal source  prepared by trypsinizing fresh tissues and seeding into culture vessels  short-lived but generally suitable for a wide range of viruses

2. Semi-continuous Cell Line  capable of a limited number of passages before undergoing senescence  easy to maintain  prepared from human lung embryo 3. Continuous Cell Line  immortalized cells that can be passaged without limit, meaning it can be subcultured indefinitely  can be maintained through an indefinite number of generations  susceptible to fewer viruses  prepared from: human cervical cancer cells(Hela) human rhabdomyosarcoma (RD cell lines) Virus growth in tissue culture is recognized by: o CPE - refers to pronounced morphologic changes induced in individual cells or group of cells by virus infection, which are easily recognized under a light microscope  simplest and most widely used criterion for infection  inclusion bodies, rounding, fusion, aggregation, shrinkage, lysis, necrosis o HEMADSORPSTION- refers to the ability of red blood cells to attached specifically to virus-infected cells  useful assay for detecting infections by viruses that causes little or known CPE’s o IMMUNOFLOURESENCE Identification of Viruses Isolated o Neutralization tests o Hemagglutination-inhibition o Immunofluorescence o Electron Microscopy 2. Chick Embryo (2nd system) provides a variety of differentiated tissues which serve as a substrate for growth of a wide variety of viruses Main Routes of Inoculation o chorioallantoic membrane o allantoic cavity o amniotic cavity Virus Growth is Recognized by: o appearance of pock (virus lesion) on the chorioallantoic membrane o hemagglutin-inhibition with standard antiviral sera

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3. Laboratory Animals (3rd system) newborn of suckling rodents often provide the best hosts virus can be isolated by inoculation of laboratory animals presence of virus can be recognized by observing for signs of disease or death viruses are identified by testing for neutralization of their pathogenicity for animals by antiviral sera Virus Isolation Requirements Proper collection of appropriate specimens o respiratory diseases: nasopharyngeal swab or washings or sputum o hemorrhagic fevers: serum, blood Storage o If the specimen could not be used right away, freeze at -60 degrees Celsius except for whole blood for antibody determination and tissue for organ or cell culture or urine for cytomegalovirus (CMV) Transport o the samples are kept cold using dry ice/ordinary ice Inoculation of suitable cell cultures, susceptible animals, or embryonated eggs o Done when new epidemic occurs, serological tests overlap, or to confirm a presumptive diagnosis DIRECT DEMONSTRATION OF VIRUS OR ANTIGEN IN MATERIALS FOR PATIENTS Widely used and fast method of diagnosis o Serological  Immunoassay - most popular  enzyme immunoassay  radioimmunoassay o Electron Microscopy - detection of virus particles **Inclusion bodies - virus-induced masses seen in the nucleus of cytoplasm of infected cells ex. Negri bodies SEROLOGY easiest and most accessible important for viruses that cannot be readily cultured of for which culture is slow or otherwise impractical, like dengue, rubella, mumps, rabies viruses demonstration of virus antibody needs a paired serum sample, for example, specimen: acute and convalescent sera

Serological tests compliment fixation test (CFT) Immunofluorescence Hemagglutination- Inhibition Test Enzyme Immunoassay (EIA) Radio Immunoassay (RIA) Detection of IgM Recent infection can be diagnosed by the following: Rising titer - increase in the level of virus antibody at least 4 fold from acute to convalescent Detection of IgM - it is the earliest anitbody to appear High Stationary Titer - if virus antibody is higher than that found in the population VI. VACCINES The Major goal of immunization against viruses is the prevention or modification of disease. Most viral vaccines prevent or modify disease without necessarily preventing infection. Types 1. ATTENUATED LIVE VIRUS VACCINES Reduced pathogenicity by repeated passage to provide immune response without disease may be naturally occurring produces long-lived immunity Advantages activation of all components of the immune systems, which equates to good immunogens o balanced systemic and local immune response o balanced humoral and cell mediated response stimulate an immune response to each of the protective antigens of a virus o this obviates the difficulties that arise from selective destruction of one of the protective antigens that may occur during preparation of a non-living virus vaccine, which can result in disease potentiation immunity induced by live virus vaccines id generally more durable and more effective low cost of production and ease of administration Disadvantages biochemically unstable for live virus; genetically unstable since it can revert back to virulence contamination is possible, can contain adventitious agents can cause illness directly or lose their attenuation during manufacture or during 6

replication in vaccines by reversion or by second site compensatory mutations can spread to contacts can lose infectivity during storage, transport, or use 2. INACTIVATED OR NON-LIVING VIRUS VACCINE the virus is then inactivated with a denaturing agent such as formalin, or disrupted with a detergent(vaccine for influenza) Advantages immunization with little or no risk of infection better immunogens that attenuated vaccines the preparation of vaccines free of nucleic acid prevent the possibility of long term adverse effect associated with infection or integration of viral nucleic acid into the host genome Disadvantages not possible for all viruses; denaturation may lead to loss of antigenicity several non-living virus vaccines have potentiated disease rather that prevented it antigenic changes can occur weaker induction of CTL response; not effective as live viruses may not protect for a long period of time

3. SUBUNIT VACCINES the newest type; completely safe, except for rare adverse reactions they tend to be the least effective Types o Synthetic vaccines - not very effective, none currently in use o Recombinant vaccines - better than synthetic vaccines; an example is the Hepa B vaccine derived from yeast o Virus vectors - the idea is to utilize a well understood, attenuated virus to present antigens to immune system; so far no success in producing this type Disadvantages relatively poor antigenicity (especially short peptide vaccines) vaccine delivery: (carriers/adjuvant needed)

END OF TRANSCRIPTION Sorry for the transcription, PowerPoint was not given so pictures weren’t placed in this transcription. “The Lord is loving and merciful, slow to become angry and full of constant love.” Psalm 145:8

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