Published on March 9, 2014
PowerPoint Presentation: General Properties of Viruses Virology Virology is the bioscience for study of viral nature,and the relationship between viruses and hosts Definition of Virus Viruses may be defined as acellular organisms whose genomes consist of nucleic acid, and which obligately replicate inside host cells using host metabolic machinery and ribosomes to form a pool of components which assemble into particles called VIRIONS History: History Smallpox was endemic in China by 1000BC. In response, the practice of variolation was developed. Recognizing that survivors of smallpox outbreaks were protected from subsequent infection, variolation involved inhalation of the dried crusts from smallpox lesions like snuff, or in later modifications, inoculation of the pus from a lesion into a scratch on the forearm of a child. Viral Properties : Viral Properties Viruses are inert (nucleoprotein ) filterable Agents Viruses are obligate intracellular parasites Viruses cannot make energy or proteins independent of a host cell Viral genome are RNA or DNA but not both Viruses lack the enzymes necessary for protein and nucleic acid synthesis Viruses do not have the genetic capability to multiply by division Viruses occupy the twilight zone that separates the ‘living’ from the ‘nonliving’ PowerPoint Presentation: Cellular organisation Growth on inanimate media Binary fission Both DNA and RNA Ribosome Sensitivity to antibiotics Sensitivity to Interferons Bacteria + + + + + + - Mycoplasma + + + + + + - Rickettsiae + - + + + + - Chlamydiae + - + + + + + Viruses - - - - - - + Properties of bacteria and viruses PowerPoint Presentation: Medical importance of viruses Viral diseases range from minor ailments such as the common cold to terrifying diseases such as rabies or AIDS They may be sporadic like mumps, endemic like infectious hepatitis, epidemic like dengue fever or pandemic like influenza They may be localised to circumscribed areas (as some arbovirus diseases) or worldwide (as Herpes simplex) Viruses can cause cancer in animals and birds, as well as in humans PowerPoint Presentation: Morphology Size Methods of analysis Passing through collodion membrane filters of graded porosity (gradocol membranes) 2. Ultracentrifuge 3. Electron microscope 4. X-ray crystallography PowerPoint Presentation: Size of Viruses A small virus has a diameter of about 20 nm. Example: Parvovirus A large virus have a diameter of up to 400 nm. Example: Poxviruses PowerPoint Presentation: Structure and shape Virion The complete infectious unit of virus particle Capsid The protein shell, or coat, that encloses the nucleic acid genome. Functions: a. Protect the viral nucleic acid. b. Participate in the viral infection. c. Antigenic and specific for each virus type d. Provides structural symmetry to the virus particle Nucleocapsid The capsid with the enclosed nucleic acid Virion: Virion Capsid Viral core envelope VIRUS GENOMES: VIRUS GENOMES DNA RNA Single Stranded Double Stranded Circular + or - Segmented Double Stranded Segmented PowerPoint Presentation: Symmetry of Nucleocapsid Cubical/Icosahedral (Adeno virus, Coxsackie virus, CMV, EBV, Hepatitis virus, HSV, Polio virus, Rubella virus) Helical (Influenza, Rubeola, Mumps, Rabies, Hanta, Corona viruses) Complex (Bacteriophage, Pox viruses) PowerPoint Presentation: Cubic or icosahedral symmetry An icosahedron is a polygon with 12 vertices or corners and 20 facets or sides Two types of capsomers constitute the icosahedral capsid Petagonal capsomers at the corners (pentons) 2. Hexagonal capsomers making up the sides (hexons) PowerPoint Presentation: Helical symmetry Complex symmetry T4 bacteriophage Pox virus PowerPoint Presentation: Envelope A lipid-containing membrane that surrounds some viral particles It is derived from the plasma membrane of the host cell during there release by budding from the cell surface Viruses-encoded glycoproteins are exposed on the surface of the envelope (peplomers) A virus may have more than one type of peplomer Envelopes confer chemical, antigenic and biological properties on viruses Not all viruses have the envelope, and viruses can be divided into 2 kinds: enveloped virus and nonenveloped (naked) virus PowerPoint Presentation: Enveloped Virus Naked Virus Cubic Helical PowerPoint Presentation: Shape of viruses Overall shape of the virus particle varies in different groups of viruses Filament-shape Brick-shape Tadpole-shape Spherical-shape Rod-shape Bullet-shape PowerPoint Presentation: Viral Hemagglutination A large number of viruses agglutinate erythrocytes from different species Hemagglutination by the influenza virus is due to the presence of hemagglutinin spikes on the surface of the virus In the haemagglutination test RBCs are added to serial dilutions of viral suspension, the highest dilution that produces haemagglutination provides the haemagglutination titer (HA units) The haemagglutination test can be carried out in test tubes or special plastic trays The test is a convenient method for detection and assay of the influenza virus and also serves to titrate killed influenza vaccine PowerPoint Presentation: Elution In myxoviruses, neuraminidase (RDE) acts on the receptor and destroys it and this leads to the reversal of hemagglutination and the release of the virus from the red cell surface As hemagglutination is specifically inhibited by the antibody to virus, hemagglutination inhibition assay is useful for determining antiviral antibody PowerPoint Presentation: Replication of viruses The viral multiplication cycle can be divided into six sequential phases, though the phases may sometimes be overlapping 1. Adsorption or attachment 2. Penetration 3. Uncoating 4. Biosynthesis 5. Maturation 6. Release PowerPoint Presentation: Adsorption Virions may come into contact with cells by random collision but adsorption takes place only if there is an affinity between the two Differences in susceptibility to viral infection are to a large extent based on the presence or absence of receptors on cells Penetration Virus particles may be engulfed by a mechanism resembling phagocytosis, a process known as ‘viropexis’ In the case of enveloped viruses, the viral envelope may fuse with the plasma membrane of the host cell and release the nucleocapsid into the cytoplasm PowerPoint Presentation: Uncoating Process of stripping the the virus of its outer layers and capsid so that the nucleic acid is released into the cell With most viruses, uncoating is effected by the action of lysosomal enzymes of the host cell Biosynthesis During this phase, viral nucleic acid, capsid protein, enzymes necessary in the various stages of viral synthesis, assembly and release will be synthesised Certain ‘regulatory proteins’ which serve to shut down the normal cellular metabolism and direct the sequential production of viral components are also synthesised The site of viral synthesis depends on the type of virus PowerPoint Presentation: Biosynthesis consists essentially of the following steps Transcription of mRNA from the viral nucleic acid Translation of the mRNA into ‘early proteins’. These are enzymes which initiate and maintain synthesis of virus components Replication of viral nucleic acid Synthesis of late or structural proteins, which are the components of daughter virion capsids The critical step in viral biosynthesis is the transcription of mRNA from the viral nucleic acid The mechanisms of nucleic acid synthesis differ in the different type of viruses PowerPoint Presentation: Maturation Assembly of daughter virions follows the synthesis of nucleic acid & proteins Virion assembly may take place in the host cell nucleus (Herpes and adenoviruses) or cytoplasm (picorna and poxviruses) Naked viruses are present intracellularly as fully developed virions but in the case of enveloped viruses, only the nucleocapsid is complete Release Progeny of bacterial viruses release by the lysis of the infected bacterium. In the case of animal viruses, release usually occurs without cell lysis Myxoviruses are released by budding from the cell membrane over a period of time In the case of some viruses (varicella), transmission occurs directly from cell to cell and poliovirus causes profound damage to the host cell and may be released by cell lysis PowerPoint Presentation: Stages in the infection of a host’s cell and replication of a virus PowerPoint Presentation: Abnormal replicative cycles Incomplete viruses Incomplete viruses are seen in large proportions, when cells are infected with a high dose (MOI) of the influenza virus This is the result of defective assembly The virus yield will have a high hemagglutinin titer but low infectivity and this is known as the ‘von Magnus phenomenon’ Abortive infection In nonpermissive cells, viral components may be synthesised but maturation or assembly is defective, and either no release occurs or the progeny is noninfectious PowerPoint Presentation: Defective viruses Some viruses are genetically defective in that when they infect cells, they are unable to give rise to fully formed progeny Yield of progeny virions occurs only if the cells are simultaneously infected with a helper virus, which can supplement the genetic deficiency (eg; hepatitis D, adeno-associated satellite viruses, which replicate only in the presence of their helper viruses – hepatitis B and adenoviruses, respectively) PowerPoint Presentation: Cultivation of viruses Systems for the propagation of viruses Human volunteers Animal inoculation Embryonated eggs Tissue culture a) Organ culture b) Explant culture c) Cell culture PowerPoint Presentation: Animal inoculation Mice, Infant (suckling) mice, rats, monkeys, chikens, guinea pigs, rabbits, ferrets are used for inoculation Growth of the virus in inoculated animals may be indicated by death, disease or visible lesions Animal inoculation is also used for the study of pathogenesis, immune response, epidemiology and oncogenesis Disadvantages of animal inoculation are that immunity may interfere with viral growth and that animals often harbour latent viruses PowerPoint Presentation: Embryonated eggs The embryonated egg offers several sites for the cultivation of viruses Inoculation on the chorioallantoic membrane (CAM) produces visible lesions (pocks); pock-forming viruses eg: variola or vaccinia Inoculation into the allantoic cavity provides a rich yield of influenza and some paramyxo viruses; hence employed for vaccine production Inoculation into the amniotic sac is employed for the primary isolation of the influenza virus Yolk sac inoculation is used for the cultivation of some viruses, chlamydiae and rickettsiae PowerPoint Presentation: Embryonated egg showing different routes of inoculation PowerPoint Presentation: Small pox (variola) virus pocks on CAM of embryonated egg PowerPoint Presentation: Tissue culture Organ culture Small bits of organs can be maintained in vitro for days and weeks, preserving their original architecture and function Useful for the isolation of some viruses which appear to be highly specialised parasites of certain organs (eg: Tracheal ring culture – corona virus, a respiratory pathogen) Explant culture Fragments of minced tissue can be grown as ‘explants’ embedded in plasma clots or may be cultivated in suspension (eg: adenoid tissue explant cultures – adenoviruses) PowerPoint Presentation: Cell culture Although embryonated eggs and laboratory animals are very useful for isolation viruses, cell culture is the sole system for virus isolation in most laboratories Based on their origin, chromosomal characters and the number of generations through which they can be maintained, cell cultures are classified into three types Primary cell cultures Diploid cell cultures (semi-continuous) Continuous cell lines PowerPoint Presentation: Primary cell cultures Prepared directly from animal or human tissues and can be subcultured only once or twice (eg: monkey kidney, human embryonic kidney cell cultures) Useful for the isolation of viruses and their cultivation for vaccine production Diploid cell cultures Derived from human fetal tissue and can be subcultured 20-50 times (human diploid fibroblasts – MRC-5, WI-38 (derived from normal embryonic lung tissue) They are useful for the isolation of some fastidious pathogens They are also useful for the production of viral vaccines (eg: poliomyelitis, rubella, rabies, CMV, VZV) PowerPoint Presentation: Continuous cell lines These are cells of a single type, usually derived from cancer cells and capable of continuous serial cultivation indefinitely (eg: HeLa, HEp-2, Vero, KB cell lines) HeLa Human carcinoma of cervix cell line HEp-2 Human epithelioma of larynx cell line Vero Vervet monkey kidney cell line KB Human carcinoma of nasopharynx cell line McCoy Human synovial carcinoma cell line BHK-21 Baby hamster kidney cell line Some continuous cell lines in common use PowerPoint Presentation: Detection of virus growth in cell cultures Cytopathic effect Many viruses cause morphological changes in cultured cells, these changes can be readily observed by microscopic examination and these changes are known as ‘cytopathic effects’ (CPE) Cytopathic effects may be cytocidal (cell death) or non-cytocidal Non-cytocidal effects include acidophilic or basophilic inclusion bodies in the nucleus or cytoplasm, or both; cell fusion, and transformation Cytopathic effects can be so characteristic of individual viruses that they can often be used to identify viruses (Measles – syncytium formation; adenovirus – large granular clumps) PowerPoint Presentation: Cytoplasmic inclusion body caused by rabies virus in brain tissue Syncytium formed by cell fusion due to infection by measles virus Normal cell CPE PowerPoint Presentation: Metabolic inhibition When viruses grow in cell cultures, cell metabolism is inhibited and there is no acid production Hemadsorption When hemagglutinating viruses grow in cell culture, their presence can be indicated by the addition of guinea pig erythrocytes to the cultures Interference The growth of a non-cytopathogenic virus in cell culture can be tested by the subsequent challenge with a known cytopathogenic virus Transformation Oncogenic viruses induce cell transformation and loss of contact inhibition, so that growth appears in a piled-up fashion producing microtumors Immunofluorescence PowerPoint Presentation: Viral assay The virus content of a specimen can be assayed in two ways 1. Total virus particles: Electron microscopy and haemagglutination 2. Assay of infectivity (with reference to the infectious virions only) Quantal assays Only indicate the presence or absence of infectious viruses but using serial dilutions of virus suspensions and with the aid of statistical methods, reasonably accurate estimates of infectivity can be obtained End points used for infectivity titration are the death of the animal, production of hemagglutinin in allantoic fluid or the appearance of CPE in cell cultures The titers are expressed as the ‘50 percent infectious dose’ (ID 50 ) or LD 50 PowerPoint Presentation: b) Quantitative assays Similar to the estimation of bacterial viable counts by colony counting Two methods are available; Plaque assay A viral suspension is added to a monolayer of cultured cells in a bottle or petri dish, and after adsorption, the medium is removed and replaced with a solid agar gel Each infectious viral particle gives rise to a localised focus of infected cells that can be seen with the naked eye. Such foci are known as ‘plaques’ 2. Pock assay PowerPoint Presentation: Plaque assay Pock assay PowerPoint Presentation: Viral Genetics Two main mechanisms for genetic modification in viruses are mutation and recombination Mutation The frequency of mutation in viruses is about 10 -4 to 10 -8 Mutations may be lethal or sub lethal, may occur spontaneously or induced by mutagens Mutations affecting virulence, host range, antigenicity and pock plaque morphology are of clinical and laboratory interest Mutants which are able to grow under certain conditions (permissive conditions) and can not grow or lethal under some specific conditions (restrictive conditions) are called as ‘conditional lethal mutants’ PowerPoint Presentation: Interaction between viruses (Genetic) Reassortment Found in viruses which have segmented genomes ( eg : influenza, bunya and arena viruses) In a single cell infected with two related viruses, there is an exchange of segments with production of reassortants It is probably one of the ways by which the pandemic strains of the influenza virus originate in nature Reactivation when a cell is infected with an active virus and a different but related inactive virus, recombination may occur and as result of this the progeny possessing one or more genetic traits of inactive virus may be produced. This process is called cross reactivation or marker rescue PowerPoint Presentation: Interaction between viral gene products (nongenetic) Phenotypic mixing When two different viruses infect the same cell, some mix up may take place during assembly, so that progeny genome of one virus may be surrounded by a capsid belonging partially or entirely to other virus This altered phenotype is not a stable variation When the genome of one virus is surrounded by the entire capsid of the other virus, it is called ‘transcapsidation’ PowerPoint Presentation: 2. Genotypic mixing It occurs when more than one complete genomes are accidentally surrounded by a single virus capsid There is no recombination between the different genomes so that the two kinds of viral progeny are formed on passage 3. Complementation One virus provides the gene products to the second virus in which the latter is defective, this allows the second virus to replicate PowerPoint Presentation: 4. Interference Infection of a cell by one virus inhibits simultaneous or subsequent infection by other virus The most important mediator of interference is interferon Interference may also be produced by destruction of cell receptors by an active or inactive virus Viral interference has been applied in the field in controlling poliomyelitis outbreaks by introducing vaccine strain into the population 5. Enhancement Mixed infection of cells may sometimes lead to increased virus yield or greater CPE PowerPoint Presentation: Classification and nomenclature of viruses Viruses are classified on the basis of biological, physical and chemical properties Viruses are broadly classified into DNA and RNA viruses and then further divided into families, subfamilies, genera and species PowerPoint Presentation: DNA viruses Poxviridae Herpesviridae Adenoviridae Papovaviridae Parvoviridae Hepadnaviridae PowerPoint Presentation: RNA viruses Picornaviridae Orthomyxoviridae Paramyxoviridae Togaviridae Flaviviridae Bunyaviridae Arenaviridae Rhabdoviridae Reoviridae Coronaviridae Retroviridae Caliciviridae Filoviridae PowerPoint Presentation: Viroids Viroids are single-stranded circular RNA molecules that lacks a protein coat and they are mainly plant pathogens Prions Prions are infectious proteins without any detectable nucleic acid They are highly resistant to physical and chemical agents They produce slow infections with very long incubation period Diseases caused by prions in animals include, scrapie of sheep and goats,mink encephalopathy, bovine spongiform encephalopathy Responsible for Kuru, Creutzfeldt-Jakob disease and some other chronic neurological degenerative diseases of humans PowerPoint Presentation: Thank you for your attention !!!