General Virology PDF

General Virology Robel Mekonnen(MSc, Asst. Prof)  Learning Outlines: History of virus Definition and property of virus Virus structure Viral classification Viral replication Pathogenesis and Epidemiology of viral infections Laboratory diagnosis Viral vaccine...

General Virology Robel Mekonnen(MSc, Asst. Prof)  Learning Outlines: History of virus Definition and property of virus Virus structure Viral classification Viral replication Pathogenesis and Epidemiology of viral infections Laboratory diagnosis Viral vaccine 2  Objectives: at the end of the sessions, the students will be able to : List general properties of viruses Describe morphological characteristics of viruses List & Explain basic classification of viruses Describe replication cycle of virus and viral pathogenesis Explain methods of viral cultivation Mention common diagnostic methods in virology 3  INTRODUCTION TO MEDICAL VIROLOGY As a science, virology evolved later than bacteriology- comparatively larger size of bacteria made them visible. The Latin word virus means “poisonous fluid”. Plant infections transmitted by bacteria-free filtrates- indicating living agents smaller than bacteria cause a wide range of plant and animal diseases virus. 4  Poliomyelitis Through the Ages 1200 BC 1968 5  INTRODUCTION TO MEDICAL VIROLOGY Evolutionary origin of viruses How did viruses become independent genetic entities? There are 3 theories exist: 1. Regressive theory Viruses are degenerate forms of intracellular parasites e.g. Leprosy bacillus, rickettsiae & chlamydia Lack their own rRNAs or protein synthesis machinery Controversy on how RNA virus evolved??? 6 2. Progressive theory Viruses originated from cellular RNA & DNA components Normal cellular nucleic acids that gained the ability to replicate autonomously & hence to evolve DNA viruses came from plasmids or transposable elements ►They then evolved protein coat & transmissibility Retroviruses derived from retro-transposon & RNA virus from mRNA 7 3. Co - evolution theory Viruses coevolved with life (free living cells) No convincing reason to think that RNA viruses have evolved in the same way as DNA viruses 8  Virology is the study of viruses  What are viruses? Viruses are very tiny, simple particles  In fact, they are so tiny that they can only be seen with a special, very powerful microscope called an "electron microscope," and they are so simple that they are technically not even considered "alive’’  The structure of a virus is extremely simple and is not sufficient for an independent life  A virus is not able to metabolize, grow, or reproduce on its own, but must take over a host cell that provides these functions; therefore a virus is not considered "living" 9  General Properties of Viruses Viruses are unique in nature Are the smallest of all organisms Viruses are acellular organisms – do not fit the three domain system Viruses can infect all forms of life (bacteria, plants, protozoa, fungi, insects, fish, reptiles, birds, and mammals) – Viruses that infect only bacteria are called bacteriophages – Viruses that infect only fungi are termed mycophages Viruses are Obligate intracellular parasites, usually infect one type of cells 10  General Properties of cont’d Viruses are not unicellular But microorganisms may be unicellular e.g. Bacteria, protozoa and some fungi Viruses do not have cellular organization Viruses are composed of a single nucleic acid, RNA or DNA - never both. Most viruses lack enzymes needed for protein or nucleic acid synthesis For this reasons, they dependent on host living cells (use host machinery) Since they are obligate intra-cellular parasites inert extra-cellularly. 11  General Properties of cont’d Viruses are infectious agents with both living and non- living characteristics Living characteristics of viruses: – They reproduce at a fantastic rate, but only in living host cells; – They can mutate Non-living characteristics of viruses: – They are acellular, that is, they contain no cytoplasm or cellular organelles; – they carry out no metabolism on their own and must replicate using the host cell's metabolic machinery 12  General Properties……..  Viruses replicate or multiply Viruses do not grow.  Multiply by budding rather than by binary fission  Unaffected by antibiotics. Why ????  Smallest living units measuring from 20nm –300nm –Largest virus is Poxvirus (300nm) –Smallest virus is Polio and foot & mouth disease virus (20nm) 13 Size comparison of viruses - how big are they?  Viral Structure There are two major structures of viruses called the naked nucleocapsid virus and the enveloped virus NAKED Enveloped 15  Viral Structure cont’d … Complete virus particle - entire infectious unit is called Virion Virion contains DNA or RNA which is surrounded by protein coat called capsid Some viruses’ capsid may be covered by a lipid envelope; they are called enveloped viruses 16 Viral Structure cont’d … – Virion -- nucleic acid & capsid (protein coat) – Capsid with nucleic acid – nucleocapsid Capsid protects nucleic acid and used for introduction of viral genome into host cells – Capsid contain morphological units of polypeptide  capsomers – Capsomers are arranged in symmetric fashion 17  Structure and chemical compositions 1. Viral core – Site: in the centre of the virus – Compositions: viral nucleic acid, either DNA or RNA, may be double or single stranded – Functions: control the viral heredity and variation, responsible for the infectivity 18  Structure and chemical … 2. Capsid Accounts for most of the virion mass – It is a complex and highly organized entity which gives form to the virus – Subunits called protomeres aggregate to form Capsomers which in turn aggregate to form the capsid Site: surround the viral genome, outer of the viral core Compositions and arrangement: – Protein in nature and consists of Capsomers – Symmetry in arrangement: helical symmetry; cubic or icosahedra symmetry; complex symmetry Functions of viral capsid: 1. protects the viral nucleic acid 2. participates in the viral infection 3. shares the antigenicity 19  Structure and chemical … 3. Envelope this is an amorphous structure composed of lipid, and glycoprotein – lies to the outside of the capsid – It contains a mosaic of antigens from the host and the virus Site: on the surface of some viruses Composition: lipid, protein and carbohydrate Functions: antigenicity, infectivity and resistance; some viruses possess neuraminidase 20  Structure and chemical … 4. Spikes These are glycoprotein projections which have enzymatic and/or adsorption and/or hemagglutinating activity They arise from the envelope and are highly antigenic 21  Structure and chemical … Simple viron (naked virus) - contain NA & capsid Complex viron (enveloped virus) - contain NA, capsid & envelope +ssRNA: same as viral mRNA -ssRNA: as a template transcribe complimentary mRNA 22  Viral Structure... Fig. 1. From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005,  Viral Symmetry - shape Three types – 1. Icosahedral (cubic) 2. helical 3. complex Cubic symmetry, the capsid is 20 triangular facets & 12 corners e.g. Adenovirus Helical symmetry- helical nucleocapsid e.g. Influenza virus Complex symmetry -- neither cubic nor helical symmetry may be brick shaped. poxvirus 24 Capsid symmetry Icosahedral Helical Naked capsid Enveloped Matrix Lipid Glycoprotein 25 Structure of DNA viruses The structure of RNA viruses  Classification of viruses Classification Structure Chemical composition Genetic makeup Host relationship Type of disease Baltimore Classification of Viruses 28  Classification of viruses … Criteria used for viral classification includes: 1. Classified mainly based on nucleic acid A. Type of nucleic acid (DNA or RNA) B. Physical construction (single / double stranded / linear / circular / segmented) C. Polarity of viral genome (positive sense / negative sense) 2. Symmetry of nucleocapsid (Icosahedral, Helical. Complex ) 3. Presence / absence of a lipid envelope 29  Baltimore Classification of Human Viruses "Group" Family Genome Genome size (kb) Capsid Envelope dsDNA Poxviridae dsDNA, linear 130 to 375 Ovoid Yes Herpesviridae dsDNA, linear 125 to 240 Icosahedral Yes Adenoviridae dsDNA, linear 26 to 45 Icosahedral No Polyomaviridae dsDNA, circular 5 Icosahedral No Papillomaviridae dsDNA, circular 7 to 8 Icosahedral No ssDNA Anellovirus ssDNA circular 3 to 4 Isometric No Parvoviradae ssDNA, linear, (- or +/-) 5 Icosahedral No Retro Hepadnaviridae dsDNA (partial), circular 3 to 4 Icosahedral Yes Retroviridae ssRNA (+), diploid 7 to 13 Spherical, rod or cone shaped Yes dsRNA Reoviridae dsRNA, segmented 19 to 32 Icosahedral No ssRNA (-) Rhabdoviridae ssRNA (-) 11 to 15 Helical Yes Filoviridae ssRNA (-) 19 Helical Yes Paramyxoviridae ssRNA (-) 10 to 15 Helical Yes Orthomyxoviridae ssRNA (-), segmented 10 to 13.6 Helical Yes Bunyaviridae ssRNA (-, ambi), segmented 11 to 19 Helical Yes Arenaviridae ssRNA (-, ambi), segmented 11 Circular, nucleosomal Yes Deltavirus ssRNA (-) circular 2 Spherical Yes ssRNA (+) Picornaviridae ssRNA (+) 7 to 9 Icosahedral No Calciviridae ssRNA (+) 7 to 8 Icosahedral No Hepevirus ssRNA (+) 7 Icosahedral No Astroviridae ssRNA (+) 6 to 7 Isometric No Coronaviridae ssRNA (+) 28 to 31 Helical Yes Flaviviridae ssRNA (+) 10 to 12 Spherical Yes Togaviridae ssRNA (+) 11 to 12 Icosahedral Yes Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Table 2.1 30  Nomenclature of viruses I. Named after disease: e.g. pox virus, herpes viruses II. On acronyms: e.g. Picornavirus (pico, small; rna, ribonucleic acid) III. On their morphology: e.g. coronavirus, that possess corona of spikes IV. Named after place they were first isolated: e.g. Coxsackie, Marburg V. Named after discoverers: e.g. Epstein-Barr virus 32  Host Range of viruses Most viruses infect specific host cells  host specific Host specificity is due to: – specific attachment sites on the host cells called receptors Receptor sites for bacteriophage are found in bacterial cell walls or fimbrae or flagella Animal cell membranes contain receptors for animal viruses e.g. CD4 for HIV – Availability of cellular factors required for viral multiplication in the host cells 34  Replication of viruses NA is necessary genetic information for replication Depend on host cell enzymes for replication Six phases in replication 1. Adsorption or Attachment 2. Penetration 3. Uncoating 4. Biosynthesis 5. Maturation/ assembly 6. Release 35  1. Adsorption /attachment Virion acquire contact with host cell – Affinity between virus & host cell viruses can attach specific receptor sites  host cells Examples: o Influenza virus (haemagglutinin)  glycoprotein receptor sites of host cell o HIV – gp120  CD4+ cells o Polio virus -- lipoprotein receptor of host cells Difference between Naked Vs. Enveloped virus?? 36 Enveloped virus Naked virus 2. Penetration Virus enter the cells Ways of viral entry into the cells: a - Endocytosis  for entry of naked viruses b - Simple fusion  Enveloped viruses * coat of enveloped viruses fuse with host cell member Viral NA is released (Release the virus nucleo- capsid into host cytoplasm) 39 Endocytosis Membrane Fusion  3. Uncoating Stripping of viral outer layers From capsid,  NA is released into host cell – Lysosomal enzymes of host cell enhance uncoating 41  4. Biosynthesis Synthesis of NA & capsid Production of enzymes needed for viral synthesis, assembly & release Regulator proteins are synthesized – Regulator proteins shut down normal host cell function 42  Enzymes encode for: - DNA / RNA polymerase - Other replication enzymes  NA replication produces new viral genomes dsDNA viruses:  Except Poxviruses, dsDNA viruses replicate in the nucleus using host cell dependent RNA polymerase to synthesize their mRNA  RNA viruses mainly in cytoplasm 43 RNA viruses : 3 kinds 1. +ssRNA +ssRNA virus itself can direct as viral mRNA 2. -ssRNA Use the virion RNA polymerase to synthesize viral mRNA 3. Retro viruses Use virion reverse transcriptase to synthesize a DNA copy of the viral RNA genome Use the host cell RNA polymerase to synthesize viral mRNA 44 45  5. Maturation Assembly of daughter virions into capsids  in host cell nucleus or cytoplasm Assembly of viral nucelo - capsid may take place in: Nucleus………………DNA viruses Cytoplasm…………...RNA viruses At cell surface….........Avian Influenza virus Herpes and Adenoviruses  host cell nucleus Picorna and Poxviruses assembly occur in cytoplasm of host cell  Accumulation of virions at sites of assembly may form inclusion bodies 46  6. Release Release of new intact infectious virions May occur by: Virus budding (A-D)  Budding through cell membrane A B C D e.g: Enveloped viruses  Lysis of infected host cells e.g: Naked viruses 47  Release Progeny virions released from host cells by budding from cell membrane 1. Cell to cell fusion 2. Rupture of host cell A Magnified View of Viral Budding - ENVELOPED VIRUSES 48 General replication scheme for an animal virus  Binding Reverse transcription Fusion Integration Endocytosis Transcription Nuclear localization Splicing Uncoating RNA export Lysosome Maturation Genomic RNA Assembly Modification mRNA Budding Translation Stages of replication - DNA virus Attachment, penetration, uncoating, transfer of DNA to cell nucleus Viral DNA integrated in host DNA transcription Replication translation mRNA Viral replication enzymes Synthesis of more viral DNA translation Viral proteins, Capsid etc Integration of viral proteins in host cell membrane Assembly Release: Budding thru membranes (knoppskyting) Cancer cell Lysis Oncogenic viruses  Stages of replication - RNA virus (+) stranded RNA = mRNA Alternative A: (-) RNA virus More difficult to treath RNA virus infect. 3' 5' (-) stranded RNA More mutation - Less repair mech. than with DNA Viral RNA replicase Protein 5' 3' mRNA (+) stranded RNA Alternative B: (+) RNA virus (≠Retrovirus) Alternative C: Retrovirus; (+) RNA virus (+) stranded RNA (+) stranded RNA 5' 3' 5' 3' Viral RNA replicase 3' 5' (-) stranded RNA 3' 5' (-) DNA strand 5' 3' Viral RNA replicase Retroviral Reverse transcriptase 5' 3' 3' 5' 5' 3' Protein 5' 3' (+) stranded RNA 3' 5' incl. mRNA Further replication 5' 3' as a DNA virus  Products of viral replication (Unusual multiplication) Virion: the complete infectious unit of virus particle 1. Abortive virus/ infection Virus infects a cell but can not complete the full replication cycle – can not produce a Virion 2. Defective virus Lacks one or more functional proteins (genes) required for replication Can not replicate with out a helper virus that provide missing genes e.g: HDV requires helps from HBV 3. Interference Infection of cell by a virus results in that cell becoming resistant to infection by other viruses 54 Unconventional viruses Are sub viruses: infectious agents smaller than normal viruses 1. Viroid & virusoid  Contain RNA only  Associated with plant disease only 2. Prions  Infectious particles encoded by gene of normal host cells  Composed of proteins only  Cause of certain ‘slow virus disease called transmissible apongiform encephalopathies’ in animals & humans 55  Bacteriophages/phages Viruses that infects bacteria Close association with bacteria Phages are responsible for transduction Bacteriophage have –Polyhedral head –Helical tail –Fibers for attachment Are considered either LYTIC or TEMPERATE Are often associated with virulence genes in bacteria EX. diphtheria toxin in Clostridium diphtheriae - also Bo-Tox from C. botulinum 56 Bacteriophages/phages T-even bacteriophage penetrate the host cell by specifically binding and injecting their DNA into the host cell. After replication, bacteriophage release lysozyme, weaken/destroy/rupture cell and release numerous virions  Pathogenesis of Viral Infection Interaction between viruses and host cells – Presence of receptors on surface of host cell determines whether virus can adsorb – Temperature and biochemical activity of host cell also play important role in initiating infection 58  Viral infection Cont’d... Important events in pathogenesis To cause disease, the virus must 1. Invade the host 2. Replicate in susceptible cells at site of inoculation 3. Overcome local defenses, for example lymphocytes, macrophages and interferon 4. Spread from site of inoculation to other areas, often via blood stream 59  Viral infection Cont’d... 5. Undergo further replication in its target areas, it may be localized (e.g. Adenovirus conjunctivitis) or generalized (e.g. measles) 6. Exit from the host in numbers large enough to infect other susceptible hosts and to ensure its own survival 60  Viral infection Cont’d... Determinants of Viral Pathogenesis 1. Interaction of Virus with Target Tissue – Access of virus to target tissue – Stability of virus in the body – Ability to cross skin or mucous epithelial cells (e.g., cross the gastrointestinal tract into the bloodstream) – Ability to establish viremia – Ability to spread through the reticuloendothelial system – Target tissue Specificity of viral attachment proteins Tissue-specific expression of receptors  Viral infection Cont’d... 2. Cytopathologic Activity of the Virus Efficiency of viral replication in the cell – Optimum temperature for replication – Permissiveness of cell for replication Cytotoxic viral proteins Inhibition of cell's macromolecular synthesis Accumulation of viral proteins and structures (inclusion bodies) – Altered cell metabolism (e.g., cell immortalization)  Viral infection Cont’d... 3. Host Protective Responses – Nonspecific antiviral responses Interferon Natural killer cells and macrophages – Antigen-specific immune responses T-cell responses Antibody responses – Viral mechanisms of escape of immune responses  Viral infection Cont’d... 4. Immunopathology – Interferon: Flu-like systemic symptoms – T-cell responses: Delayed-type hypersensitivity – Antibody: Complement, antibody-dependent cellular cytotoxicity, immune complexes – Other inflammatory responses Viral pathogenesis 1. Cycle of infection A. Entry B. Primary site replication C. Spread within the host D. Shedding E. Transmission 2. Effects on cells 66  Cycle of infection Entry Shedding Primary site Local Lymphatic Spread Neuronal Blood (viremia) Secondary sites Shedding 67  Mechanism of viral Cytopathogenesis Mechanism Examples Inhibition of cellular protein synthesis Polioviruse, herpes simplex virus, togavirusese, poxviruses Inhibition and degradation of cellular DNA Herpes virus Alteration of cell membrane structure Enveloped viruses Glycoprotein insertion All enveloped viruses Syncytia formation HrSV, VZV, paramyxoviruses, HIV Disruption of cytoskeleton Non-enveloped viruses (accumulation), HrSV Permeability Togaviruses, herpesvirus Inclusion bodies Negri bodies (intracytoplasmic) Rabies Toxicity of virion components Adenovirus fibers, reovirus NSP4 protien  Important mechanisms of viral cytopathogenesis (Effects on cells) 1. Inhibition of cellular protein synthesis oPoliovirus, Herpes simplex virus, Pox 2. Inhibition & degradation of cell DNA »Herpes viruses 3. Alteration of cell membrane structure »Enveloped viruses 4. Inclusion bodies formation 69  Some important target organs Skin: rash or exanthema e.g. Rubella, Measles, Herpes simplex virus, Chicken pox virus Lung: Infection may occur after local spread or as part of general infection e.g. Measles, varicella Liver: Target of Hepatitis viruses, yellow fever Kidney: cytomegalovirus, hantavirus CNS: poliovirus, arbovirus encephalitis, Herpes simplex, rabies, varicella-zoster 70 71  Viral Epidemiology Mechanisms of Viral Transmission 1) Aerosols 2) Food and water 3) Fomites (e.g., tissues, clothes) 4) Direct contact with secretions (e.g., saliva, semen) 5) Sexual contact, or at birth 6) Blood transfusion or organ transplant 7) Zoonosis (animals, insects  arboviruses]) Route of Viral Transmission  Viral Epidemiology ……..  Cause a large number of human diseases  May follow epidemiological patterns Sporadic infection – mumps Endemic disease – rabies Epidemic disease – measles Pandemics - influenza and HIV  May also cause cancer (Oncogenic viruses) 74 Laboratory Techniques for Diagnosing Viral Infections 1. Cell Culture (Virus isolation and growth) 2. Cytological examination 3. Electron microscopy 4. Detection of viral proteins (antigens and enzymes) 5. Detection of viral genomes 6. Serology  Cultivation and Replication In vivo methods 1. Laboratory animals 2. Embryonic bird tissues In vitro methods Cell or tissue culture  Cultivation of viruses Viruses cannot grow in cell free media Grow in cell cultures, egg inoculation & animal inoculation Cell culture refers maintenance of animal cells in vitro – Cultured cells currently provide the most widely used and most powerful hosts for cultivation and assay of viruses Three types of cell cultures 1. Primary cultures 2. Secondary cultures/cell strains 3. Continuous cell lines/cell lines 77  1. Primary cell cultures Primary cell cultures are derived directly from the source animal Primary cell cultures cannot be passaged for not more than few passages (sub cultures) Examples I. Chick Embryo Fibroblast Cell Culture, II. Primary Human Embryonic Kidney Cells, III. Primary Monkey Kidney Cell Cultures 78  2. Secondary cell cultures Can be passaged up to 50 passages (sub-culturings) Secondary cell cultures have undergone a change that allows their limited culture Retain their normal chromosome pattern Examples – I. WI-38 (human embryonic lung cell strain) II. HL-8 (rhesus embryo cell strain) 79  3. Continuous cell lines Cultures capable of more prolonged, indefinite growth  malignant tissues Examples – I. HEp-2 (human epithelioma of larynx cell line), II. HeLa (human carcinoma of cervix cell line), III. Vero (Vervet monkey kidney cell line), IV. McCoy (human synovial carcinoma cell line), V. KB (human carcinoma of nasopharynx cell line), VI. Detroit-6 (sternal marrow cell line) 80  B A B Cell culture in a petri dish and test tube  B A B Inoculation into the amniotic cavity of the chick embryo (B) and into the yolk sac (A).  Detection of virus infected cells Recognition of Viral Growth in Culture Two principal methods exist: 1. Cytopathic effect Virus infected cells can be identified by development of cytopathic effect (CPE) – These effects are cytological changes CPE produced by each virus varies from each other 83  CPE is composed of two different phenomena A) Morphologic Changes – induced in individual cells or groups of cells by virus infection – easily recognizable under a light microscope Rounding, Shrinkage, Increased Refractility, Fusion, Aggregation, Loss of Adherence, Or Lysis 84  CPE is composed of two different phenomena B) Inclusion Bodies are more subtle alterations to the intracellular architecture of individual cells Refers to the observation of intracellular abnormalities specific to an infected cell and Discernable by light microscopy 85  Inclusion bodies….. The presence of a specific type of inclusion body can be diagnostic of a specific virus infection Inclusion bodies can be found in– – Cytoplasm (pox virus) – Nucleus (herpesvirus) – In both (measles virus) – Intra-cytoplasmic inclusion in nerve cells (Rabies virus) 86  Inclusion bodies – Negri bodies (Intra-cytoplasmic):  Rabies virus – Owl’s eye (Intra nuclear):  Cytomegalovirus – Cowdry type A (intra nuclear):  HSV, Measles – Intra nuclear basophilic:  Adenoviruses – Intra cytoplasmic acidophilic:  Poxviruses – Peri-nuclear cytoplasmic acidophilic:  Reoviruses 87  Cytopathic Effect Syncytium formation in cell culture caused by RSV (top), and measles virus (bottom). From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-1. 88 2. Hemadsorption: Refers to the ability of red blood cells to attach specifically to virus-infected cells Many viruses synthesize cell attachment proteins, which carry out their function wholly or in part by binding substituants (e.g., sialic acid) that are abundant on a wide variety of cell types, including erythrocytes viral proteins are expressed on the surface of the infected cell cluster of infected cells can be easily detectable to the naked eye as areas that stain red after exposure to an appropriate preparation of red blood cells Hemadsorption can be a particularly useful assay for detecting infections by viruses that cause little or no CPE 89  Hemadsorbtion refers to indirect measurement of viral protein synthesis in infected cells detected by adsorption of erythrocytes to the surface of infected cells It is due to presence of virus encoded haemagglutinin in the cell surface Haemadsorption may be positive even before the appearance of CPE in infected cell Cytopathic effect is the simplest and most widely used criterion for infection  By polymerase chain reaction (PCR) it is possible to detect viral nucleic acid in infected cells rapidly 90  Electron Microscopy Viruses may be detected in the following specimens using EM: Faeces  Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus Vesicle Fluid  HSV, VZV Skin scrapings  Papillomavirus, orf Molluscum contagiosum  Diagnostic methods in Virology In general, diagnostic tests can be grouped into 3 categories 1. direct detection 2. indirect examination (virus isolation), and 3. serology In direct examination, the clinical specimen is examined directly for the presence of virus particles, virus antigen or viral nucleic acids In indirect examination, the specimen inoculated into cell culture, eggs or animals in an attempt to grow the virus: this is called virus isolation The majority of common viral infections can be diagnosed by serology 92 Direct Examination of Specimen 1. Electron Microscopy morphology / immune electron microscopy 2. Light microscopy histological appearance - e.g. inclusion bodies 3. Antigen detection immunofluorescence, ELISA etc. 4. Molecular techniques for the direct detection of viral genomes 93 Indirect Examination 1. Cell Culture - cytopathic effect, haemadsorption 2. Animals inoculation Serology – Detection of rising titers of antibody 94  Viral vaccines Purpose of viral vaccines is to utilize immune response of the host to prevent viral disease Several remarkably effective – Small pox eradicated – Reducing annual incidence several viral diseases 95 Making the Influenza vaccine  Viral Vaccines Virus Vaccine Components Who Should Receive Vaccinations Polio Inactivated (inactivated polio vaccine, Salk Children vaccine) Attenuated (oral polio vaccine, Sabin vaccine) Children Measles Attenuated Children Mumps Attenuated Children Rubella Attenuated Children Varicella-zoster Attenuated Children Influenza Inactivated Adults, especially medical personnel and the elderly Attenuated (nasal spray) Older children, adults