logging in or signing up Piatak Lucianna Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINTLite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 391 Category: Entertainment License: All Rights Reserved Like it (1) Dislike it (0) Added: October 23, 2007 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript General Characteristics of Viruses Michael Piatak, Jr, PhD AIDS Vaccine Program SAIC Frederick, Inc. National Cancer Institute, Frederick: General Characteristics of Viruses Michael Piatak, Jr, PhD AIDS Vaccine Program SAIC Frederick, Inc. National Cancer Institute, FrederickSlide2: HIV ! Avian Flu ! SARS ! Ebola ! West Nile ! Smallpox ! Hepatitis A,B,C,D,E ! Polio ! HPV ! Viruses infect all living things: Viruses infect all living things Animal kingdom Plant kingdom Microbes Specificity/selectivity for host Manifestations of Viral Infections: Manifestations of Viral Infections Asymptomatic Acute viral syndrome (influenza, rhinovirus, etc) Persistent viral syndrome (EBV) Chronic infection Reactivating (HSV-1/2, VZV (chicken pox/shingles)) Progressive (HBV, HCV, HIV) Cancer (EBV, HPV-16, HBV, HCV, KSHV) Death (HIV, et al) Acute death (smallpox, Ebola, SARS)Viral pathogenesis --- disease: Viral pathogenesis --- disease Virus replication Targeted cells Host responseWhat is a virus?: What is a virus? What are the essential characteristics of a virus? Physical Biological What is a virus?: What is a virus? Physical Characteristics Genetic Material Nucleic acid RNA (ssRNA, dsRNA, segmented) DNA (ssDNA, dsDNA) Protein coat (subunit structure) Nucleoprotein Capsid Capsomeres, Geometrical constraints Envelope (some)Slide8: Adenoviruses Rhabdoviruses (rabies)Size of Viruses: Size of VirusesWhat is a virus?: What is a virus? Genetic material limited 10^3 to 10^5 bases long Codes for unique proteins Polymerases – RNA replication Kinases Proteases Structural proteins Accessory proteins Targets for antiviral agents - limited What is a virus?: What is a virus? Biological Charateristics Viruses as parasites Parasites Not capable of independently completing a full replication cycle Cellular parasites Subcellular “parasites” Viruses Viroids PrionsWhat is a virus?: What is a virus? Are viruses alive? Characteristics of living organisms? What is a virus?: What is a virus? Biological Characteristics Viral replication strategies Viral replication cycles Binding/entry Uncoating Genome replication (w/wo integration) Protein production Assembly Budding Host cell tropism (receptors) Lytic vs. non-lytic infectionSlide14: Vaccinia Poliovirus Adenovirus HerpesvirusHow to characterize and study viruses: How to characterize and study viruses Structural & Morphological: X-ray crystallography, Electron microscopy Biochemical: Gel electrophoresis, filtration, metabolic labeling, Enzymatic assays (Reverse Transcriptase, Neuraminidase) Cell/Virus interaction: Hemagglutination, Titration, Focus forming, Plaque assay, Syncytia, In vivo labeling Immunological: ELISA, Western blot, IFA, Plaque-reduction neutralization Physical: Differential centrifugation, Solvent resistance Nucleic acid based: PCR, Sequencing, Hybridization, In vivo labeling, etc… Host/Virus interaction: Host range, LD50, Vaccination-challenge, Suckling mice, Epidemiology studiesMorphological Assays: Morphological Assays Electron microscopy Isolated virus or infected cells Nonenveloped or enveloped (alternate method to determine if an envelope is present?) Core structureBiochemical - Density Gradient Separation: Biochemical - Density Gradient SeparationCell/virus –titrate infectious units: Cell/virus –titrate infectious units Plaque assays are usually over layered with agarose Uninfected Infected Plaque Assay Syncytia AssayTitration: TCID50 Assay: Titration: TCID50 Assay 8/8 = 100% 8/8 = 100% 8/8 = 100% 8/8 = 100% 8/8 = 100% 8/8 = 100% 6/8 = 63% TCID50 2/8 = 25% 0/10 = 0% 0/10 = 0% 0/10 = 0% 0/10 = 0% Host/virus interactions – LD50Metabolic Labeling : Metabolic Labeling Proteins: Radioactive amino acids, [35S]Methionine Nucleic Acids: Radioactive nucleotides, [32P]ATP Immunological ELISA Western Blot Antibody Neutralization Slide22: ELISA – antigen capture of HIV-1 capsid protein p24 Color light Western Blot Assays HIV/SIV Example: Western Blot Assays HIV/SIV Example membrane gp120 gp41 < SIV anti-gp41 < HIV anti-gp41 Molecular Assays: Molecular Assays Is the genetic material isolated from the virus infectious? Assay for viral genome RNA verses DNA Northern blot & RT/PCR, Southern blot and PCR Sequence analysis Mutation analysisMolecular Assays – Quantitative PCR: Molecular Assays – Quantitative PCRSIV infection in Rhesus macaques: SIV infection in Rhesus macaques SIVsmE660 SIVmac239 CD8 PMPA + FTC Weeks Plasma SIV RNA (Copy/Eq/mL) SIVsmE660Epidemiology: Epidemiology Tracking prevalence of viruses in host populations Blood components and tissue samples Virus culture In vitro (tissue culture) In vivo (susceptible animal model) Molecular testing PCR methods Southern blot ELISA (enzyme linked immunosorbent assay) Assay for antigen (viral protein) Assay for antibody directed against virusAcknowledgements: Acknowledgements Jeff Lifson, MD Director, AIDS Vaccine Program Bruce Crise, PhD AIDS Vaccine Program SAIC/NCI-Frederick You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
Piatak Lucianna Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINTLite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 391 Category: Entertainment License: All Rights Reserved Like it (1) Dislike it (0) Added: October 23, 2007 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript General Characteristics of Viruses Michael Piatak, Jr, PhD AIDS Vaccine Program SAIC Frederick, Inc. National Cancer Institute, Frederick: General Characteristics of Viruses Michael Piatak, Jr, PhD AIDS Vaccine Program SAIC Frederick, Inc. National Cancer Institute, FrederickSlide2: HIV ! Avian Flu ! SARS ! Ebola ! West Nile ! Smallpox ! Hepatitis A,B,C,D,E ! Polio ! HPV ! Viruses infect all living things: Viruses infect all living things Animal kingdom Plant kingdom Microbes Specificity/selectivity for host Manifestations of Viral Infections: Manifestations of Viral Infections Asymptomatic Acute viral syndrome (influenza, rhinovirus, etc) Persistent viral syndrome (EBV) Chronic infection Reactivating (HSV-1/2, VZV (chicken pox/shingles)) Progressive (HBV, HCV, HIV) Cancer (EBV, HPV-16, HBV, HCV, KSHV) Death (HIV, et al) Acute death (smallpox, Ebola, SARS)Viral pathogenesis --- disease: Viral pathogenesis --- disease Virus replication Targeted cells Host responseWhat is a virus?: What is a virus? What are the essential characteristics of a virus? Physical Biological What is a virus?: What is a virus? Physical Characteristics Genetic Material Nucleic acid RNA (ssRNA, dsRNA, segmented) DNA (ssDNA, dsDNA) Protein coat (subunit structure) Nucleoprotein Capsid Capsomeres, Geometrical constraints Envelope (some)Slide8: Adenoviruses Rhabdoviruses (rabies)Size of Viruses: Size of VirusesWhat is a virus?: What is a virus? Genetic material limited 10^3 to 10^5 bases long Codes for unique proteins Polymerases – RNA replication Kinases Proteases Structural proteins Accessory proteins Targets for antiviral agents - limited What is a virus?: What is a virus? Biological Charateristics Viruses as parasites Parasites Not capable of independently completing a full replication cycle Cellular parasites Subcellular “parasites” Viruses Viroids PrionsWhat is a virus?: What is a virus? Are viruses alive? Characteristics of living organisms? What is a virus?: What is a virus? Biological Characteristics Viral replication strategies Viral replication cycles Binding/entry Uncoating Genome replication (w/wo integration) Protein production Assembly Budding Host cell tropism (receptors) Lytic vs. non-lytic infectionSlide14: Vaccinia Poliovirus Adenovirus HerpesvirusHow to characterize and study viruses: How to characterize and study viruses Structural & Morphological: X-ray crystallography, Electron microscopy Biochemical: Gel electrophoresis, filtration, metabolic labeling, Enzymatic assays (Reverse Transcriptase, Neuraminidase) Cell/Virus interaction: Hemagglutination, Titration, Focus forming, Plaque assay, Syncytia, In vivo labeling Immunological: ELISA, Western blot, IFA, Plaque-reduction neutralization Physical: Differential centrifugation, Solvent resistance Nucleic acid based: PCR, Sequencing, Hybridization, In vivo labeling, etc… Host/Virus interaction: Host range, LD50, Vaccination-challenge, Suckling mice, Epidemiology studiesMorphological Assays: Morphological Assays Electron microscopy Isolated virus or infected cells Nonenveloped or enveloped (alternate method to determine if an envelope is present?) Core structureBiochemical - Density Gradient Separation: Biochemical - Density Gradient SeparationCell/virus –titrate infectious units: Cell/virus –titrate infectious units Plaque assays are usually over layered with agarose Uninfected Infected Plaque Assay Syncytia AssayTitration: TCID50 Assay: Titration: TCID50 Assay 8/8 = 100% 8/8 = 100% 8/8 = 100% 8/8 = 100% 8/8 = 100% 8/8 = 100% 6/8 = 63% TCID50 2/8 = 25% 0/10 = 0% 0/10 = 0% 0/10 = 0% 0/10 = 0% Host/virus interactions – LD50Metabolic Labeling : Metabolic Labeling Proteins: Radioactive amino acids, [35S]Methionine Nucleic Acids: Radioactive nucleotides, [32P]ATP Immunological ELISA Western Blot Antibody Neutralization Slide22: ELISA – antigen capture of HIV-1 capsid protein p24 Color light Western Blot Assays HIV/SIV Example: Western Blot Assays HIV/SIV Example membrane gp120 gp41 < SIV anti-gp41 < HIV anti-gp41 Molecular Assays: Molecular Assays Is the genetic material isolated from the virus infectious? Assay for viral genome RNA verses DNA Northern blot & RT/PCR, Southern blot and PCR Sequence analysis Mutation analysisMolecular Assays – Quantitative PCR: Molecular Assays – Quantitative PCRSIV infection in Rhesus macaques: SIV infection in Rhesus macaques SIVsmE660 SIVmac239 CD8 PMPA + FTC Weeks Plasma SIV RNA (Copy/Eq/mL) SIVsmE660Epidemiology: Epidemiology Tracking prevalence of viruses in host populations Blood components and tissue samples Virus culture In vitro (tissue culture) In vivo (susceptible animal model) Molecular testing PCR methods Southern blot ELISA (enzyme linked immunosorbent assay) Assay for antigen (viral protein) Assay for antibody directed against virusAcknowledgements: Acknowledgements Jeff Lifson, MD Director, AIDS Vaccine Program Bruce Crise, PhD AIDS Vaccine Program SAIC/NCI-Frederick