General Characteristics of Viruses Michael Piatak Jr Ph

General Characteristics of Viruses Michael Piatak, Jr, Ph. D AIDS Vaccine Program SAIC Frederick, Inc. National Cancer Institute, Frederick

HIV ! Avian Flu ! SARS ! Ebola ! West Nile ! Smallpox ! Hepatitis A, B, C, D, E ! Polio ! HPV !

Viruses infect all living things • • • Animal kingdom Plant kingdom Microbes • Specificity/selectivity for host

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 • • • Virus replication Targeted cells Host response

What is a virus? What are the essential characteristics of a virus? • Physical • Biological

What is a virus? • Physical Characteristics – Genetic Material • Nucleic acid – RNA (ss. RNA, ds. RNA, segmented) – DNA (ss. DNA, ds. DNA) – Protein coat (subunit structure) • Nucleoprotein • Capsid – Capsomeres, Geometrical constraints – Envelope (some)

HIV-1, SIV and Other Retroviruses Rhabdoviruses (rabies) Adenoviruses

Size of Viruses

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? Biological Charateristics • Viruses as parasites • Parasites – Not capable of independently completing a full replication cycle – Cellular parasites – Subcellular “parasites” • Viruses • Viroids • Prions

What is a virus? • Are viruses alive? • Characteristics of living organisms?

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 infection

Vaccinia Poliovirus Adenovirus Herpesvirus

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, Plaquereduction neutralization • Physical: Differential centrifugation, Solvent resistance • Nucleic acid based: PCR, Sequencing, Hybridization, In vivo labeling, etc… • Host/Virus interaction: Host range, LD 50, Vaccinationchallenge, Suckling mice, Epidemiology studies

Morphological Assays • Electron microscopy • Isolated virus or infected cells • Nonenveloped or enveloped (alternate method to determine if an envelope is present? ) • Core structure

Biochemical - Density Gradient Separation

Cell/virus –titrate infectious units Uninfected Infected Plaque Assay Plaque assays are usually over layered with agarose Syncytia Assay

Titration: TCID 50 Assay 1 8/8 = 100% 103 8/8 = 100% 104 8/8 = 100% 105 8/8 = 100% 106 8/8 = 100% 107 6/8 = 63% TCID 50 108 2/8 = 25% 109 0/10 = 0% 1010 0/10 = 0% 1011 0/10 = 0% 1012 0/10 = 0% Host/virus interactions – LD 50

Metabolic Labeling • Proteins: Radioactive amino acids, [35 S]Methionine • Nucleic Acids: Radioactive nucleotides, [32 P]ATP Immunological • • • ELISA Western Blot Antibody Neutralization

ELISA – antigen capture of HIV-1 capsid protein Color light p 24

Western Blot Assays SIV (gp 32+gp 41) SIV (gp 32) HIV-1 Experimental Mol. Wt. Std. s HIV/SIV Example gp 120 gp 41 < SIV anti-gp 41 membrane < HIV anti-gp 41 SIV, anti-gp 41 HIV-1, anti-gp 41 Antibody recognizes extracellular domain Antibody recognizes intracellular domain

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 analysis

Molecular Assays – Quantitative PCR

Plasma SIV RNA (Copy/Eq/m. L) SIV infection in Rhesus macaques PMPA + FTC Weeks SIVsm. E 660 CD 8 SIVsm. E 660 SIVmac 239

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 virus

Acknowledgements Jeff Lifson, MD Director, AIDS Vaccine Program Bruce Crise, Ph. D AIDS Vaccine Program SAIC/NCI-Frederick