MICR 201 Microbiology for Health Related Sciences Lecture 6: Viruses, viroids, and prions Edith Porter, M.D. MICR 201 Microbiology for Health Related Sciences

Lecture Outline Viruses Viroids Prions General characteristics Viral structure Taxonomy Isolation, cultivation and identification Viruses and disease Viroids Prions

Viruses

What is a Virus? Virus is Latin for poison Initially, viral disease described as disease that could be transmitted with poisonous fluid that had been passed through a “sterile” filter Acellular infectious agent Exceptionally complex aggregation of non-living chemicals OR Exceptionally simple living organism

Size of viruses in comparion with bacteria and eukaryotic cells Prions are not viruses

General characteristics of viruses Obligatory intracellular agents Require a cellular host for proliferation Multiply inside living cells by using entirely the synthesizing machinery of the cell Cause the synthesis of specialized structures that can transfer the viral nucleic acid to other cells Most viruses infect only specific types of cells in one host Host range is determined by specific host attachment sites and cellular factors Contain single type of nucleic acid (DNA or RNA) Contain a protein coat Some are enclosed by an envelope Some viruses have spikes

Bacteria and viruses compared

Basic Viral Structure Contain a single type of nucleic acid Either DNA or RNA Single or double stranded Protein coat surrounding nucleic acid Capsid Composed of capsomer subunits Protects Vehicle for transmission May contain a very limited number of enzymes Virion: complete virus with nucleic acid and protein coat

Main Appearance Forms of Viruses Polyhedral (spherical, icosahedral) Helical (filamentous) Complex

Additional virus structures Spikes Glycoproteins projecting from surface Can clump cells Hemagglutination Envelopes Originate mainly from host membranes Lipid, protein, carbohydrate Some virus encoded proteins

Viral taxonomy Based on nucleic acid composition, replication mode, morphology Family names end in –viridae (Herpesviridae) Genus names end in –virus (Herpesvirus) Viral species: A group of viruses sharing the same genetic information and ecological niche (host). Common names are used for species (Herpes simplexvirus) Subspecies are designated by a number (Herpes simplexvirus 2) Often abbreviated names HSV HIV CMV

Fits only to specific structures (receptors) on the host cell Viral hosts In principle, any cell can be infected by a virus A particular virus can infect only a specific host cell type (receptor mediated entry) Prokaryotic cells Bacterial viruses (phages) Archaeal viruses Eukaryotic cells Fungal cells Algae Protozoa Plant cell viruses Insect cell viruses Animal viruses Mastadenovirus Fits only to specific structures (receptors) on the host cell

How to Culture Viruses? Need a living host cell Bacteriophages Bacteria Plant virus Plant cells Animal viruses Living animal Embryonated eggs Large scale production Cell culture diagnostics

Viral cytopathic effects in cell culture Virus induced changes of eukaryotic cell morphology Cell rounding Cell aggregation Inclusion bodies Cell fusion Transformation Continous growth of eukaryotic cell Can be used for diagnostic Normal Cell rounding

Virus identification Cytopathic effects Hemagglutination Monkey kidney cells Foreskin cells Fibroblasts Hemagglutination Serology (look for patient antibodies) PCR (polymerase chain reaction, detects virus specific nucleic acid sequences) RFLP (restriction fragment polymorphism)

How to Quantify Viruses Electron microscope Count Plaque Assay Bacterial lawn Add virus Add agar on top to immobilize virus Incubate Count plaques lack of bacterial growth where 1 virus had been

Viral multiplication : one-step growth curve Eclipse is the period immediately after penetration during which not a single intact virus is present In vitro, no new host cells are provided and number of virions decrease over time

Viral multiplication in bacteria Typically double stranded DNA viruses Lytic cycle Rapid large scale production of viruses Host cell lysis and death Lysogenic cycle Host cell survives Viral genome incorporated into host cell genome Replication with host cell No active virion production V V V V V V V V V V V DEAD V V V V

Lytic cycle of virus multiplication (1) Attachment Penetration Biosynthesis Maturation and assembly Host lysis and virion release V V V V V V V V V V V V DEAD

Lytic cycle of virus multiplication (2)

Lysogenic cycle of virus multiplication Attachment Penetration Phage DNA integrates into bacterial host genome by recombination Virus now: prophage Host cell: lysogenic bacterium Lysogenic conversion: bacterium produces virus encoded proteins Prevent superinfection with similar phage Some are toxins (e.g. diphteria toxin by C. diphteriae) Switch to lytic cycle can be induced by UV light Specialized transduction: accidentally, bacterial host DNA is cut out too V V V

Lysogenic and lytic cycle of bacteriophage l in E. coli

Transduction Virus serves as vector for bacterial DNA During virus assembly a segment of bacterial DNA is accidentally packed into virus capsids Specialized transduction: a segment of bacterial DNA along with the proper viral DNA Generalized transduction: only bacterial DNA is packaged into the capsid

Specialized transduction

Animal viruses DNA or RNA viruses Single or double stranded Negative or positive sense Unique viral biosynthesis pathways RNA viruses require enzymes not present in eukaryotic cells

Key steps in the multiplication of animal viruses Attachment Entry Uncoating Biosynthesis Early genes for replication Late genes for structural elements Assembly (maturation) Release Host rupture: non-enveloped viruses Budding: enveloped viruses

Entry and exit of animal viruses

Bacteriophage and animal virus multiplication compared

Retroviruses RNA viruses Include HIV Carry reverse transcriptase RNA-dependent DNA polymerase Synthesize DNA from RNA Used in molecular biology (RT-PCR)

Multiplication of a retrovirus

The course of viral diseases Acute Infection Unspecific: fever, muscle and joint aches Specific: depend on target host cell Latent Infections Virus retreats in host cells Herpesviridae in neurons (fever blisters) Persistent Infections Slow virus disease Gradual increase of symptoms Subacute sclerosing panenecephalitis after measles infection Cancer Chicken leukemia virus, Epstein Barr virus (lymphoma), HPV (cervix carcinoma) HBV (liver cancer) http://pathcuric1.swmed.edu/PathDemo/gbp3/gbp340.jpg

Examples for viral diseases Caused by various virus genera Conjunctivitis Diarrhea Encephalitis “Flu” Influenza SARS Avian flu Hepatitis Diseases with virus specific symptoms Measles Rubella Herpes AIDS

Viroids RNA only Short piece of naked RNA RNA does not code for protein Similarities between introns and viroids Often found in plant diseases

Prions Protein only Normal protein variant exists (PrPc) Proteinaceous infectious particle Infectivity can be reduced with protease treatment Infects central nervous system Normal protein variant exists (PrPc) Prion protein (PrPsc) induces conformation change of normal variant and aggregation Snow ball effect Damage in central nervous system due to loss of cell function and inflammatory host response Neurological disease Mad cow disease (with limited human transmission) Scrapie Creutzfeldt-Jakob disease

Multiplication of prions

Important to remember Viruses Viroids Prions Acellular, requires a host cell to mulitply Protein capsid and 1 type of nucleic acid (RNA or DNA) Spherical, helical and complex structure Can have envelopes and carry a few enzymes Lytic and lysogenic multiplication in bacteria Uncoating and budding is part of animal virus multiplication Viroids RNA only Plant diseases Prions Protein only Neurological diseases