The

Infec)ous Cycle
Lecture 2 Biology W3310/4310 Virology Spring 2012

The Infec)ous Cycle

Virologists divide the infec>ous cycle into steps to facilitate their study, but no such ar>cial boundaries occur

Some important deni)ons

A suscep)ble cell has a func>onal receptor for a given virus - the cell may or may not be able to support viral replica3on A resistant cell has no receptor - it may or may not be competent to support viral replica3on A permissive cell has the capacity to replicate virus - it may or may not be suscep3ble A suscep)ble AND permissive cell is the only cell that can take up a virus par>cle and replicate it
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Animal viruses, though discovered in early 1900s, could not be rou>nely propagated in cultured cells Most viruses were grown in laboratory animals

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Studying the infec)ous cycle in cells

Not possible before 1949 (animal viruses) Enders, Weller, Robbins propagate poliovirus in human cell culture - primary cultures of embryonic >ssues Nobel prize, 1954

hRp://www.virology.ws/2009/02/09/the-amazing-hela-cells-of-henrieRa-lacks/
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Virus cul)va)on

primary human mouse broblast cell line foreskin broblasts (3T3)

human epithelial cell line (HeLa)

con>nuous cell lines diploid cell strains (e.g. WI-38, human embryonic lung)
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cytopathic eect (CPE)

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Forma>on of syncy3a

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How many viruses?

Infec>vity Physical: virus par>cles and their components

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Plaque assay

1930s: used to study mul>plica>on of bacteriophages

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Plaque assay

1952, Renato Dulbecco showed that animal viruses form plaques in a similar way as bacteriophages Nobel Prize, 1975

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Plaque assay

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How many viruses are needed to form a plaque?

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Plaque purica)on

A method for producing clonal virus stocks. Usually done 3 >mes. Why?
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Endpoint dilu)on assay

10-2 -3 10 10-4

TCID50

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Par)cle-to-PFU ra)o

Number of virus par>cles in sample/number of infec>ous par>cles ~1 for many bacteriophages High for many animal viruses Complicates study of animal viruses

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Par)cle-to-PFU ra)o

A single par>cle can ini>ate infec>on (how do we know this?) High par>cle-to-pfu ra>o: not all viruses are successful. Why not?

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damaged par>cles muta>ons complexity of infec>ous cycle: failure at any one step prevents comple>on
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One-step growth cycle

Ellis & Delbruck, 1939, studies on E. coli bacteriophages Adsorb Dilute culture Sample Assay

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Synchronous infec)on - key to one-step growth cycle

Need to infect all cells How do we know?

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Mul)plicity of infec)on (MOI)

Number of infec>ous par>cles ADDED per cell Not the number of infec>ous par>cles each cell receives Add 107 virions to 106 cells - MOI of 10 - each cell does NOT receive 10 virions

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MOI

Infec>on depends on the random collision of virions and cells When suscep>ble cells are mixed with virus, some cells are uninfected, some receive one, two, three or more par>cles The distribu>on of virus par>cles per cell is best described by the Poisson distribu3on

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P(k) = e-mmk/k! P(k): frac>on of cells infected by k virus par>cles m: mul>plicity of infec>on (moi) uninfected cells: P(0) = e-m cells receiving 1 par>cle: P(1) = me-m cells mul>ply infected: P(>1) = 1-e-m(m+1) [obtained by subtrac>ng from 1 {the sum of all probabili>es for any value of k} the probabili>es P(0) and P(1)]
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Examples: If 106 cells are infected at an moi of 10: 45 cells are uninfected 450 cells receive 1 par>cle the rest receive >1 par>cle If 106 cells are infected at an moi of 1: 37% of the cells are uninfected 37% of the cells receive 1 par>cle 26% receive >1 par>cle If 106 cells are infected at an moi of .001: 99% of the cells are uninfected 0.1% of the cells receive 1 par>cle (1000) 0.000001% receive >1 par>cle
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Physical measurements of virus par)cles

Hemagglu>na>on Electron microscopy Viral enzymes Serology Nucleic acids

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Hemagglu)na)on - measurement of virus par)cles

GK Hirst, 1941 First rapid, quan>ta>ve assay for eukaryo>c viruses

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Measurement of viral enzyme ac)vity

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Immunostaining

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ImmunobloLng (western blot analysis)

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Enzyme-linked immunosorbent assay (ELISA): detec)ng viral an)gens or an)bodies

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Green uorescent protein

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Southern blot hybridiza)on

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Polymerase chain reac)on (PCR)

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Pyrosequencing

400-600 million bases per 10 hr run

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Viruses and viral sequences

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In vivo viral infec)ons usually begin at exposed epithelial surfaces

Simple Squamous: thin cells, blood vessels

Three topological surfaces: Apical: presented to outside (top) Basal: presented to inside (boRom) Lateral: side-to-side cell contacts

Simple columnar: thick, mucous secre>ng; GI tract

Transi)onal: Dis>nct layers: expandable

Stra)ed Squamous: Epidermal layer of skin, mouth, genitals

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