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VIROLOGY

(The study of Viruses)

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Dr. Kaveh Haratian Department of Microbiology and Immunology

Alborz University of Medical Sciences

Fall 1394

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OUTLINE

• introduction to viruses

structure and classification

• basic virology

• clinical virology

In VIROLOGY we notice:

different structure

different method of replication

implications for

diagnosis

treatment

prevention

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CONTROL METHODS

INVOLVE KNOWLEDGE OF:

RESERVOIRS

MODE OF TRANSMISSION

METHODS TO INACTIVATE VIRUS OF INTEREST

VACCINES

ANTI-VIRAL DRUGS

DEVELOPMENT OF DRUG RESISTANCE

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EMERGING VIRAL DISEASES

Some new global examples:

HIV/AIDS

Hantavirus pulmonary syndrome (HPS)

West Nile encephalitis (WNV)

Severe acute respiratory syndrome (SARS)

Monkey pox

Human metapneumovirus

Ebola hemorrhagic disease

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Consequences of viral infections

50% of all absenteeism

Children:

7 or more viral infections per year that

involve a visit to a physician

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Consequences of viral infections

Suffering, followed by recovery

Persistent disease

Fatal disease

Congenital disease

Contributory factor in cancer

Contributory factor in other diseases

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SOME ARE ASYMPTOMATIC!

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VIRUSES CAN BE USEFUL

VACCINE DEVELOPMENT

GENE THERAPY

TOOLS TO INVESTIGATE HOST CELLS

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WHAT ARE VIRUSES?

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“A PIECE OF BAD NEWS

WRAPPED UP IN A PROTEIN”

WHAT ARE VIRUSES?

NUCLEIC ACID GENOME:

DNA OR RNA

PROTEIN COAT

PROTECTION, ENTRY

LIPID ENVELOPE IN SOME VIRUSES

SMALL

(20-400nm)

OBLIGATE INTRACELLULAR PARASITES

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Virus particle =

virion

White, DO and Fenner, FJ.

Medical Virology, 4th Ed. 1994

14 Koneman et al. Color Atlas and Textbook of Microbiology 5th Ed. 1997

Virus versus Virion

Virus is a broad general term for any aspect of the infectious agent and includes:

• the infectious or inactivated virus particle

• viral nucleic acid and protein in the infected cell

Virion is the physical particle in the extra-cellular phase which is able to spread to new host cells; complete intact virus particle

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Growth

on

artificial

media

Division

by

binary

fission

Contain

DNA and

RNA

Contain

protein

synthesi

s

machine

ry

Contain

muramic

acid

Sensitiv

e to

antibioti

cs

Bacteria

often

yes

yes

yes

often

yes

Viruses

never

no

Either

DNA or

RNA

no*

no

no

* The arenavirus family appears to ‘accidentally’ package ribosomes, but these

appear to play no role in protein synthesis.

CONSEQUENCES

NO BROAD RANGE ANTIBIOTICS

HEAVILY PARASITIC ON HOST CELL

NEED TO LOOK FOR WEAK LINK

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HOST RANGE

MAY BE WIDE OR NARROW

MAY BE INSECT/ANIMAL,

INSECT/PLANT

DO NOT CROSS EUCARYOTE

/ PROCARYOTE BOUNDARY

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FACTORS AFFECTING HOST RANGE

- CELL SURFACE RECEPTORS

FACTORS AFFECTING HOST RANGE

CELL SURFACE RECEPTORS

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• AVAILABILITY OF REPLICATION MACHINERY

• ABILITY TO GET OUT OF CELL AND SPREAD

• HOST ANTI-VIRAL RESPONSE

VIRAL STRUCTURE : SOME

TERMINOLOGY

virus particle = virion

protein which coats the genome = capsid

capsid usually symmetrical

capsid + genome = nucleocapsid

may have an envelope

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ICOSAHEDRAL SYMMETRY

20 faces

12 vertices

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http://www.tulane.edu/~dmsander/WWW/Video/Video.html

ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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Adenovirus

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Adenovirus

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ICOSAHEDRAL SYMMETRY

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SYMMETRY OF

NUCLEOCAPSID

ICOSAHEDRAL

HELICAL

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TOBACCO MOSAIC VIRUS

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adapted from:

Klug and Caspar Adv. Virus Res. 7:225

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Helical symmetry

Length controlled by nucleic acid

Helix may be stiff or flexible

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COMPLEX SYMMETRY

44 POXVIRUS FAMILY

ENVELOPE

OBTAINED BY BUDDING THROUGH A CELLULAR

MEMBRANE (except poxviruses)

POSSIBILITY OF EXITING CELL WITHOUT KILLING IT

CONTAINS AT LEAST ONE VIRALLY CODED PROTEIN

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ENVELOPE

OBTAINED BY BUDDING THROUGH A CELLULAR MEMBRANE (except poxviruses)

POSSIBILITY OF EXITING CELL WITHOUT KILLING IT

CONTAINS AT LEAST ONE VIRALLY CODED PROTEIN

ATTACHMENT PROTEIN

LOSS OF ENVELOPE RESULTS IN LOSS OF INFECTIVITY

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ENVELOPE

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5 BASIC TYPES OF VIRAL STRUCTURE

49 HELICAL ENVELOPED HELICAL

ENVELOPED ICOSAHEDRAL

COMPLEX

ICOSAHEDRAL

Adapted from Schaechter et al., Mechanisms of Microbial Disease

nucleocapsid icosahedral nucleocapsid

nucleocapsid

helical nucleocapsid

lipid bilayer

lipid bilayer

glycoprotein spikes = peplomers

UNCONVENTIONAL AGENTS

VIROIDS

RNA only

Small genome

Do not code for protein

So far, only known viroids are in plants

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• hepatitis delta agent

- some viroid, some virus features

UNCONVENTIONAL AGENTS

PRIONS

protein only?

do not contain any nucleic acid?

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LIVING OR DEAD?

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CLASSIFICATION

BASIC STRUCTURE AND MOLECULAR BIOLOGY

particularly important as diagnostic and therapeutic abilities expand

past schemes

host range

tissue infected

type of cell infected

mode of transmission

disease caused

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Arboviruses

(arthropod borne)

CLASSIFICATION

NUCLEIC ACID

CAPSID

PRESENCE OF ENVELOPE

REPLICATION STRATEGY

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CLASSIFICATION

NUCLEIC ACID

RNA or DNA

segmented or non-segmented

linear or circular

single-stranded or double-stranded

if single-stranded

is genome mRNA (+) sense or complementary to mRNA (-)

sense

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symmetry

icosahedral, helical, complex

enveloped or non-enveloped

number of capsomers

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CLASSIFICATION CAPSID

CLASSIFICATION

ENVELOPE

REPLICATION STRATEGY

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HERPESVIRIDAE

HEPADNAVIRIDAE

ENVELOPED

PAPILLOMAVIRIDAE

POLYOMAVIRIDAE (formerly grouped together as the

PAPOVAVIRIDAE)

CIRCULAR

ADENOVIRIDAE

LINEAR

NON-ENVELOPED

DOUBLE STRANDED

PARVOVIRIDAE

SINGLE STRANDED

NON-ENVELOPED

POXVIRIDAE

COMPLEX

ENVELOPED

DNA VIRUSES

Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991

All families shown are

icosahedral except for

poxviruses

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FLAVIVIRIDAE TOGAVIRIDAE

RETROVIRIDAE

ICOSAHEDRAL

CORONAVIRIDAE

HELICAL

ENVELOPED

ICOSAHEDRAL

PICORNAVIRIDAE CALICIVIRIDAE

NONENVELOPED

SINGLE STRANDED positive sense

BUNYAVIRIDAE ARENAVIRIDAE

ORTHOMYXOVIRIDAE PARAMYXOVIRIDAE

RHABDOVIRIDAE FILOVIRIDAE

SINGLE STRANDED negative sense

REOVIRIDAE

DOUBLE STRANDED

RNA VIRUSES

ENVELOPED

HELICAL ICOSAHEDRAL

NONENVELOPED

Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991

Adenovirus

Herpesviridae

Influenza Virus

Smallpox Virus

Virus Classification

Historically based on:

Host preference: Plant, insect, animal, human

Target organ: respiratory, hepatic, enteric, etc.

Vector: arboviruses

Overlapping, inconsistent

Currently based on molecular biology of genome and biophysical

structure

Virus Classification

Viruses with similar structural, genomic &

replication properties are grouped into

families (suffix: viridae) e.g. Herpesviridae

Families subdivided into genera (suffix:

virus) e.g. Herpes simplex virus,

Cytomegalovirus, Varicella zoster virus

Subtypes based on nucleotide sequence

and antigenic reactivities e.g. Herpes

simplex virus type 1, Herpes simplex virus

type 2

Virus Classification

Viruses

Nucleic acid: DNA RNA

Envelope: Yes No

Symmetry: Cubic Helical

(Icosahedral) (Cylindrical)

Classification of Some Common Viruses

Family

Viruses

Type of

Nucleic Acid

Envelope

Capsid

Symmetry

Picornaviridae Enteroviruses,

polio, hep. A

ss (+) RNA No I

Caliciviridae Norwalk virus ss (+) RNA No I

Togaviridae Rubella ss (+) RNA Yes I

Rhabodoviridae Rabies ss (+) RNA Yes H

Paramyxoviridae Parainfluenza,

RSV, measles,

mumps

ss (-) RNA Yes H

Orthomyxoviridae Influenza ss (-) RNA Yes H

Retroviridae HIV 1,2, HTL I,II ss (+) RNA Yes I

Hepadnaviridae Hepatitis B ds DNA Yes Unknown

Parvoviridae Parovirus B - 19 ss (+) or (-) DNA No I

Adenoviridae Adenovirus ds DNA No I

Herpesviridae HSV, CMV, EBV,

VZV, HHV6

ds DNA

Yes

I

I = icosahedral, H = helical

Virus Classification

(Common)

DNA RNA

Hepatitis B Human Papilloma Virus

Parvovirus B19 Adenovirus

Herpesviridae Polyomaviruses

Influenza RSV

Parainfluenza Hepatitis A, C, D, E

Enteroviruses Encephalitis viruses

Measles, Mumps, Rubella Norwalk, Rotavirus Virtually all others

BASIC STEPS IN VIRAL LIFE

CYCLE

ADSORPTION

PENETRATION

UNCOATING AND ECLIPSE

SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN

ASSEMBLY (maturation)

RELEASE

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ADSORPTION

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ADSORPTION

TEMPERATURE INDEPENDENT

REQUIRES VIRAL ATTACHMENT PROTEIN

CELLULAR RECEPTORS

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PENETRATION

- ENVELOPED VIRUSES

•FUSION WITH PLASMA MEMBRANE

•ENTRY VIA ENDOSOMES

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PENETRATION

73 herpesviruses, paramyxoviruses, HIV

PENETRATION

- ENVELOPED VIRUSES

•FUSION WITH PLASMA MEMBRANE

•ENTRY VIA ENDOSOMES, FUSION WITH ACIDIC ENDOSOME MEMBRANE

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PENETRATION

- ENVELOPED VIRUSES

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from Schaechter et al, Mechanisms of Microbial Disease, 3rd ed, 1998

VIRUS UPTAKE VIA

ENDOSOMES

CALLED

VIROPEXIS / ENDOCYTOSIS / PINOCYTOSIS

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PENETRATION

NON-ENVELOPED VIRUSES

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PENETRATION

NON-ENVELOPED VIRUSES

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entry directly across

plasma membrane:

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UNCOATING

NEED TO MAKE GENOME AVAILABLE

ONCE UNCOATING OCCURS, ENTER ECLIPSE PHASE

ECLIPSE PHASE LASTS UNTIL FIRST NEW VIRUS PARTICLE FORMED

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SYNTHESIS OF VIRAL NUCLEIC

ACID AND PROTEIN

MANY STRATEGIES

NUCLEIC ACID MAY BE MADE IN NUCLEUS OR CYTOPLASM

PROTEIN SYNTHESIS IS ALWAYS IN THE CYTOPLASM

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ASSEMBLY AND MATURATION

NUCLEUS

CYTOPLASM

AT MEMBRANE

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smallpox virus cytoplasmic

assembly and maturation

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F. A. Murphy, School of Veterinary Medicine, University of California, Davis. http://www.vetnet.ucdavis.edu/fam_graphics/download.html

RELEASE

LYSIS

BUDDING THROUGH PLASMA MEMBRANE

NOT EVERY RELEASED VIRION IS INFECTIOUS

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HIV budding and maturation

87 Hsiung, GD et al., Diagnostic Virology 1994 p204 (D. Medina)

HIV – mature form

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Briggs JA et al. Structure. (2006) 14:15-20

Viral Replication

i) adsorption (attachment)

ii) entry

iii) uncoating

iv) transcription

v) synthesis of virus components

vi) assembly

vii) release

Viral Replication

i) Adsorption (attachment):

random collision

interaction between specific proteins on viral surface and specific receptors on target cell membrane (tropism)

not all cells carrying a receptor for a particular virus can be productively infected by that virus

Viral Replication

i) Adsorption (attachment):

some viruses may use more than one host cell receptor (e.g. HIV)

able to infect a limited spectrum of cell types (host range)

most neutralizing antibodies are specific for virion attachment proteins

Viral Replication

ii) Entry (penetration):

2 mechanisms - endocytosis - fusion of virus envelope with cell membrane

iii) Uncoating:

release of viral genome

cell enzymes (lysosomes) strip off the virus protein coat

virion can no longer be detected; known as the “eclipse period”

Viral Replication

iv) Transcription/Translation/Synthesis:

a) DNA viruses:

• replicate their DNA in host cell nucleus mediated

by viral enzymes

• synthesize capsid and other proteins in cytoplasm

using host cell enzymes

• new viral proteins move to nucleus where they

combine with new DNA to form new viruses

• Exception - Poxviruses synthesize their parts in

host cell’s cytoplasm

Viral Replication

iv)

Transcription/Translation/Synthesi

s:

b) RNA viruses:

–“+” sense RNA acts as mRNA - viral

proteins are made immediately in

cytoplasm mediated by viral

enzymes

–“-” sense RNA (e.g. influenza) - lst

makes a “+” sense RNA copy via

viral enzyme

Viral Replication

iv) Transcription/Translation/Synthesis:

• Retroviridae (e.g. HIV)

• Contain enzyme “Reverse

transcriptase” • “+” sense Viral RNA cDNA integrated into host cell

chromosome

• mRNA (for viral proteins) and progeny

virion RNA are synthesized from

integrated viral DNA by host cell

enzymes (RNA polymerases)

Viral Replication

v) Synthesis:

Protein synthesis - 2 types

• structural

• non-structural (enzymes for replication)

Nucleic acid synthesis

• new virus genome

• most often by a virus - coded polymerase or replicase; with some DNA viruses a cell enzyme carries this out

Viral Replication

vi) Assembly:

may take place in cell nucleus, cytoplasm or (with most enveloped viruses) at the plasma membrane

vii) Release:

sudden rupture of cell

gradual extrusion (budding) of enveloped viruses through the cell membrane

may occur together with assembly

Enveloped Virus Entry via Fusion

Non-enveloped Virus Entry via Endocytosis

Outcome of Viral Infections

Virus-host cell interaction may result in:

1. Cell death (lytic) - due to cytopathic

effect of virus

2. Cell transformation - cell converted to

malignant or cancerous cell

3. Latent infection (occult) - persistent

infection in quiescent state which may

reactive anytime to produce disease;

continuous or intermittent shedding

4. Cell fusion to form multinucleated cells

Persistent Viral Infections

3 types of persistent viral infection (some

overlap):

1. Chronic carrier - eg. Hepatitis B; results

in chronic illness

2. Latent infection - eg. Herpesviridae;

result in symptomatic or asymptomatic

shedding

3. Slow virus infections - due to prolonged

incubation period (eg. Measles virus and

SSPE)

Host - Organism Relationship

• Interaction between host and organism

affecting the development and outcome of

an infection includes:

– Host’s primary physical barriers

– Host’s immunologic ability to control and eliminate the

invading organisms

– Organism’s ability to evade, destruction/virulence

– Ability of organism to spread in the body

Virulence of Viruses & Evasion of the

Immune Response

Poorly understood processes:

Antigenic variation

Some viruses encode receptors for various

mediators of immunity (eg. IL1 & TNF) thus

blocking their ability to interact with receptors on

their intended targets

Some viruses (eg. HIV) reduce expression of class

I MHC proteins, thus reducing ability of cytotoxic T

cells to kill the virus-infected cells

Direct cell-to-cell propagation

Attenuated viruses (eg. Vaccine strains)

Definitions

Exposure: contact with a potentially

infectious agent

Infection: persistence on or within another

living organism

Disease: end product (damage) resulting

from an infectious process

Incubation: time from infection to

development of symptoms /

disease

Virus: Incubation Times

Hours to 1-2 days:

Respiratory viruses

GI viruses

1 to 3 weeks:

Measles/Mumps/Rubella

VZV, HSV

Chlamydia

Enteroviruses, Polio

WNV

Weeks to months:

• Hepatitis viruses

• HIV

• EBV

• Rabies

Months to years:

• Prions

Routes of Transmission

Horizontal transmission:

Direct contact (secretions, blood etc.)

Respiratory (aerosol)

Contaminated inanimate objects

Insect vector (mosquitoes, ticks, etc.)

Zoonoses

Vertical transmission:

Mother to fetus [Transplacental (Congenital), Perinatally]

Viruses - Transmission

Can occur - with or without disease

- during asymptomatic shedding

- during incubation period

Transmission results in primary infection disease; reactivation results in secondary disease

Viruses - Epidemiology

mode of transmission

age

gender

ethnic background / country of origin

travel history

occupation

season

underlying medical condition(s)

DEFINITIONS - VIRAL

PROTEINS

STRUCTURAL PROTEINS

ALL PROTEINS IN A MATURE VIRION

NON-STRUCTURAL PROTEINS

VIRALLY CODED PROTEINS WHICH ARE NOT PACKAGED IN

THE VIRION

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EFFECTS ON HOST

MAY INHIBIT HOST DNA, RNA OR PROTEIN SYNTHESIS

DETAILS AND MECHANISM VARY

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CYTOPATHIC EFFECT

ANY DETECTABLE CHANGES IN THE HOST CELL

MORPHOLOGICAL CHANGES

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114 Hockley et al. J Gen Virol 69:2455-2469

uninfected HIV infected

HIV infected

(at higher magnifcation)

CYTOPATHIC EFFECT

ANY DETECTABLE CHANGES IN THE HOST CELL

MORPHOLOGICAL CHANGES

DEATH

APOPTOSIS

INDEFINITE GROWTH

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tissue culture cells

118 epithelial epithelioid fibroblastic slides from CDC

epithelial cells - adenovirus

119 uninfected early infection late infection slides from CDC

epithelial cells - respiratory syncytial virus

120 uninfected respiratory syncytial virus slides from CDC

fibroblastic cells - herpes simplex virus

121 uninfected early infection late infection slides from CDC

fibroblastic cells - poliovirus

122 uninfected early infection late infection slides from CDC

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PLAQUE ASSAY PLAQUE ASSAY

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PLAQUE ASSAY PLAQUE ASSAY

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PLAQUE ASSAY PLAQUE ASSAY

126 Diluted 10 fold Diluted 100 fold Diluted 1000 fold

PLAQUE FORMING UNIT

P.F.U.

pfu

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SOME POINTS TO REMEMBER

INFECTIVITY

NOT EVERY RELEASED PARTICLE IS INFECTIOUS

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• ASSAYS

– detect every particle (e.g. electron microscope)

– detect infectious particles only (e.g. plaque assay)

GLOSSARY

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