MICR 3330 Midterm

Introduction

Smallpox

• Most dreadful infectious disease in history

• May have killed Pharaoh Ramses V

• Pivotal role in conquest of North America

  • Jeffrey Amherst (1763) suggested germ warfare by infected blankets

  • Captain Simeon Ecuyer distributed blankets to Indians (not confirmed)

  • Also evidence in French-Indian war

Influenza

• 1st recorded by Hippocrates 412 BC

• Origin

  • Domesticated animals (likely)

• Initially believed as bad air

• Seasonal epidemics

  • Flu shot

• Spanish flu

  • 1918-1919

Poliomyelitis & Infantile Paralysis

• Transmission

  • Oral-fecal route

Ebola

• Transmission

  • Direct contact w body fluid, contaminated objects

• Origin

  • Fruit bat or primates (likely)

HIV/AIDS

• Attack immune system

  • Death from secondary infections

• Transmission

  • Body fluid (sex, injection, blood transfusion, etc)

SARS

• Outbreak 2002

Common & Manageable Human Viral Diseases

• Herpesvirus 1 (cold sore)

  • Herpes simplex virus 1

  • Re-activation caused by stress (mental, UV, etc)

• Infectious mononucleosis

  • Epstein-Barr virus

  • In multiple autoimmune systems

• Human papillomaviruses

  • Cause of most STDs

  • Age 50, ~80% women (USA) at least 1 type HPV

    • High-risk 15% in females

  • High risk types can cause cervical cancer

Other notable viruses

• Tobacco mosaic virus

• Papaya ringspot virus

  • Caused papaya devastation

• Grapevine leafroll associated viruses

Virus Pathogenicity

• Humans are biggest & most effective "vector" for emerging & re-emerging virus

• Most viruses not lethal

  • Co-exist w host through genetic mut'n & co-adaption

Good Things from Viruses

• Molecular bio from studying viruses

• DNA (in phage) or RNA (TMV) is genetic material

• Polyadenylation of mRNA transcripts

  • Polyomavirus

• 5' cap of euk mRNAs

  • Reovirus & alphavirus

• Defiance of central dogma, reverse txn, & RTase

• Splicing of pre-mRNAs in euk systems

  • Adenovirus

• Nuclear localization signals for prtn targeting

• 1st complete genome

  • φX174

• 1st "mammalian source" genome sequenced

  • SV40

• RNA silencing

  • RNAi or post-txn'l gene silencing

• Viruses as vectors in gene therapy & cancer treatment

• Viruses as VIGS vectors for functional genomics

• Nature's utility of viruses to maintain the ecosystem

Applications of viruses

• Gene therapy

• Prtn expression

• Oncolytics

• Recombinant vaccines

• Functional genomics

• Revival of phage therapy

  • Control bact'l infections w multi-drug resistance

• Nanoparticles

Questions in Virology

• Definition of a virus?

• Are viruses living or non-living?

• Origin of viruses?

• Why can't vaccination programs eradicate all viruses?

• How to viruses perpetuate over eternity?

• How viruses maximize coding capacity of their genes?

• Can viruses be benificial?

• How to control viruses (& diseases)?

• How to eliminate all viruses?

  • Is this necessary?

Discovery, Morphology, & Composition

TMV: First Virus Discovered

Adolf Mayer

• 1879

  • Extracts from tobacco w mosaic disease were infectious

  • Fungal pathogen not involved

  • No culture on Petri dish

• 1882

  • Initial conclusion: 'soluble, enzyme-like contagium'

• 1886

  • Publication: 'unknown bacterium'

D. Ivanovsky

• 1892

  • Passing tobacco extract through bacteria-proof filter

  • Filtrate still infectious

  • Reports: Filter must still be infectious

M. Beijerinck

• 1998

  • Filter extract -> dilute filtrate -> inoculate healthy tobacco -> replenish

  • 'Contagium vivum fluidum'

Later work on nature of TMV

• 1935: Stanley

  • Crystallization of TMV particles

    • Purified from tobacco

  • Conclusion: 'virus is proteinaceous in nature'

• 1936: Bawden & Pirie

  • TMV particle also contains RNA (5%)

• 1933: Ernst Ruska

  • Invented EM

• 1939: Helmut Ruska

  • Revealed TMV

FMDV

• 1st Animal Virus Discovered

• Loeffler & Frosch (1898)

  • Virus from foot-and-mouth disease remained infectious after filtering

• Highly contagious in cloven-hoofed animals

  • Fatal in calves

• Repeated vaccination required

Yellow Fever Virus

• 1st Human Virus Discovered

• Natural hosts: monkeys & mosquitos

• Symptoms

  • Damage to liver

  • Jaundice

• Slave brought to America

• Philadelphia epidemic (1793)

  • Killed ~15% of city pop

• Discovery: 1901

  • Human volunteers for vector transmission studies

    • 19 tested

    • 8 infected

    • 3 died

Discovery of Bacteriophages

• 1915: Frederick Twort

  • Attempts to grow vaccinia

  • Petri dish contamination

• 1915: Felix d'Herelle

  • Found virus that killed Shigella bacteria (causes dysentery)

    • Killed bact & plaque formation among soldiers

  • Wanted phage as therapy to cure bact'l diseases in humans

• 1939: Delbruck & Ellis

  • One-step growth experiment

• 1940: Luria & Delbruck

  • Make phage workshops

• Studies of phages & E. Coli

  • Foundation for molecular bio & bact'l genetics

DNA as Genetic Material in Phage T2

• 1952: Hershey & Chase Experiments

  • Label T2 with ³⁵S or ³²P

  • Mix with bact

  • Blend

  • Let culture grow

  • Centrifuge

  • Measure radioactivity

  • Results after centrifugation

    • ³⁵S

      • (Labelled prtn capsule)

      • No sulphur detected in cells

    • ³²P

      • (Labelled viral genetic material)

      • Phosphorus detected

Composition

• Nucleic acids

  • RNA or DNA as genetic material

    • Not both

    • Majority RNA

• Prtns

  • Structural & non-structural

• Lipids

  • Enveloped viruses only

  • Derived from cellular lipid layer

• Carbohydrates

  • In glycoprtns & glycolipids

  • Recognizing cell receptors & attachment to host cells

---

• RNA viruses

  • dsRNA

    • Almost always segmented

  • ssRNA

    • Segmented or non-segmented

• DNA viruses

  • dsDNA & ssDNA

    • Linear or circular

---

• Naked virus

  • Nucleic acids inside a capsid

• Enveloped virus

  • Enveloped w glycoprtn spikes

  • Nucleic acids inside nucleocapsid

--

• Hepadnaviridae (dsDNA) & Retroviridae (ssDNA)

  • Reverse txn to finish replication

  • Vertebrates viruses

• Nanoviridae (ssDNA)

  • Reverse txn to finish replication

  • Plant virus

    • No true dsDNA plant virus discovered

Morphology & Dimensions

1. Rigid rods / Flexuous filaments

   1. (14 x 71 nm - 80 x 14,000 nm)

      • TMV: 18 x 300 nm

      • Filoviridae (Ebola): 80 x 650-1400 nm

      • Closteroviridae: 12 x 2200 nm

2. Spherical / Isometric

   1. (17 - 300 nm diameter)

      • Parvoviridae: 25 nm

      • Picornaviridae: 30 nm

      • Adenoviridae: 80 - 110 nm

      • Herpesviridae: 120 - 300 nm

3. Irregular / Complex morphology

   1. T-even bacteriophages

      • Spherical head: contains DNA

      • Tail: Helical sheath, tail fiber, & tail baseplate

   2. Baculoviruses (insects)

      • Occluded virion (OV): Survival when released into env'nt

      • Budded virion (BV): For spread w/in insect

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• Brick Shaped

  • Vaccinia & pox viruses

Terminology

• Virion

  • Complete viral particle

• Capsid (coat)

  • Prtn shell encasing the viral genome

• Nucleocapsid (core)

  • Nucleic acid + prtn

  • In virion of enveloped viruses

• Subunit (promoter)

  • Single, folded polypeptide

  • Individual capsid prtn

• Structural unit (capsomer)

  • Basic unit for building capsid or nucleo-capsid

  • 1+ subunits

• Envelope

  • Lipid mem enclosing nucleo-capsid

  • (enveloped viruses only)

• Structural prtn

  • Prtns part of virion structure

• Non-structural prtn

  • Encoded by a virus but not part of virion

  • Enzymes needed for viral replication, movement, & infection

Helical Symmetry

• Rod-shaped & filamentous viruses

• Capsid prtn subunits have equal binding to another

  • Except those at both ends

• 'Open' structure: Unlimited packing capacity (for insertion of foreign DNA/viral vectors)

• P (pitch of helix) = µ (# of subunits per helical turn) x p (the axial rise per subunit)

• TMV

• Paramyxovirus (Sendai virus)

Icosahedral Symmetry (& linkage to geodesic dome)

• More complex structure

• 'Closed' structure: packaging of only limited genome sizes

• Watson & Crick (1956)

  • Proposed spherical viruses were cubic structures (proven wrong)

• Basic design of spherical capsids & nucleocapsids:

  • Icosahedral symmetry; 20 triangular faces

• Pillars:

  • Triangulation number (T): define possible icosahedral surface lattice

  • Quasi-equivalence: describe the nearly identical bonding rel'nship among subunits in a spherical capsid

  • Spontaneous self-assembly of individual CP subunits into virus-like particles (VLPs) to identify the assembly processes

• Simplest cases

  • Capsid built from 60 copies of 1 capsid prtn arranged into 20 triangular faces

  • Ex. Satellite tobacco necrosis virus, parvoviruses, øX174

  • All capsid subunits in equivalent bonding rel'nship

  • 3 Types of rotational symmetry: 2-, 3-, and 5-fold

    • 2 axes in 2-fold

    • 20 axes in 3-fold

    • 12 axes in 5-fold

• In larger isometric viruses

  • Triangulation # (T): # of triangles w/in ea of the 20 triangular faces

  • Only certain multiples of 60 subunits allowed (T= 1, 3, 4, 7)

  • Quasi-equivalence: in icosahedrons w T>1

    • Rel'nships b/w capsid prtn subunits similar (NOT identical)

---

• β-barrel jelly roll fold

  • 3D structure of capsid prtns

    • Shared by viruses w icosahedral symmetry (conserved)

  • Similar fold in 'phaseolin' (storage prtn) of beans

Interactions among CP subunits

• CP subunits small in most viruses (20-70 kDa)

  • # of subunits must incr to let them to exist in a quasi-equivalent position in viruses w large capsids

• CP subunits spontaneously assemble into larger structures w or w/out help of viral genome

  • Ex. Structural units, intact capsid shell

  • CP subunits stabilized by the max # of non-covalent bonds b/w them

    • -> lowest free energy state

  • All sub-sub & sub-RNA bonds are weak

    • Mainly hydrophobic & van der Waals

  • Secondary structures in viral RNA = packaging signals

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• T x 60 = # of prtn copies in capsid/nucleocapsid

  • Exceptions - Do not contain predicted # of subunits

    • Polyomaviruses

    • Adenoviruses

    • Reoviruses

Phage lambda

• Icosahedral head

• Helical tail made of tail cone (sheath), tail plate, & attachment fibres

• Ea part is assembles individually, followed by assembly into an intact virion

Members of the Adenoviridae family

• 1500 expected copies of subunits (based on T=25)

  • Actual: 780 copies

• Prtn II: 720 copies/virion; forming hexons (240 hexons/virion)

• Prtn III: 60 copies; forming pentons (12 pentons/virion)

Simian virus 40

• 5 VP1 subunits interlock into a pentamer

• C-term arms of ea pentamer inserts into neighbouring pentamers

  • Stabilizes capsid

• 2 spatial arrangements = deviation from icosahedral symmetry rule

  • 12 pentamers at 5-fold axes, ea surrounded by 5 pentamers

  • 60 pentamers at remainder of capsid, ea surrounded by 6 pentamers

Recap

• TMV

  • 1st virus identified (1898) via bacteria-proof Chamberland filter candle

  • Rod-shaped particle

    • 95% prtn (capsid), 5% RNA (genome)

  • Major role in understanding genetic info, etc.

• FMDV & YFV

  • Foot-and-mouth disease virus

  • 1st animal virus

• YFV

  • Yellow fever virus

  • 1st human virus

• Virus composition

  • Prtn & nucleic acids (DNA or RNA, not both)

  • Some also have lipids & carbohydrates in form of glycoprtns & glycolipids

• Virus morphology

  • Sometimes genome split into multiple segments packed into:

    • Same virion (influenza)

    • Different virions (some plant viruses)

• Categories of viruses

  • Naked (have capsids)

  • Enveloped (nucleocapsid w/in a lipid envelope)

• Symmetry

  • Helical

    • Rigid rods or flexible filaments

  • Icosahedral

• Triangulation # and quasi-equivalence

  • Describe icosahedral design of viruses w isometric capsids or nucleocapsids

  • Capsid prtn subunits self-assemble into structural units & intact virions (VLPs)

    • VLPs= basis for subunit vaccines for human & animal diseases

Classification, Taxonomy, & Nomenclature

Linnaean hierarchical system (1700s): Plants & animals

3 domains system today: Bacteria, Archaea, Eukarya

Homes (1948)

• Failed attempt to classify & name viruses using Linnaean syst

• Order: Virales

  1. Phaginae (viruses of bact)

  2. Phytophaginae (viruses of plants)

  3. Zoophaginae (viruses of animals & humans)

Classification & Taxonomy

• Pre-1930

  • Based on diseases, signs, & symptoms

  • No distinction b/w disease & its causative agent

    • Hep A: Picornaviridae

    • Hep B: Hepadnaviridae

    • Hep C: Flaviviridae

  • Mosaic virus cause mosaic symptoms in plant leaves

    • TMV: Virgaviridae

    • Cauliflower MV: Caulimoviridae

    • Cucumber MV: Bromoviridae

    • Turnip MV: Tymoviridae

    • Soybean MV: Potyviridae

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• Virus: agent that causes an infection or disease

• Disease: outcome & manifestation of an infection resulting from interactions b/w a virus & its host

• Infection: can lead to disease (not necessarily the cause)

---

• 1930-1966

  • Emphasis on virus over disease

  • Based on:

    • Morphology

    • Capsid structure

    • Chemical composition

    • Type of genome

  • Viruses classified in 'groups' initially

    • Herpesvirus group

      • dsDNA

      • Icosahedral heads

      • Large virion

      • Has envelope

    • Poxvirus group

      • dsDNA

      • Complex & irregular virion

      • 200 nm or larger

      • Enveloped

    • Myxovirus group

      • ssRNA

      • Spherical virion

      • Helical nucleocapsids

      • Enveloped

• 1966-present

  • International Committee on the Taxonomy of Viruses (ICTV)

  • 2 approaches considered:

    • Monothetic: 1 characteristic at a time

      • Nature of viral genome, symmetry of capsid, presence/absence of envelope, etc…

      • Problem: assumes all members of a group originate from same ancestor; can't reflect diversity

    • Polythetic: Considers multiple characteristics

      • Individuals share most (not all) of a set of common characteristics

      • Not assume all viruses share same ancestor

      • Ex. family Closteroviridae

        • Long & filamentous virion

        • Large (+)ssRNA genome

        • HSP70h (viral homolog of cellular HSP70)

        • Tropism for phloem

Defining a virus species

• Pre-2013: "A polythetic class of viruses that constitute a replicating lineage and occupy a particular ecological niche"

• Post-2013: "A monophyletic group of viruses whose properties can be distinguished from those of other species by multiple criteria"

• Practical:

  • Defined by relatedness in seq'ces of a specific gene, set of genes, or entire genome

  • Values of seq'ce identity used for species distinction vary among families

• Taxon pre-2019: Order, family, (subfamily), genus, & species

  • Subspecies designations: Strains, serotypes, genotypes, subtypes, variants, etc

Naming Viruses

• Bacterial: Specific code: Qβ, φX, λ, T1, T2…

• Plant: Host in which virus identified then descriptor of key symptoms

  • Eg. Tobacco mosaic virus, beet yellow virus, etc

• Mammalian: Based on diseases & symptoms

  • Eg. Hepatitis virus, measles virus, SARS-CoV, etc

• Insect: Latin name of host & effect of infection on virus

  • Eg. Autographa californica multiple nucleopolyhedrovirus

Characteristics for classifying genus/family

• Nature & organization of genomes

  • DNA or RNA

  • Strandedness (ss or ds)

  • Polarity (+/-)

  • Segmented or non-segmented

  • Topology (linear/circular; closed/open circle)

• Virion morphology (structure of capsids & nucleocapsid)

  • Helical, icosahedral, complex

  • Shape, size, surface features

  • Envelope (yes/no)

• Genome structure, strategies for genome replication expression

• Enzymes (Pol, RTase, protease, integrase, etc.)

Characteristics for defining species

• Natural host range

• Cell & tissue tropism

• Pathology (host) & cytopathology (cell culture)

• Mode of transmission

• Physico-chemical properties of virions

• Antigenic properties of viral prtns

• Seq'ce relatedness of individual genes & whole genomes

Phylogenetic Analysis & Viral Taxonomy

• Phylogeny: prediction of evolutionary relatedness among viruses based on comparison of their seq'ces using computer & mathematical algorithms

• Analysis based on NT or AA sequences (or both)

• Dif methods to generate phylogenetic trees:

  • Neighbor joining (NJ)

  • Maximum likelihood (ML)

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• 9th Report of ICTV (2011)

  • Families not yet assigned to an order = 78

  • Genera not yet assigned to a family = 13

Virus Nomenclature & Taxonomy

• 1991-2018: 5 rank structure

• Since 2019: 15 rank structure

  • 1st attempt to adopt the Linnaean syst to classify all viruses

  • Intro of 8 more lvls for higher taxa

• Changes in 2021

  • Binomial system

  • Abolition of type species in all genera

  • Increased scope: include viruses, viroids, & satellites

  • Realms ex.

    • Adnaviria: DNA viruses from Archaea hosts

    • Duplodnaviria: dsDNA viruses

    • Varidnaviria: large dsDNA viruses (nucleocytoplasmic)

    • Riboviria: RNA viruses (including retroviruses)

Types of genome viruses

• (+)ssRNA genome viruses

  • Genome 3-31 kb

  • Linear genomes (no circular)

  • Most lack an envelope

  • Examples

    • Closteroviridae (Helical)

    • Coronaviridae (Helical)

• (-)ssRNA genome viruses

  • Helical symmetry

  • Linear genomes

  • Some v pathogenic human viruses

  • May have envelope

• ssDNA genome viruses

  • Small genomes 2-9 kb

  • Naked (no envelope)

  • Icosahedral symmetry (except Inoviridae)

  • Circular genomes (except Parvoviridae)

• dsDNA genome viruses

  • Large variation

Baltimore Classification System: Transcription considered most important aspect for a virus

Virus Replication Cycle

Cell culture system

• Enders, Weller, Robins (1949)

  • Primary embryonic cell cultures (from mouse) for measles virus & polio virus

• Cultured on plastic surface or as liquid suspensions

• Widely used in research & vaccine dev'nt

Types of cell cultures

1. Primary cell cultures

   • From live tissues/organs

   • Multiple cell types

   • Finite capacity in cell division (5-20x)

   • Ex. Monkey kidney (for polio vaccine), chicken embryo, mouse embryo

2. Diploid cell strains

   • Single cell; epithelial or fibroblast

   • Normal morphology & # of chromos

   • Cell division up to 100x

   • Ex. WI-38 (from female embryonic lung)

3. Immortal (continuous) cell lines

   • Homogeneous in cell type

   • Infinite capacity of cell division

   • Abnormal chromo morphology & #

   • Loss of contact inhibition

     • Detach from surface, piling up, form 'focus'

   • Tumorigenic

   • Source: tumors, transformed cell strains, cells mutated by oncoviruses or mutagens

   • Ex.

     • HeLa: cervical tumor (Henrietta Lacks, HPV)

     • Vero: kidney of African green monkey

     • BHK-21: Baby hamster kidney

     • BY2: right yellow, tobacco

Cytopathic effects (CPE)

• Morphological alterations of a cell due to viral infection

• Cell death

• Rounding up of cells -> detach from surface

• Syncytium = large cell w a lot of nuclei (bc of fusion)

• Abnormality in morphology & # of chromosomes

• Inclusion bodies:

  • Polyhedron inclusion bodies (PIBs)

  • Negri bodies (in rabies-infected cells)

  • Inclusion bodies in plant viruses

    • X-bodies, pinwheels, etc

One-step growth cycle

• Synchronous infection of all cells w virus

  • High MOI: 5-10

• Time intervals

  • Eclipse period: from absorption to 1st intra-cellular virion

  • Latent period: from absorption to 1st extra-cellular virion

  • Burst size: sum of virions produced in 1 cell

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• MOI: Multiplicity of infection: # of infectious virions added per cell

  • Not all cels in dish receive same # of viruses

  • MOI of 5-10 commonly used for synchronous infection

• Allocation of virions among cells is calculated by Poisson distribution

  • B/c random collision b/w viral particles & cells

1. Attachment (adsorption)

• Collision b/w viral particles & cells

  • Via Brownian motion

• Weak contact

  • Via interaction b/w viral particle & -ve charges on cell surface

• Specific attachment achieved via interactions b/w:

  • Attachment prtn (on virus)

    • Eg. HA in influenza virus; fiber in adenoviruses; surface structure 'canyon' in poliovirus

    • Some attached to mem by TMD

    • Some anchored indirectly to PM via fatty acids/alcohol

      • Types of anchors for these prtns:

        • Myristic acid

        • Farnesyl

        • Glycosyl phosphatidyl inositol-linked prtns

Only external surface of PM

• Receptor(s) (on host cell surface)

  • Eg. Sialic acid for HA; Icam-1 for major group rhinoviruses

  • ~500,000 molecules/cell

  • Plasma mem has 'lipid rafts' w distinctive structure & fxns

  • Viruses hijack surface molecules & lipid rafts for entry & replication

• Stronger w more interactions b/w attachment prtns & receptors

• Naked viruses examples

  • Attachment via surface features on virion

    • Polioviruses & rhinoviruses have canyon surrounding ea pentamer - attachment site for cell receptors

  • Attachment via fibers on virion surface

    • Adenoviruses & picornaviruses

    • Fiber is a homo-trimer at ea of the 12 penton bases

    • Terminal knob on fiber w depression - attachment site for 'Car'

• Enveloped viruses examples

  • Glycoprtns on viral envelope are responsible for attachment

  • Hemagglutinin of influenza A & B viruses binds sialic acid

  • Surface glycoprtn (HIV-1) binds CD4 of T-helper cells & macrophages

    • Co-receptor needed for viral entry

Image: Well-studied receptors & co-receptors

• Receptors extra info

  • For most viruses are unknown

  • Viruses w carbohydrate receptors tend to have broader host range

    • Presence of receptor (& co-receptor) determines, in part, the host range & tissue tropism of a virus

2. Entry & Uncoating

• Basic tricks

  • Drilling hole at PM

    • Eg. Picornaviruses

  • Fusion b/w viral envelope & PM

    • At neutral pH

    • Needs fusion prtns/peptides

    • Eg. Paramyxoviruses, retroviruses, coronaviruses, baculoviruses

  • Receptor-mediated endocytosis, followed by uncoating @ an intracellular mem (endosomal or nuclear)

    • Low pH (endosome, lysosome)

    • Eg. Orthomyxoviruses, adenoviruses

• Models of poliovirus entry

  • @ plasma or endosomal mem

  • Steps in entry & uncoating

    • Attachment to multiple receptor Pvr

    • N-term of VP1 exposed, inserted into PM (forms channel)

    • Viral RNA released into ctpsm

• HIV-1

  • Viral entry needs binding to both receptor (CD4) & co-receptor

    • Triggers exposure of fusion peptide -> fusion b/w viral envelope & cell mem

• Receptor-mediated endocytosis

  • Selective import of extracellular molecules (ligands) into a cell via receptor & mem invagination

  • Formation of clathrin-coated pits needs ATP hydrolysis

  • Decrease in pH from early endosome -> late endosome -> lysosome

• Ex. Influenza

  • Receptor mediated endocytosis & uncoating @ endosomal mem

    1. HA attaches to sialic acid

    2. Endocytosis

    3. Fusion pep emerges from HA under low pH

    4. H+ into virion via M2 channel

    5. Matrix layer dismantles, viral envelope fuses w endosomal mem

    6. Release RNPs into cytosol

    7. RNPs enter the nucleus

• Ex. Adenoviruses

  • Receptor-mediated endocytosis & stepwise uncoating

    1. Fiber binds to Car

    2. Penton base interacts w integrin, leading to endocytosis

    3. Losing fibers in endosome

    4. Penton bases dismantle in late endosome

    5. Broken virion release into cytosol

    6. Free ride on microtubule

    7. Dock at nuclear pore

    8. Tug-of-war b/w dynein & kinesin releases viral DNA

    9. Viral DNA enters nucleus

• DNA viruses & retroviruses nucleus entry

  • Adenoviruses:

    1. Partially dissembled virion transported by microtubules

    2. Dock onto nuclear port

    3. Pulling by kinesin break up virion

    4. DNA enters

  • Herpesviruses:

    1. Release of pressure in inner capsid injects DNA

  • Polyomaviruses:

    1. Remodeling of nuclear envelope & lamina allows DNA entry

  • HIV-1:

    1. PIC docks onto nuclear pore due to nuclear localization signals (NLS) on the capsid & integrase proteins

    2. Integration into host chromosome

• Phages T4 (dsDNA)

  1. Replication < 30 mins

  2. Tail fibers recognize/bind receptors (LPS & OmpC) on bact'l cell surface

  3. Causes changes in base plate structure

  4. Tail sheath contracts, exposes inside tube

  5. Lysozymes from phage dissolve cell wall

  6. DNA injected into ctpsm

• Plant viruses

  • Most don't need receptors

  • Many transmitted through insect vectors

  • Mechanical transmission: via minor wounds/abrasions

  • Vertical transmission: via reproductive organs (pollen/seeds)

  • Via ex:

    • Vegetative propagation materials

    • Grafting b/w rootstock & scion

3. Biosynthesis

• Biosynthesis: Syn of all viral components needed for building next gen of viruses

• Transcription: Production of mRNAs from genome (DNA or RNA) (most important aspect of a virus)

• Reverse transcription: Generation of cDNA using RNA as template (only in retroviruses & retro-like viruses)

• Translation: Production of polypeptides using mRNA; relies on cell tln machinery

• Genome replication: Production of nascent viral genomes (DNA or RNA)

• Class I: DNA viruses

  • ssDNA: Parvoviridae (linear), Circoviridae (circular), Geminiviridae

  • dsDNA: Polyomaviridae (circular), Baculoviridae (circular), Adenoviridae (linear), Herpesviridae (linear)

• Class II: RNA viruses

  • dsRNA: Reoviridae, Birnaviridae, Partitiviridae, Chrysoviridae

  • (+)ssRNA: Picornaviridae, Togaviridae, Betaflexiviridae, Closteroviridae

  • (-)ssRNA: Orthomyxoviridae, Paramyxoviridae, Filoviridae, Bornaviridae

• Class III: viruses needing reverse txn

  • Retroviridae (RNA; integrates genome into host chromo)

  • Caulimoviridae (RNA; doesn't integrate)

  • Hepadnaviridae (DNA; doesn't integrate)

• Sites for biosynthesis

• Exceptions:

  • Orthomyxoviridae: Need nuc's for txn & rep'n

  • Poxviridae: Everything in ctpsm of infected cell

  • NCLDV: need nuc's & ctpsm for rep'n

DNA Viruses

• Infect cells that are either dividing or they force dormant cells to enter S phase

• Potential oncogenicity

  • Adenovirus A & C

  • Herpesviruses: Epstein-Barr virus; Burkitt's lymphoma

  • Polyomaviruses: SV40

  • Papillomaviruses: HPV type 16 & 18

• Expression

  • Immediate early (IE) genes

    • Expressed right after infection

    • Functions of IE prtns:

      • Rendering cells toenter S phase

      • Induce expression of other viral genes

      • Inhibit host mechanism & biosynthesis

    • Eg. Adenovirus E1A & E1B bind p23, blocking apoptosis & forcing cell to enter S phase

  • Early (E) genes

    • Enzymes & accessary factors required for genome replication

  • Replication

    • In nucleus (except Poxviridae - in ctpsm - virions carry all needed prtns/enzymes)

    • Requires dNTPs, enzymes, host machinery

    • Strategies vary among families

  • Late (L) genes

    • Structural prtns required for assembly

  • Very late genes

    • For few viruses (polyhedron, baculoviruses)

RNA Viruses

• All of replication cycle in ctpsm

  • Exceptions: Orthomyxoviridae & Bornaviridae)

• Must assoc w intracellular mems

  • ER - TMV, BYV, PVX, etc.

  • Endosomal mem - Sindbis virus (Alphavirus, Togaviridae)

  • Vesicular mem - Poliovirus (Picornaviridae)

  • Peroxisome mem - Tomato bushy stunt virus (Tombusviridae)

  • Mitochondria - Grapevine leafroll-associated virus (Ampelovirus)

  • Chloroplast - Turnip yellow mosaic virus (Tymoviridae)

• (+) strand RNA viruses

  • Txn & rep'n: RNA-dependent RNA Pol (RdRP)

  • Genomic RNA is 1st (or only) mRNA

  • Replicase has conserved domains (RdRP, helicase, etc)

  • 5' proximal ORF(s) translated directly on genomic RNA

  • ORF1 encodes 126 kD prtn w MTR & helicase domains

  • ORF2 encodes RdRp as part of 183 kD prtn via suppression of leaky stop codon

• (-) strand RNA viruses

  • Genome exists as ribonucleoprotein (RNP) complex and not as naked RNA

    • Must 1st be transcribed before tln occurs

      • Virion carries not only RNA but also enzymes

  • Influenza viruses

    • RNPs enter the nucleus -> replicate & transcribe

    • Needs splicing of transcripts made from genome segments

Retroviruses & para-retroviruses

• Recall:

  • Retroviridae (RNA; integrates genome into host chromo)

  • Caulimoviridae (RNA; doesn't integrate)

  • Hepadnaviridae (DNA; doesn't integrate)

• Discovery of RTase went against central dogma

• 2 identical (+)RNA molecules, RTase (50-100 copies), integrase

• Reverse txn starts in virion on route of entry into host cell

• cDNA inserts into host chromo

  • Becomes part of host genome

  • Establishes latency

  • Source of persistent infections

• Host cell enzymes transcribes viral mRNAs including gRNA

Assembly & Egress (release)

1. Formation of individual structural units from 1+ structural prtns

   • de novo process

   • Spontaneous; results from interactions among capsid/nucleocapsid subunits

   • AA sequence decides interlocking among structural prtn subunits

   • Assembly of capsids occurs in special compartment of infected cell

     • High [prtns] ensures assembly is correct, efficient, & directional

   • In ctpsm

     • RNA viruses (except 1 group), DNA viruses of Poxviridae

   • In nucleus

     • DNA viruses, retroviruses, orthomyxoviruses

   • Examples:

     • Adenoviruses

       • Formation of penton requires 2 prtns (fiber, penton base)

       • Formation of hexon trimer by prtn II needs chaperone

     • Poliovirus:

       • Single polyprotein precursor & cleavage

2. Assembly of capsid shell w structural units

   • ssRNA viruses

     • Genome RNA involved in virion assembly (eg. TMV)

     • Poliovirus RNA may be involved in assembly & final cleavage during virion maturation

   • Large DNA viruses & DNA phages

     • Procapsids formed w help of scaffold prtns

     • Collapse & removal of scaffold structures

     • Genome DNA inserted into procapsid

     • Needs ATP hydrolysis for energy

   • Poliovirus

     • Proteolytic cleavage of VP0 makes virion infectious

       • Process may involve genomic RNA

3. Selective packaging of viral genome & other virion components

4. Acquisition of a lipid envelope (mostly from PM)

5. Exit the infected cell

   • Naked viruses

     • Lysis of infected cells - cytopathic effects

       • Shutdown of biosynthesis

       • Destroy cell structure

       • Mem alteration

       • Chromo breakup

       • Syncytium

       • Inclusion bodies

     • Ex. Adenovirus E1B: disrupts nuclear lamina & breakdown of intermediate filaments

   • Enveloped viruses

     • Source of envelope determined by glycoprtns of the virus

     • Budding & pinching off at PM

6. Maturation of virions

   • Only some viruses

   • Needs proteolytic cleavage

---

• Host range: Range of hosts that can be infected by a virus

• Tissue tropism: A virus' preference for certain types of cell & tissue in hosts

• Susceptible cells: Allow attachment & entry of virus b/c of suitable receptor(s)

• Permissive cells: Permit replication of a virus, have all factors needed. May/may not be susceptible

Summary

• Replication cycle (AEBAE):

  • Attachment

  • Entry/uncoating

  • Biosynthesis

  • Assembly

  • Exit

• Attachment

  • Via interactions of specific prtns/cell features on incoming virion & receptors on cell surface

  • Some viruses need receptor & co-receptor

  • Factor of host range & tissue tropism

• Entry/uncoating

  • Entry: virion enters cell via PM or internal mem

    • Direct entry through pore at PM

    • Direct fusion b/w viral envelope & PM (needs fusion peptide)

    • Receptor-mediated endocytosis

  • Uncoating: viral genome released into ctpsm of cell

    • During or after entry

• Biosynthesis

  • All macromolecules needed for viral rep'n are synthesized

  • Includes:

    • Tsl to make structural (& non-structural) prtns

    • Txn to make mRNAs (& other RNAs w reg'ry fxn)

    • Genome rep'n

• Assembly

  • Reversal of uncoating

  • Nascent capsids, nucleocapsids, & intact virions get assembled

  • de novo process (spontaneous & doesn't need template)

  • Subunits => structural units => (nucleo)capsids

    • Viruses w helical symmetry (eg. TMV) - gRNAs involved in virion assembly

    • Large DNA viruses - empty shells assembled -> packaging of gDNA via insertion of DNA through specialized portal

• Egress

  • Naked viruses

    • Exit upon lysis of the infected cell

  • Enveloped viruses

    • Budding at PM or internal organelle mem

TMV

(+)ssRNA supergroups

• Based on phylogenetic rel'nship of RdRP (Pol):

  • Alphavirus-like (III)

    • TMV

  • Picornavirus-like (I)

    • Polio, FMDV

  • Flavivirus-like (II)

    • Yellow fever, Hep C

• Class 4

  • Revised Baltimore system

TMV

• Genus: Tobamovirus

• Family: Virgaviridae

• Easily transmissible; persists for decades

Research Milestones

• 1898: TMV as first filterable virus conceptualized (contagious living fluid)

• 1955: Reconstitution of TMV & RNA as genetic material

• 1971: Role of CP double disks as structural unit in capsid assembly

• 1978: Discovery of movement prtn in TMV

• 1986: Infectious RNA transcript from full-length cDNA clones

• 1986: Coat prtn-mediated resistance against TMV infection

Genome structure & expression strategies

• Small, (+)ssRNA, 6.4 kb

• gRNA is 1st mRNA to translate replication-related enzymes

• 5' end: cap structure

• 3' UTR: secondary & tertiary structures (3 pseudo-knots)

• 3' end: tRNA-like structure

  • No Poly-A tail

  • Aminoacylated w His

• Suppression of leaky stop codon of ORF1 leads to continued tsl of ORF2

  • 126 kDa prtn (has MTR & HEL)

  • 183 kDa prtn (MTR, HEL + RdRP)

*MTR = methyltransferase domain

*HEL = helicase domain

Reconstitution Experiment

• Proves RNA is genetic material (not prtns)

• Fraenkel-Conrat

  1. CP alone assembles into virion: Not infectious

  2. CP & RNA assembles into virion: Infectious

  3. RNA alone: Infectious

• Conclusion

  • RNA iis genetic material, not capsid prtn

Local lesion assay to quantify TMV

• Francis Holmes (1929)

• Process:

  1. Spread abrasives on leaf surface

  2. Rub leaf w viral stock dilution

  3. Wait for infection & symptoms

  4. Count # of local lesions

  5. Calculate the titer of OG viral stock

• Like plaque assay for bacteriophages & animal viruses

Assembly of capsids & genome packing

• Structural unit for TMV: CP double disk

• 16.3 copies of CP per helical term

• 2,1300 CP copies per virion

• Building-up as double discs

• RNA involved in virion assembly

• 5' genome segment threads through the interior of the elongating helix

• One of 1st models on virion assembly

CP-mediated protection in crop plans against viral diseases

• 1st demonstration of transgenic resistance against viruses by genetic engineering

  • Transgenic plants expressing TMV CP exhibited delayed onset of disease

• Theory: "Coat prtn-mediated protection"

  • Excess CP from the transgene blocks virus disassembly = virus resistance

Stages of TMV in plant (virus travels through phloem)

1. Intracellular movement

   • Viral rep'n complexes (VRCs) form in assoc'n w ER

   • Move to other parts of infected cell

   • Produces multiple VRCs in same cell

2. Intercellular (cell-to-cell) movement

   • VRC docks @ plasmodesma traverses it w help of movement prtn (MP)

   • Completes new rep'n in neighbouring cells

3. Long distance movement

   • Virions gain entry into sieve elements of phloem

   • Move rapidly to distal parts of plants

   • Causes systemic infection

Viruses move b/w cells via plasmodesmata

• Size exclusion limit

  • Allows passive diffusion (1-7 kDa)

• Active process (w ATP) for larger molecule transport through PD

• Move w help of MP

Movement prtns (MP)

• 1st discovered in TMV, encoded by all plant viruses

  • Temp sensitive TMV mutant, Ls1

  • Complementation by transgenic MP

  • Most plant viruses encode 1 MP (some 2+)

• Common properties

  • Binds RNA

    • Forms v thin & long RNPs

  • N-term region increases SEL of PD

  • Interacts w ER, actin filaments, & microtubes

  • MPs encoded by non-related viruses may compliment defective MP

  • Bonds p38 (cell wall assoc'd prtn @ PD, receptor for MP)

• Earlier model of TMV cell-cell movement

  • MP complexes w TMV RNA to form thin thread

    • Moves on actin filament

    • Interacts w p38 -> increases SEL

    • Squeezes through central cavity of PD

  • Phos'n by host kinase releases MP

    • Frees viral RNA in 2nd cell for tsln

• TMV MP assoc'd w peripheral ER in plants

  • Immuno-staining w anti-Bip antibody shows ER in red

    • BiP: luminal prtn of ER, marker for ER network

Composition & structure of VRC

• Components:

  • Replicase prtns

  • MP

  • Viral genomic RNA

  • ER mem

  • Other viral & cellular prtns

• Helicase domains self-interact

  • Forms hexameric ring-like structures when over-expressed in bact'l cells

  • Bind ssRNA

  • Act as ATPase & helicase

    • Hydrolyzes ATP & unwinds dsRNA

  • Ea hexamer likely comprised of 5 molecules of 126 kD & 1 molecule 183 kD

VRC aligns w & moves on actin filaments

• 126:GFP fusion forms punctate structures in cells

• DsRed:Talin marks actin filaments (MF) in red

• 126:GFP co-localizes w DsRed:talin

  • Suggests localization to microfilaments

• VRC (shown by MP:GFP) also aligns w actin filaments

• Conclusion: 126 kD aligns on & moves along actin filaments

Recent research in TMV cell-to-cell movement

• Aim: test if it takes same amount of time for TMV to replicate in initially infected cells compared to that in secondary infected cells

• Tracking of TMV movement w time lapse microscopy in initially infected cells vs. movement in additional cells suggest:

  • TMV moves as VRC & not as thin MP-RNA threads

  • Much shorter time to complete same process in neighboring cells

Current model of cell-to-cell movement

TMV as vectors for VIGS & prtn expression in plants

• VIGS: virus-induced gene silencing

• RNA-based defence syst for gen reg'n & defence against viruses

• PSY (phytoene synthase) & PDS (phytoene desaturase) protect chlorophyll

• TMV engineered to produce mRNA for PDS or PSY

  • Triggers RNA silencing in tobacco

• Infected leaves exhibit photo bleaching upon light exposure

Summary of TMV

• (+)ssRNA

  • Representative member of the Alphavirus supergroup

• Prototype member of genus Tobamovirus in family Virgaviridae

• Rod shaped capsid

• 2130 subunits

• Helical symmetry

  • 1 turn = 16.3 subunits

• RNA genome

  • 6400 nts

  • 5' cap

  • No Poly-A tail

  • V compact

    • 5'UTR

    • 4 ORFs

    • 3' UTR

  • ORF1&2

    • Near 5' end

    • Directly translated from gRNA brought by virion upon entry

  • ORF1

    • Translated into 126 kDa prtn

      • Has conserved methyltransferase (MTR) & helicase (HEL) domains

  • ORF2

    • Encodes viral RNA-dependent RNA Pol (RdRp)

    • Translated as an extension to the 126 kDa via suppression of stop codon (translational read-through)

      • Ribosome "reads-through" stop codon instead of stopping & makes extension of prtn

      • Makes 183 kDa prtn

        • N-term: 126 kDa

        • C-term : RdRp)

  • ORF3&4

    • Expression via translation of 2 sgRNAs

      • Viral replicase enzymes transcribe sgRNAs -> ea sgRNA has own promoter of the ORF it will be translated into -> sgRNAs used as template for tsln -> production of the prtns

  • ORF3

    • Encodes MP (non-structural)

  • ORF4

    • Encodes CP (structural)

      • Used to construct rod-shaped virion

• Infection

  • Via temporary wound on leaf surface

  • Immediately after entry, ORF 1&2 occur

    • Makes 2 polypeptides that are needed for rep'n

  • Viral rep'n in special compartment in ctpsm of infected cell

    • Viral Replication Complex (VRC)

    • Made of mem from ER, replicase prtns, gRNA, MP, host factors (unidentified)

  • Intercellular movement of VRC

    • Through plasmodesma (PD) (microchannel b/w adjacent cells)

    • MP interacts w RNA & PD components

      • Increases size exclusion limit of PD, allowing VRC to go through

        • Early theory (disproven): RNA-MP thin threads move through

    • TMV enters vasculature (phloem)

      • Reaches distal parts of the plants

      • Results in systemic infection

• Recent TMV study

  • TMV promising in improvement of energy storage & lithium-ion batteries

Picornaviridae

Supergroup I of (+)ssRNA - Picornaviruses

• Features

  • No 5' cap

  • 3' Poly(A) tail

  • No envelope

  • V stable

  • 22-30 nm diameter

  • Icosahedral symmetry

    • 60 copies ea of 4 structural prtns (VP1-4)

      • VP-3 on surface

      • VP-4 hidden under

  • Oral-fecal transmission

  • Genomic RNA is the only mRNA

    • Translated into a polyprtn (precursor)

  • IRES= start site for viral tsln

  • Triangulation # of pseudo 3

  • β-barrel jelly roll conserved in icosahedral viruses of plants, insects, animals, humans

    • Topological family of prtns

Diseases caused by Picornaviridae

• Poliomyelitis

  • Infantile infection w multiple epidemics in North America

• Common cold

  • Upper respiratory tract

  • ~50% of common colds

• Hep A

  • Acute liver infections

  • Sporadic outbreaks via food & drinks

• Heart infections

  • Myocarditis

  • Dilated cardiomyopathy (DCM)

    • Caused by group B Coxsackieviruses

• Diabetes & pancreatic disorders

  • Coxsackieviruses

  • Encephalomyocarditis virus

• Foot-and-mouth disease

  • Quarantinable

Poliomyelitis

• Most polio infections were inapparent & self limiting

• Paralytic form most feared

• Major infantile & childhood disease in the 1st half of the 20th century

  • The hygiene hypothesis

    • Early exposure to microorganisms helps build immune system

• United States

  • 1894

    • 1st reported case

  • 1916

    • 27 000 cases

    • 6000 deaths (1/3 from NYC)

  • 1930s

    • FDR declares national war against polio

  • National Foundation for Infantile Paralysis

    • Later, March of Dimes

  • 1952 epidemic

    • 58 000 children infected

    • 21 269 (36%) displayed paralysis of various severity

    • 3145 (5.4%) deaths

• Canada

  • 1910

    • 1st reported case (Hamilton, ON)

  • 1937

    • 4000 cases nationally (>50% in ON)

    • 119 deaths (4.7%)

    • Only single iron lung available - hospital staff rush to make more

  • 1953

    • ~9000 cases

    • 500 deaths (5.5%)

  • Panic - social distance, isolation, quarantine, etc.

• Iron lung

  • Only treatment & hope of severe polio patients w lung complications

  • Stay in lung until immune syst heals body

  • Ex. Barton Hebert

    • Stayed in iron lung for last 50 yrs of life

• Salk vaccine

  • Jonas Salk (1955)

    • 1st highly effective inactivated polio vaccine

    • Tested on self & family to convince others

    • Large clinical trials in USA

Key discoveries made w picornaviruses

• FMDV

  • Loeffler & Frosch (1898)

  • 1st animal virus discovered shortly after TMV

• Isolation of PV

  • Landsteiner (1909)

  • Via transmission experiment to monkeys

• Cell culture

  • Enders, Robbins, & Weller (1949)

• Plaque assay

  • Dulbecco (1952)

• IPV (inactivated vaccine)

  • Jonas Salk (1955)

• OPV (weakened live virus vaccine)

  • Albert Sabin & Hilary Koprowski (1960)

• RdRP

  • Baltimore (1963)

  • From polio-infected cell

• Polyprtn

  • Summers & Maizel (1968)

• Infectious cDNA clone

  • Racaniello & Baltimore (1981)

• IRES

  • Pelletier & Sonenberg (1988)

Genome structure & expression strategies

• (+)ssRNA

• 7500 - 8450 nts

• 5' end

  • VPg (virion prtn, genome-linked, 22-24 aa residues)

  • 5' UTR: v long secondary structures

• Middle

  • Single large ORF encoding 1 polyprtn as precursor

• 3' end

  • 47-125 nts UTR

  • Poly(A) tail essential for infectivity

Polyprtn & proteolytic processing

• Viral RNA is only mRNA

• Single large ORF encodes a polyprtn

  • Polyprtn cut by proteases into 11-12 functional prtns needed for rep'n

• 2A^pro cuts once

  • Separates P1 from rest of polyprtn

• 3C^pro cuts at 8 places

  • Produces all final prtns needed for rep'n

• Polio infection shuts down tsln of cellular mRNAs

  • Tsln machinery re-directed for viral prtns only

  • Prevention of PIC formation at cap of host mRNAs

• Cap-dependent tsln

  • All mRNAs, PIC assembles at 5' cap

    • Brings 2 ends of mRNA together and scan for AUG

    • Large ribosome subunit joins & tsln starts

  • Prtns in tsln:

    • eIF-4F: tripartite structure (eIF -4A, -4E, and -4G)

    • eIF-4G: euk initiation factor 4G

• Picornaviruses block tsln of host mRNA

  • Doesn't block own tsln

  • Inhibit PIC formation at 5' cap via:

    • 2A^Pro (polio) or L protease (FMDV) cleaves eIF-4G

    • Dephosp'n of 4E-BP1, binds eIF-4E tight, sequestering eIF-4E (used mt FMDV, not PV)

  • How PV prtns are translated

    • Secondary structures= stem-loop (5' UTR)

    • Tertiary structures= pseudoknots (5' UTR)

    • AUG Is downstream of IRES

    • Pyrimidine-rich seq'ce upstream of AUG

    • PIC binds directly at IRES -> lands on AUG -> initiates tsln

      • No need for cap

Poliovirus replication cycle

• Attachment

  • Canyon: poliovirus, rhinovirus

  • Surface loop: FMDV

  • Receptors: PVR, ICAM-1

• Uncoating

  • Sphingosine in hydrophobic pocket of pentamer helps VP1 penetrate mem (forms pore)

• Biosynthesis

  • Genome rep'n & IRES-based prtn syn

• Assembly

• Maturation

  • VP0 cleavage into VP2 & Vp4

Control of poliomyelitis

• Inactivated polio virus vaccine (Salk, 1955)

• Live attenuated vaccines (Sabin, 1960)

  • Dif Sabin vaccine strains possess mut'ns at multiple sites

The Cutter Incident

• April 1955

• Patient inoculated in buttock w Cutter vaccine

  • 9 days later, admitted to hospital for flaccid paralysis in both legs

• Stats

  • ~400 000 children given same vaccine in 10-day period

  • Within 2 months:

    • 94 cases of polio among vaccinees

    • 166 cases among family & community contacts

• 1995, Stalk succeeded with killed polio vaccine

Reconstruction of poliovirus

• US department of defence

• Syn DNA fragments as oligos

• Assembly into genome fragments

• Clone vector under T7 promoter

• In vitro txn to get viral RNA

• Assay for infectivity in mice expressing receptor Pvr

VRC: sites of poliovirus rep'n

• Viral rep'n on vesicles from ER

• Viral tsln, vesicle formation, & RNA syn are couples

VPg as prtn primes for genome rep'n

• Viral 3AB attaches to ER mem

• Tyr in 3B undergoes uridylylation

• Inserted 'UU' anneals poly(A) tail of viral genome

• VPg (3B) cleaved off from 3AB by 3C^Pro

• Syn (-)RNA

• Replicative intermediate (RI)

• Replicative form (RF)

Summary

• Picornaviridae: fam of (+)ssRNA viruses

  • Order: Picornavirales

  • Realm: Riboviria

  • Kingdom: Orthornavirae

• Belong to picornavirus-like supergroup of RNA viruses

• Members cause various diseases in humans & livestock

  • Poliomyelitis

  • Common cold

  • Heart diseases in humans

  • FMDV

• Polio

  • Effects

    • One of most damaging viral diseases of 20th century (after atomic bomb)

    • Causes epidemics of flaccid paralysis & death in infants due to lack of protecting antibodies in mothers as a result of the practice of better personal hygiene

    • Development & global use of vaccines (OPV & IPV) resulted in near worldwide eradication

  • Prototype of Enterovirus genus & family Picornaviridae

    • RNA genome:

      • V long 5' UTR

      • Single ORF

        • Tsln via cap-independent mechanism by cellular machinery using unique structure designated to the Internal ribosomal entry site (IRES)

        • Translated into a polyprtn, which is cleaved by proteases (encoded by the virus) into final prtn products

        • 2A: cleaves off P1 (b/w P1 & P2)

        • 3CD & 3C: cleave remaining sites

        • 2A & L cleave the euk tsln initiation factor (eIF-4G) -> shuts down tsln of host RNA upon infection -> infected cell now a factory for progeny viruses

      • 3' UTR

      • N-term: VPg polypeptide

        • Prtn primer for syn of gRNA & complimentary RNA (uridylylation)

• Research discoveries

  • Identification of RNA-dependent RNA Pol (RdRP)

  • Establishment of non-neuronal cell cultures

  • Plaque assay

  • Inactivated polio vaccine (IPV) (Salk)

  • Attenuated oral vaccine (OPV) (Sabin)

  • Polyprtn & proteolytic processing

  • Internal ribosome entry site (IRES) for initiation of tsln of viral DNA

  • 1st infectious viral clone for an animal virus

  • 1st synthetic polio viral clone from synthetic biology approach

Flaviviridae

• Supergroup of (+)RNA viruses

Phylogeny of Flaviviridae

• Based on the helicase domain of NS3

Classification of Flavivirus genus

• Viruses transmitted by & replicated in arthropods

• Principal hosts & natural reservoirs

  • Birds, rodents, monkeys, pigs

Defining features

• Density map in virion:

  • Envelope prtn layer: 180 copies of E & M prtns (T=3)

  • Lipid bilayer (viral envelope)

  • Nucleocapsid core: icosahedral (T=3), 25-30 nm, CP basic

• E prtns lie on top of, & in parallel w, lipid mem

  • Smooth surface

• (+)ssRNA genomes, 10-11 kb

• Genome expression: a single ORF, poly-prtn, & proteolytic processing

• Many (not all) members are transmitted by arthropod vectors (mosquito or tick) in which they also replicate (hence arboviruses)

Arboviruses

• Viruses of humans & animals that are transmitted by, & replicate in, arthropod vectors

• Members of the genus Flavivirus transmitted by mosquitos or ticks

• Viral families that are also vectored by/replicate in insects:

  • Flaviviridae

  • Togaviridae

  • Bunyaviridae

  • Arenaviridae

  • Rhabdoviridae

• 2 Modes of Transmission

  • Jungle cycle

    • Primates

  • Urban cycle

    • Humans (epidemic)

Yellow fever

• Origin: Africa

  • America & Europe via slave trades

• 1648: 1st recorded epidemic

• 1881: 1st suggested transmission by mosquito (Dr. Finlay)

• 1901: 1st human virus discovered

  • Experimentally confirms yellow fever transmission by mosquito

• Frequent epidemics in US 1700-1800s

• Re-emergence by urbanization & suspension of mosquito control

• 15% infections develop severe disease

  • Mortality: 20-50% in severe epidemics

• Established by yellow fever commission:

  • Serum contains the 'virus'

  • Infectious agent is filterable

  • Mosquitoes transmit disease

• Symptoms

  • Asymptomatic

  • Mild flu-like symptoms

  • Fulminant infections

  • Fatal

• Stages of infections w sever outcomes

  • Period of infection

    • 3-6 days after catching infection

    • Fever, chills, myalgia, back pain

    • Contagious

  • Period of remission

  • Period of intoxication

    • Jaundice, vomiting, viral rep'n in liver, no viremia

  • Hemorrhagic fever

    • Renal fever, hemorrhage, shock, multiple organ failure

• Control & prevention

  • Max Theiler (1930s)

  • Attenuated vaccine strain 17-D

  • Isolation via passages in monkeys, followed by 176 passages in primary cell cultures

  • 17-D provides immunity in monkeys & humans

  • Consensus vaccine strains differ from Asibi by 32 aa & 4 nts

Dengue fever

• Most prevalent vector-borne viral infection in the world

• Spread during WWII

• Primary infections for 1-2 weeks

• 25% of hospitalized patients develop prolonged fatigue & depression

  • Similar to mononucleosis & long covid

• 4 genotypes: DENV-1, -2, -3, & -4

  • Vary by 20-40% in E prtn

  • Asymptomatic infections

  • Dengue fever (DF): mild & self-limiting (DENV-4)

  • Dengue hemorrhagic fever (DHF)

  • Dengue shock syndrome (DSS)

• Secondary Dengue infections & antibody-dependent enhancement

  • 2nd infection in person who previously infected w dif serotype may lead to higher viremia & worse outcomes (DHF & DSS)

  • Main reason for lack of effective vaccines against DENV

• Proposed mechanism:

  • Cross-reactive antibodies present in the patient from earlier infection bind to virions of new serotype

    • Assists viral entry into large # of cells expressing Fc receptors for IgG

  • Instead of conferring partial immunity against secondary infections, pre-existing antibodies help virus enter large # of cells during a new infection

    • Leads to severe outcomes (DHF & DSS)

• Sanofi Pasteur - live attenuated vaccine

West Nile

• 1937: 1st reported case

• Infection of central nervous system (CNS) -> encephalitis (mid-fatal) -> paralysis -> death

• 1999: Introduction in NYC

• 2000: Re-appeared in mosquito season

• 2002: Blood transfusion shown to cause infection in recipients

• Humans/mammals = 'dead-end' carriers

  • Cant transmit back to mosquito

Zika virus

• Since 2007: moving from Pacific ocean -> America

• Microcephaly in children (small head birth defect)

• Feb 2016: WHO declares it as a Public Health Emergency of International Concern

Hepatitis C (HCV)

• One of most widespread diseases globally

• Transmission: blood, blood products, organ transplants, injection drug use, body piercing

• 1989: Discovery through molecular cloning and sequencing

• Most acute infections become chronic

  • -> Liver cancer & cirrhosis (liver scarring)

• High prevalence Egypt, Asia, & Australia

  • Reuse of syringes among children when treating schistosomiasis

• Infects hepatocytes & lymphocytes

  • 50 virions/day/hepatocyte -> 10¹² produced/infected person/day

• Infection w one genotype does NOT confer immunity against another

• Chromic HCV infections asymptomatic for first decades

• Long term outcomes

  • 75-85% infected will develop chronic infection

  • 60-70% develop cirrhosis over 20-30 yrs

  • 1-5% die from cirrhosis or liver cancer

• Disease outcomes

  • Cirrhosis

    • Increased chance if alcohol or certain prescription drugs

  • Hepatocellular carcinoma

  • Liver failure

  • Death

• No vaccines available

  • Lack of effective experimental systems impeded research & antiviral development until recently

    • Pre-2005: Slow research bc low viral titer in the liver

      • Relied on full-length & mini (subgenomic) viral amplicon & humanized mice

    • Post-2005: JFH-1 isolated

      • Grows in cell culture without need for adaptive mutants

  • Treatment w antiviral drugs:

    • Old: pegylated interferon (INF) α, ribavirin (RBV)

    • New: direct acting antivirals (DAAs)

• Genome structure & expression strategies

  • No 5' Cap (HEP C ONLY, NOT ALL FLAVIVIRUSES)

    • Use IRES to initiate txn of viral polyprtn

  • No 3' Poly-A tail

    • Has many helices on 3' end

  • Size: 9.6 kb (HCV)

    • Members of genus Flavivirus: 11 kb

  • Shared properties w Flaviviridae family

    • Single large ORF encoding a protein

    • Cleavage by viral & host proteases

Virus replication cycle

• Attachment

  • E prtn binds 1+ receptors (possibly glycosaminoglycans)

• Entry

  • Receptor-mediated endocytosis

• Genome uncoating

  • Low pH dependent mem fusion w endosome mem

• Prtn synthesis

  • Polyprtn assoc's w ER

    • Cleavage into multiple functional prtns

• RNA synthesis

  • In small ER-derived vesicles

• Assembly & release

  • On ctpsmic side of ER

  • Bud into ER lumen

    • Exit via exocytosis

  • Fusion b/w transport vesicle & plasma mem releases virions

Evolution of HCV treatment using antivirals

• Traditional treatments relied on combinational therapy w interferon & ribavirin

• Problems in low compliance w antiviral treatment

  • Severe side effects (headache, nausea, fever)

• Liver transplants scarce

  • V costly

• Direct acting antiviral (DAA) drugs

  • Target key viral enzymes (protease & RdRp)

  • Nucleoside analog RdRP inhibitor drugs offer high % of SVR

  • High efficiency, short treatment duration

Sofosbuvir

• Most effective nucleoside inhibitor analog of RdRP

• Cure rate 30-70% (depends on HCV genotypes)

• V effective against multiple genotypes

• Strong barrier against emergence of resistant mutants

• Oral administration

• No-mild side effects

Forms of virion

• Virion has lipid mem, studded w envelope prtns (icosahedral symmetry; T=3)

• Immature viral particles display spikes on virion surface

  • Ea spike has 3 pairs of E & M heterodimers

• Conformational changes in E & M prtns produce smooth & infectious virions

Homodimer of envelope prtns

• Fusion peptide: Hidden by domain III

• Domain II: Interaction to form E dimers

• Domain III: Ig fold, binding to receptor

• W/in endosome:

  1. Low pH changes conformation of E prtn

  2. Exposes fusion peptide

  3. Releases core into ctpsm

Summary

• Flaviviridae: Flavivirus-like supergroup of (+)ssRNA viruses

• 3 genera:

  • Flavivirus (yellow fever virus)

  • Hepacivirus (Hep C)

  • Pestivirus

• Flavivirus transmission: arthropod vectors (arboviruses)

• Yellow fever

  • 1st human virus identified

  • In tropics & subtropics

  • Non-human primates -> humans via mosquito bites

  • To America via slave trade

  • 1793 epidemic killed 10% of Philadelphia

  • Caused impediment to Panama Canal Project

  • Max Theiler - attenuated vaccine strain 17-D (derived from v pathogenic strain)

• Hep C (HCV)

  • Often results in chronic infection

    • -> cirrhosis, liver cancer, & death

  • Transmission via blood, organ transplant, contaminated needles, etc

    • Early 1900s blood screening reduced prevalence of HCV

  • 1989: virus identified through molecular cloning

    • Cell culture system - HCV isolate (JFH-1)

      • Was able to replicate in the cell without need for adaptive mutations

  • No vaccines, treatment uses interferons & antiviral drugs (ribavirin, protease inhibitors)

  • Recently: direct acting antivirals (DAA)

    • More efficient

    • Safer

    • Shorter treatment times

    • Less/no side effects

• Structure

  • (+)ssRNA

  • 9.5-11 kb (Flavivirus)

  • 3 layers:

    • Envelope prtn layer: E & M prtns

    • Lipid mem layer: icosahedral symmetry

    • Nucleocapsid core: icosahedral symmetry

  • 1 ORF

    • Translated into single polyprtn

      • Proteases cleavage into 10 functional prtns (like Picornaviridae)

  • Flavivirus only

    • 5' cap

    • (transmission by arthropod vectors)

  • Hepacivirus only (Hep C)

    • No 5' cap (has IRES)