Virology Notes

What is Virology?

• Virology is the study of viruses and virus-like agents, including their taxonomy, disease-producing properties, cultivation, and genetics.

• Viruses are infectious agents that cannot replicate on their own.

• Viruses are obligate intracellular parasites and are ultra-microscopic in size.

What are Viruses & What Matters?

• Viruses are obligate intracellular parasites, meaning they cannot reproduce outside a cellular host.

• Viruses are not alive and have only one goal: a productive infection.

• Viruses cause more than 80% of infectious diseases.

• Understanding viral structure and the infection cycle is important for health professionals.

Definition and Properties of a Virus

• Viruses are filterable agents.

• Viruses are obligate intracellular parasites.

• Viruses cannot make energy or proteins independently of a host cell.

• Viral genomes may be RNA or DNA but not both.

• Viruses have a naked capsid or an envelope morphology.

• Viral components are assembled and do not replicate by "division."

Consequences of Viral Properties

• Viruses are not living.

• Viruses must be infectious to endure in nature.

• Viruses must be able to use host cell processes to produce their components (viral messenger RNA, protein, and identical copies of the genome).

• Viruses must encode any required processes not provided by the cell.

• Viral components must self-assemble.

Virus Structure

• An infective viral particle is called a virion.

• Viral nucleic acid is surrounded by a protein coat called a capsid.

• Each capsid is made up of capsomeres.

• Some viruses have a lipid envelope.

• Nucleocapsid: capsid + nucleic acid binding proteins.

• Enveloped Virus: Enclosed within a lipid membrane and sensitive to heat.

• Non-enveloped Virus: Lacks a bilayer lipid membrane and heat-resistant.

Virion Structure

• Virion structure must:

  • Be strong enough to protect the viral nucleic acid.

  • Be able to shed the protein coat upon entry into a host cell.

• Capsid and Envelope Protect viral genome from Chemical agents & Physical conditions (pH, dehydration, temperature etc.)

Classification Criteria

• Nucleic acid type (DNA or RNA).

• Symmetry of capsid (icosahedral, helical, complex).

• Naked or enveloped.

Families of DNA Viruses

• Poxviridae: Smallpox virus, vaccinia virus, monkeypox, canarypox, molluscum contagiosum

• Herpesviridae: Herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesviruses 6, 7, and 8

• Adenoviridae: Adenovirus

• Papillomaviridae: Papillomavirus

• Polyomaviridae: JC virus, BK virus, SV40

• Parvoviridae: Parvovirus B19, adeno-associated virus

• Hepadnaviridae: Hepatitis B virus

Families of RNA Viruses

• Paramyxoviridae: Parainfluenza virus, Sendai virus, measles virus, mumps virus, respiratory syncytial virus, metapneumovirus

• Orthomyxoviridae: Influenza virus types A, B, C and thogotoviruses

• Coronaviridae: Coronavirus, SARS virus, MERS virus, SARS-CoV-1 AND SARS-COV-2

• Arenaviridae: Lassa fever virus, Tacaribe virus complex (Junin and Machupo viruses), lymphocytic choriomeningitis virus

• Rhabdoviridae: Rabies virus, vesicular stomatitis virus

• Filoviridae: Ebola virus, Marburg virus

• Bunyaviridae: California encephalitis virus, La Crosse virus, sandfly fever virus, hemorrhagic fever virus, hantavirus

• Retroviridae: Human T-cell leukemia virus types I and II, HIV, animal oncoviruses

• Reoviridae: Rotavirus, Colorado tick fever virus

• Togaviridae: Rubella virus; western, eastern, and Venezuelan equine encephalitis virus; Ross River virus; Sindbis virus; Semliki Forest virus; chikungunya virus

• Flaviviridae: Yellow fever virus, dengue virus, St. Louis encephalitis virus, West Nile virus, hepatitis C virus

• Caliciviridae: Norwalk virus, calicivirus

• Picornaviridae: Rhinoviruses, poliovirus, echoviruses, parechovirus, coxsackievirus, hepatitis A virus

• Hepeviridae: Hepatitis E virus

• Astroviridae: Astrovirus

• Delta: Delta agent

The Capsid

• Built from identical protein units called capsomeres.

• Capsomeres bond together to give the capsid structural symmetry.

• Viruses are either helical, icosahedral, or complex.

Helical Viruses

• Two shapes:

  • Rod – straight and relatively rigid

  • Filamentous – flexible, curved, or coiled

• Example: Influenza Virus

Influenza Virus

• Enveloped helical virus.

• RNA virus with RNA segmented into 8.

• Each segment has its own helical capsid.

• Nucleoproteins keep the capsid in place.

• Matrix protein: a second layer of protein

• Envelope contains Hemagglutinin (H) for binding to and entering the host cell and Neuraminidase (N) for release from the host cell. Subtypes

  • Hemagglutinin: 17 subtypes

  • Neuraminidase: 17 subtypes

• Only two influenza A virus subtypes (i.e., H1N1, and H3N2) are currently in general circulation among people → Seasonal Flu.
  Pandemics:

• Pandemics happen when new (novel) influenza A viruses emerge which are able to infect people easily and spread from person to person in an efficient and sustained way.

How Pandemic Flu Viruses Generated?

• Antigenic Shift: Reassortment (Bird-to-human transmission)

• Antigenic Drift: Mutations

Genetic Reassortment

• Cause: Antigenic Shift (combining of viral RNA)

• Mutation accumulation

• Cause: Antigenic Drift

Coronavirus Origins

• Coronavirus (SARS and MERS) is the most prominent example of an emerging virus that has crossed the species barrier from wild animals to humans.

• The origin of SARS-CoV-2 is also suspected to be from an intermediate animal host.

• The possibility of crossing the species barrier again for the fourth time cannot be ruled out.

Point to Think

• Why do you need to get vaccinated every year?

• Human influenza A and B viruses cause seasonal epidemics of disease.

• Influenza viruses are constantly changing in two different ways.

Icosahedral Viruses

• Their shape is derived from 20 triangular faces that make up the capsid.

• There are two types of icosahedral viruses:

  • Simple

  • Complex

• Non-enveloped Simple Icosahedral Virus

Herpes Virus

• Enveloped Complex Icosahedral Virus.

Polio

• Polio is a simple icosahedral virus

• The smallest and simplest of the viruses that infects humans. 50nm - $(50 * 10^{-6})$ mm

Complex Viruses

• Their shape is not symmetrical.

• They have complicated structure

• Eg. Variola virus (human virus) and Bacteriophage (bacterial virus)

Viral Envelope

• Many viruses that infect humans and other animals are enveloped.

• Envelopes form when viral glycoproteins and oligosaccharides associate with the plasma membrane of the host cell.

• All envelopes have a phospholipid bilayer.

• Glycoproteins: integral membrane proteins, forms spikes.

• Envelopes vary in:

  • Size

  • Morphology

  • Complexity

  • Composition

Virion Structure: Naked Capsid

• Component: Protein

• Properties:

  • Is environmentally stable to temperature, acid, proteases, detergents, and drying.

  • Is released from cell by lysis.

• Consequences:

  • Can be spread easily (on fomites, from hand to hand, by dust, by small droplets).

  • Can dry out and retain infectivity.

  • Can survive the adverse conditions of the gut.

  • Can be resistant to detergents and poor sewage treatment.

  • Antibody may be sufficient for immunoprotection.

Virion Structure: Envelope

• Components: Membrane, Lipids, Proteins, Glycoproteins

• Properties:

  • Is environmentally labile-disrupted by acid, detergents, drying, and heat.

  • Modifies cell membrane during replication.

  • Is released by budding and cell lysis.

• Consequences:

  • Must stay wet.

  • Cannot survive the gastrointestinal tract.

  • Spreads in large droplets, secretions, organ transplants, and blood transfusions.

  • Does not need to kill the cell to spread.

  • May need antibody and cell-mediated immune response for protection and control.

  • Elicits hypersensitivity and inflammation to cause immunopathogenesis.

Viral Classification

• Structure: size, morphology, and nucleic acid (e.g., picornavirus [small RNA], togavirus)

• Biochemical characteristics: structure and mode of replicationª

• Disease: encephalitis and hepatitis viruses, for example

• Means of transmission: arbovirus spread by insects, for example

• Host cell (host range): animal (human, mouse, bird), plant, bacteria

• Tissue or organ (tropism): adenovirus and enterovirus, for example

Virus Classifications

• Based on the host: Animal viruses, Plant viruses, Bacterial viruses (bacteriophages/phages)

• Based on the genomic material: DNA viruses, RNA viruses

• According to the shape/capsid symmetry: Icosahedron, Helical, Complex

• Absence/Presence of envelope: Naked, Enveloped

• Double stranded (ds) vs Single stranded (ss)

• Positive sense vs negative sense

DNA Viruses (Enveloped)

• Pox

• Herpes

• Hepadna

DNA Viruses (Naked Capsid)

• Polyoma

• Parvo (ss)

• Papilloma

• Adeno

RNA Viruses (+RNA)

• Picorna (N)

• Toga (E)

• Calici (N)

• Flavi (E)

• Hepe (N)

• Corona (E)

RNA Viruses (-RNA)

• Rhabdo (E)

• Filo (E)

• Orthomyxo (E)

• Paramyxo (E)

• Bunya (E)

• Arena (E)

RNA Viruses (+/-RNA)

• Reo (Double capsid)

RNA Viruses (+RNA via DNA)

• Retro (E)

• Astro (N)

The Infection Cycle

How to produce more virions?

• Once produced, the host cells fill with virions and either bursts or releases the virions via budding.

• The result can be host cell death → Lytic Infection

• The viral genome becomes incorporated into the host cell’s DNA, forming a provirus

• New virions are not produced

• It can remain this way for an extended period

• The host cell lives → Latent infection

What Happens in Each Phase?

• Early phase

• Late phase

• Eclipse phase

• Latent phase

• Burst size

Steps in Viral Replication

1. Recognition of the target cell

2. Attachment

3. Penetration

4. Uncoating

5. Macromolecular synthesis

   • Early mRNA and nonstructural protein synthesis: genes for enzymes and nucleic acid-binding proteins

   • Replication of genome

   • Late mRNA and structural protein synthesis

   • Posttranslational modification of protein

6. Assembly of virus

7. Budding of enveloped viruses

8. Release of virus

Recognition and Attachment

• Attachment occurs when a virion binds to specific receptors on a host cell.

• Some viruses require a co-receptor to attach.

  • Without the co-receptor, there is no infection

• Bonding occur via VAPs (viral attachment proteins) or surface structures on the viral capsid.

• Viral-host cell interactions occur through random collisions.

• The number of viruses is extremely important

  • Large number of viral progeny increases the chance

When Virus Meets Host

• The host cell must be permissive for the infection to succeed

  • It must contain all of the components required to make new virions

• Viral infections at the apical (top) cell surface usually cause localized infection

• Viral infection at the basolateral (bottom) cell surface can become systemic

• Many viruses attach only to specific areas of the host cell membrane called lipid rafts

  • Lipid rafts are rich in cholesterol, fatty acids, and other lipids

  • They are more reliable for stable attachment

  • They are also the site of release for many viruses

• Some case host cell receptors or co-receptors are used for a successful attachment.

Receptor Binding

• Many different host cell molecules can be used as viral receptors

  • Some viruses use more than one type

  • Some receptors are shared by many viruses

• Receptors can determine host range of virus

• Virus-receptor binding is highly specific

• Non-enveloped viruses

  • Binding takes place between viral capsid and receptor

• Enveloped viruses

  • Binding takes place between viral envelope proteins and receptor

Receptor Examples

• Epstein-Barr virus: C3d complement receptor (CR2, CD21) on B cells

• HIV: CD4 molecule and chemokine coreceptor on helper T cells

• Rhinovirus: ICAM-1 (immunoglobulin superfamily protein) on Epithelial cells

• Poliovirus: Immunoglobulin superfamily protein on Epithelial cells

• Herpes simplex virus: Herpesvirus entry mediator (HveA), nectin-1 on Many cells

• Rabies virus: Acetylcholine receptor, NCAM on Neuron

• Influenza A virus: Sialic acid on Epithelial cells

• B19 parvovirus: Erythrocyte P antigen (globoside) on Erythroid precursors

Penetration & Uncoating

• Penetration or internalization mediated by the host cell receptors and VAPs

• Non enveloped viruses → Endocytosis or Viropexis

• Enveloped viruses → Membrane fusion

Non-Enveloped Viruses

• Use receptor-mediated endocytosis to gain entry into the host cell

• Virus is enclosed in a vesicle – the early endosome

• Early endosomes fuse with or become late endosomes

• Late endosomes fuse with the lysosome where uncoating begins

Enveloped Viruses

• Envelope fuses with the host cell membrane

• Fusion is mediated by specialized fusion proteins of the host cell

• It results in the formation of a fusion pore – an opening allowing viral entry

• For some viruses, fusion requires the presence of co-receptor molecules

Uncoating Strategies

• Once attached, the virus must gain entry to the host cell

• It must also uncoat or remove the capsid

• Uncoating can occur:

  • At the plasma membrane

  • Within endosomes

  • At the nuclear membrane

Cytoplasmic Transport of Viral Components

• Viral infection requires compartmentalization

• Viral genomes, capsids, and other viral proteins are synthesized in specific locations in the host cell

• Newly synthesized viral components are moved to other locations for assembly of viral particles

• After uncoating viral components are moved in vesicles through:

  • membrane enclosed vesicles with host cell cytoskeleton (microtubules) OR

  • direct association with cell transport mechanisms OR

  • Specialized host cell lipids or proteins(chaperons)

Transport of the Viral Genome into the Nucleus

• DNA viruses

  • dock to the nuclear membrane.

  • The viral genome is delivered into the nucleus

• RNA viruses

  • Some use reverse transcriptase to convert RNA to DNA but this applies only to retroviruses

  • Movement of the DNA copy into the host cell nucleus occurs during mitosis, when the nuclear membrane is broken down

  • The DNA copy integrates with the host DNA

The Central Dogma

• The central dogma of molecular biology is an explanation of the flow of genetic information within a biological system.

• The main thesis of molecular inheritance

• "DNA makes RNA, and RNA makes protein"

Macromolecular Synthesis

• Biosynthesis of new viral components (DNA/RNA and proteins) can be complex – depending on the type of virus

• Viral genomes are either DNA or RNA

• Both can be single-stranded or double-stranded

Replication of DNA Virus Genomes

• Replication is performed by the host cell machinery

• Rapid transcription of viral DNA is regulated by host cell proteins

• Host DNA synthesis is inhibited by the virus

• All polymerases and proteins concentrate on viral DNA synthesis

dsDNA Genome Viruses

• have a genome exactly the same as the host cell that they are infecting.

• For this reason, many host enzymes can be utilized for replication and/or protein production.

• The genome replication: dsDNA → dsDNA. Use DNA-polymerase from either the virus or the host cell.

• dsDNA → mRNA → protein RNA-polymerase producing the mRNA and the host ribosome producing the protein.

SSDNA Genome Viruses

• First dsDNA made and proceed as usual. ssDNA → dsDNA → dsDNA

• The single viral strand is converted into double strands of DNA

• This uses the host cell’s DNA polymerase

• The complementary copy is used as a template for transcription

RNA Viruses

• RNA viruses are classified using a (+) or (–) strand designation for their genomes

• Single-stranded RNA viruses are either:

  • (+) single-stranded, contain a (+) strand

  • (–) single-stranded, contain a (–) strand

• Double-stranded RNA viruses contain one (+) strand and one (–) strand

• Replication and transcription are based on:

  • Complementary base pairing

  • The use of templates

Positive and Negative Sense RNA Viruses

• The main difference between positive and negative sense RNA virus is that positive sense RNA virus consists of viral mRNA that can be directly translated into proteins whereas negative sense RNA virus consists of viral RNA that is complementary to the viral mRNA.

Double-Stranded RNA Viruses

• Genomes contain one (+) strand and one (–) strand

• During infection, the (–) strand is copied into mRNA

• This is done by a viral RNA polymerase

• The mRNA is used to produce viral proteins

• Each of the strands is used as a template to make a new double-stranded genome

(+) ssRNA Viruses

• The (+) strand is already mRNA

• It can be directly translated into viral proteins

• Genome replication has 2 steps:

  • The (+) strand is copied into (–) template

  • The template is used to make more (+) strands

(-) ssRNA Viruses

• The (–) strand cannot be used as mRNA

• It must first be copied into a (+) template strand

• This is done using a viral RNA polymerase

• The new template strand is mRNA:

  • It is used to synthesis viral proteins

  • It is also used to make new viral genomes

Retroviral Transcription and Integration

• Retroviruses are RNA viruses that contain the enzyme reverse transcriptase

• Reverse transcriptase converts RNA into DNA

• Converted viral DNA can be inserted into the host cell chromosome

Viral Control of Translation

• Viruses rely on host cell machinery for translation

  • Host cell ribosomes

  • Host tRNA

  • Host’s post translational mechanisms

• They often modify this machinery for their purposes

• The entire genome (+) strand RNA virus is read by the ribosome to make one giant polyprotein

• Host genes can inhibit RNA binding to ribosomes. Viruses have developed ways to inactivate these genes.

Virus Assembly

• Involves the movement of newly made viral components to specific sites in the host cell

• There are two steps in maturation:

  • Intracellular trafficking

  • Assembly

Intracellular Trafficking

• Some viral components are synthesized in the cytoplasm, and some in the nucleus

• They are transported through the cell by host cell microtubules to assembly sites

• Assembly sites depend on:

  • The type of genome (DNA or RNA)

  • The mechanism of genome replication

  • The presence or absence of an envelope

• Assembly sites:

  • near the host cell membrane

  • near membrane-bound organelles

  • in the host cytoplasm or host cell nucleus (mostly enveloped viruses)

• Viral proteins travel from the assembly site to the cell membrane in vesicles

• Viral protein transport involves the Golgi apparatus, which sorts the proteins

Virus Assembly (Non-enveloped viruses)

• Formation of structural subunits for the capsid

• Assembly of the capsid

• Association of the viral genome within the capsid

Virus Assembly (Enveloped viruses)

• Formation of structural subunits for the capsid

• Assembly of the capsid

• Association of viral genome within the capsid

• Assembly of viral envelope glycoproteins

• All virions must complete a common set of assembly reactions

Capsid Assembly

• Capsomeres are assembled first

• Assembly is different in DNA viruses and RNA viruses

• The number of capsomeres produced is always more than the number required

  • This maximizes the chances of capsomeres finding each other

• Capsid assembly can be assisted by host chaperone proteins

Budding & Release

• New virions can be released from the host cell in two ways:

  1. Budding

  2. Lysis

• In some viral infections, completed virus particle is non-infectious.

• Viral enzymes convert the immature virus into an infectious form after release

• Complex process of: bud formation, bud growth, fusion of the bud membrane and separation.

• Membrane fusion reactions pinch off the viral bud from the host plasma membrane.

• Some viruses gain envelope during this bud release

Spread of the Infection

• Viruses can spread from cell to cell

  • They can use tight junctions between cells

  • They can also spread through the formation of syncytia

• They allow movement through the body without exposure to the immune system

• Some viruses produce decoy virions

  • These are empty capsids or non-infectious virions

  • They confuse and distract the host defenses

• Some viruses incorporate host proteins as a type of camouflage