M25 - Intro to Viruses

HSV-1: Herpetic Whitlow

DISCUSSED LATER

Laryngeal Papillomas

HPV-6, HPV-11 Discussed Later

Model of Bacteriophage Particle.

Head (Capsid enclosing DNA) Collar (stabilizes head onto rest of phage) Core Helical Sheath (healical protein covering the body that contracts to inject viral DNA into bacterial cell) Tail Pins (used to attach to host cell receptor) Hexagonal Base Plate

Viral Hemagglutination - Antiserum

Hemagglutination assay may be modified to include the addition of an antiserum. By using a standard amount of virus, a standard amount of RBCs, and serially diluting the antiserum, one can identify the concentration of the antiserum [the greatest dilution (smallest amount) which inhibits hemagglutination]

HPV

Human Papilloma Virus Discussed Later

Negri Body

In rabies: eosinophilic intracytoplasmic inclusions in hippocampus and Cerebellar Purkinje cells - contain virus - consist of ribonuclear proteins produced by the virus

Intranuclear inclusion bodies

Indicative of viral infection, found within cells infected with cytomegalovirus

Capsomeres

Protein subunits used in capsid synthesis

Influenza Virus

RNA Virus Discussed later

Virion-Associated Polymerases

RNA-dependent RNA polymerase RNA-dependent DNA polymerase

Virion-associated enzymes

RNA-dependent RNA polymerase RNA-dependent DNA polymerase Neuraminidase Lysozyme

Cytopathic/Cytopathogenic Effect (CPE)

Refers to detectable damage/pathological changes to host cells during virus invasion - include such things as stopping mitosis, lysis of cells, formation of inclusion bodies, cell fusion, antigenic and chromosomal changes and transformation - degenerative or morphologic changes including chromosomal aberrations, membrane permeability, and protein synthesis Damage is especially associated with the multiplication of certain viruses

Viral Genome

consisting of DNA or RNA 1.) Genomic DNA size range: 1.2 x 106  dalton (3.2 kbp) genome of hepadnaviruses to 4 x 108 dalton (375kbp) genome of poxviruse 2.) Genomic RNA size range:1.7 kb genome of hepatitis delta virus (HDV) to 33 kb genome of coronaviruses.

Live Attenuated Vaccine

created by reducing the virulence of a pathogen, but still keeping it viable (or "live") - Attenuation takes an infectious agent and alters it so that it becomes harmless or less virulent

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Baltimore Classification Scheme Class VI

ssRNA, positive sense, dsDNA intermediate produced during replication (retroviruses)

Baltimore Classification Scheme Class IV

ssRNA, positive-sense (picornaviruses)

Inactivated Vaccines

vaccines that contain killed microbes

Baltimore Classification Scheme Class IIa

ssDNA, positive-sense (bacteriophage M13)

Baltimore Classification Scheme Class V

ssRNA, negative-sense (orthomyxoviruses)

Adenovirus Particles

*36-38 kbp DNA genome, and prominent penton fibers* - Adenoviruses arew the largest nonenveloped viruses. Large size emables them to be transported through the endosome (envelope fusion is not necessary).

Baltimore Classification Scheme

*Class I*: dsDNA (herpesviruses, poxviruses) *Class IIa*: ssDNA, positive-sense (bacteriophage M13) *Class IIb*: ssDNA, negative-sense (parvoviruses) *Class III*: dsRNA (rotaviruses) *Class IV*: ssRNA, positive-sense (picornaviruses) *Class V*: ssRNA, negative-sense (orthomyxoviruses) *Class VI*: ssRNA, positive sense, dsDNA intermediate produced during replication (retroviruses) *Class VII*: dsDNA, ssRNA intermediate produced during replication (hepadnaviruses)

Response of cells to infection

*Cytopathic effect, Cytopathogenic effect, CPE)* 1.) No change in cell morphology 2.) Rounding of cells/detachment 3.) Lysis 4.) Fusion

General Features of Viruses

- Acellular - Small in comparison to cellular entities - Dependent on host cells for replication - Multiply via the assembly of progeny viruses rather than the division of precursor viruses

Types of Vaccines

- Inactivated - Live attenuated - Subunit - DNA

Viral Growth Steps

1.) *Adsorption* of virus (initial phase) 2.) *Eclipse phase*: lasts for 10-12 hrs, corresponds to the period during which the input virus becomes uncoated. As a result, no infectious virus can detected during this time (any infectious virus detected is simply virus that is still stuck on the cell membrane). 3.) *Synthetic phase* Starts around 12 hours post-infection and corresponds to the time during which new virus particles are assembled. 4.) *Latent Period:* During this period, no extracellular virus can be detected. After ~18 hours, extracellular virus is detected. Ultimately, production will reach a maximum plateau level

Virus Replication

1.) *Attachment*: Virus particles bind to cell-surface receptors 2.) Penetration of virus particle into host cell 3.) Uncoating of viral genome 4.) Synthesis a.) Genome replication b.) Protein synthesis 5.) Packaging and release of progeny viruses

Viral Replication Detailed

1.) Attachment of virus to host cell at CD4 receptor 2.) Fusion of viral envelope with cell membrane --> entry of virion into cell 3.) Synthesis of dsRNA by Reverse Transcriptase 4a.) Integration of viral DNA into host cell DNA 4b.) Some viral DNA stays in cytomplasm 5.) Transcription of proviral DNA into viral genomic RNA and mRNA by host cell's RNA Polymerase 6.) Translation of viral mRNA into viral compondents 7a.) Assembly of viral components and maturation of virion 7b.) Assembly of viral envelope at cell surface membrane 8.) Budding of virion from host cell 9.) Dissemination of virion to other body sites via circulation

Baltimore classification scheme and the central dogma

1.) Baltimore Classification Scheme (Class I-VII) 2.) Virion-Associated Polymerases 3.) Infectious vs. Noninfectious genomes

Classification of Viruses

1.) Capsid structure (virion morphology) 2.) Presence or absence of envelope - Enveloped viruses possess virus-encoded envelope glycoproteins - Ether sensitivity 3.) Identity of host 4.) Effect infection has on host. 5.) Nucleic acid content 6.) Virus Families Established

Enumeration of Viruses

1.) Direct examination with electron microscope 2.) Plaque assay 3.) Focus-forming assay 4.) Fluorescent-focus assay 5.) Hemagglutination

Herpesvirus Replication Cycle

1.) Entry and Uncoating 2.) Immediate Early: Transcription and Post-Transcriptional Processing 3.) Early: DNA Replication, Assembly of new viruses --> break out and spread to other cells 4.) Late: ??? Proteins on cell membrane???

Interference with Virus Replication

1.) Interferon 2.) Antibodies that mask ligands, induce complement activity, promote ADCC a.) Vaccines - Inactivated - Live attenuated - Subunit - DNA 3.) Base analogs 4.) Inhibitors of uncoating 5.) Soluble receptors or receptor ligands

Transformation

1.) Introduction of oncogene 2.) Change in level of expression of cellular gene. - Viral promoters, enhancers - Insertional inactivation

Culturing of Animal Viruses

1.) Live animals 2.) Fertilized eggs 3.) Organ cultures 4.) Cell cultures - Primary cell cultures - Continuous cell lines 5.) Cell extracts.

Koch's Postulates

1.) The causative agent must be present in every individual with the disease 2.) The causative agent must be isolated and grown in pure culture 3.) The pure culture must cause the disease when inoculated into an experimental animal 4.) The causative agent must be re-isolated from the experimental animal and re-identified in pure culture

Virus Structure

1.) Viral Genome consisting of DNA or RNA 2.) Capsid composed of Protein 3.) Some Viruses posses an Envelope that surrounds the capsid and contains glycoproteins

Particle/PFU Ratio

1.) Virion as infectious virus particle 2.) Published particle/PFU ratios: a.) Poliovirus ~30-1000 b.) Influenza virus ~20-50 b.) Reovirus ~10 c.) Poxviridae ~1-100 d.) Herpes simplex virus~50-200

Herpesvirus Particle

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Rotavirus Particles

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Hepatitis A Virus Particles

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PFU (Plaque Forming Unit)

A measure of the number of particles capable of forming plaques per unit volume, such as virus particles. Functional measurement rather than a measurement of the absolute quantity of particles: viral particles that are defective or which fail to infect their target cell will not produce a plaque and thus will not be counted

Interferon

A protein released by infected cells, usually in response to the entry of a virus, that has the property of inhibiting virus replication by attaching to uninfected cells which stimulates the uninfected cell to synthesize another antiviral protein that inhibits viral replication. (Quizlet definition)

Penton Fibers

Adenoviruses have a unique "spike" or fiber associated with each penton base of the capsid that aids in attachment to the host cell via the receptor on the surface of the host cell - *epitope*

Continuous Cell Lines

An established or immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene - Numerous cell lines are well established as representative of particular cell types

Lysozyme (Muramidase)

Glycoside hydrolases - Damage bacterial cell walls by catalyzing hydrolysis of linkages between NAM and NAG residues

Baltimore Classification Scheme Class IIb

ssDNA, negative-sense (parvoviruses)

Capsid Composed of Protein

Capsid composed of protein - nucleocapsid: genome + capsid - capsomeres: the subunits used in capsid synthesis 1.) Polyhedral (icosahedral) 2.) Helical 3.) Binal 4.) Complex

RNA-dependent RNA Polymerase

Catalyzes the replication of RNA from an RNA template - Essential protein encoded in genomes of all RNA-containing viruses with no DNA stage that have sense negative RNA

Primary Cell Cultures

Cells that are cultured directly from a subject Have limited lifespan - After a certain number of population doublings (called the Hayflick limit), cells undergo the process of senescence and stop dividing, while generally retaining viability

HSV-1: Oral Infection

DISCUSSED LATER Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known as human herpesvirus 1 and 2 (HHV-1 and HHV-2), are two members of the herpesvirus family, Herpesviridae, that infect humans.Both are ubiquitous and contagious - Symptoms include watery blisters in the skin or mucous membranes of the mouth, lips or genitals. Lesions heal with a scab characteristic of herpetic disease. As neurotropic and neuroinvasive viruses, HSV-1 and -2 persist in the body by becoming latent and hiding from the immune system in the cell bodies of neurons. After the initial or primary infection, some infected people experience sporadic episodes of viral reactivation or outbreaks. In an outbreak, the virus in a nerve cell becomes active and is transported via the neuron's axon to the skin, where virus replication and shedding occur and cause new sores.

HIV Structure and Budding

Different in structure from other retroviruses, roughly spherical, ~60x smaller than RBC, yet large for a virus Composed of 2 copies of positive single-stranded RNA enclosed by a *conical capsid* (composed of viral protein p24) - *Matrix* of viral protein p17 surrounds the capsid - Matrix surrounded *Viral envelope* composed of phospholipid bilayer taken from membrane of host cell when a newly formed virus particle buds from the cell - Embedded in viral envelope are proteins from the host cell and ~70 copies of a complex HIV protein (Env) that protrudes through the surface of the virus particle that enables the virus to attach to and fuse with target cells to initiate the infectious cycle New HIV particles bud out of the host cell, taking plasma membrane with them.

Oral Papillomavirus Infections

Discussed Later

RNA-dependent DNA Polymerase

Enzyme used to generate complementary DNA (cDNA) from an RNA template (Reverse Transcription - RT). - Important for Retroviruses (e.g., HIV), Retroviral RT has 3 sequential biochemical activities: - RNA-dependent DNA polymerase, - Ribonuclease H, and - DNA-dependent DNA polymerase. These activities convert single-stranded genomic RNA into double-stranded cDNA which can integrate into the host genome, potentially generating a long-term infection that can be very difficult to eradicate

Nucleocapsid

Genome + Capsid

Neuraminidase

Glycoside hydrolase enzymes that cleave the glycosidic linkages of neuraminic acids - Drug target for prevention of the spread of Flu - Viral neuraminidases frequently used as antigenic determinants found on the surface of the Flu virus Catalyzes the hydrolysis of terminal sialic acid residues from the newly formed virions and from the host cell receptors - activities include assistance in mobility of virus particles through the respiratory tract mucus and in the elution of virion progeny from the infected cell

Intracellular inclusion bodies

Intranuclear Intracytoplasmic

Primary Gingivostomatitis

Most common viral infection of the mouth Combination of gingivitis and stomatitis (inflammation of oral mucosa and gingiva) - often the initial presentation during the "Primary" herpes simplex infection - Greater severity than herpes labialis (cold sores) which is often the subsequent presentations

Reovirus

Nonenveloped; dsRNA (linear, 10-12 segments); double capsid; Icosahedra - affect the GI system (e.g. Rotavirus) and Respiratory tract. Virus can be readily detected in feces, and may also be recovered from pharyngeal or nasal secretions, urine, cerebrospinal fluid, and blood - despite the ease of finding Reovirus in clinical specimens, their role in human disease or treatment is still uncertain.

Reovirus Replication

Occurs in the cytoplasm of the cell, and in the final stages of assembly the virus particles bud through the ER membrane --> Cytoplasmic sites of accumulated viral protein are stained with eosin

Central Dogma Devation

Some viruses deviate from biology's "central dogma", which states that information flows from DNA → RNA → protein E.g: *Rotaviruses* and *Picornaviruses* (such as the common cold-causing rhinoviruses), use only the RNA → protein portion of the pathway, while *Retroviruses* employ an RNA → DNA → RNA → protein sequence of information delivery.

Viral Envelope

Surrounds capsid and contains glycoproteins Derived from portions of host cell membranes (phospholipids and proteins), but include some viral glycoproteins. 1.) Used to help viruses enter host cells --> Envelope Glycoproteins bind receptor sites on the host's membrane, viral envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host 2.) Relatively sensitive to desiccation, heat, and detergents - these viruses easier to sterilize than non-enveloped viruses, have limited survival outside host environments, and typically must transfer directly from host to host 3.) *Enveloped viruses possess great adaptability and can change in a short time in order to evade the immune system. Enveloped viruses can cause persistent infections*

Virus Families

Virus families established, determined by: - Virion morphology - Genome structure, - Replication strategies At least 71 families currently recognized. - Virus family titles have the suffix *-viridae* - Genera and species end in -virus e.g.: Family Picornaviridae Genus Enterovirus Species Poliovirus-1

Rabies Virus Particles

Viruses are enveloped and have single stranded RNA genome - Transcription and replication events take place in cytoplasm inside a specialized "virus factory", the *Negri body* (2-10µm diameter, istological proof of rabies infection) Helical symmetry, infectious particles cylindrical, bullet like shape - One end is rounded or conical and the other end is planar or concave - Lipoprotein envelope carries knob-like spikes of Glycoprotein G. Spikes do not cover the planar end of the virion (virus particle). - Beneath envelope is membrane/matrix protein layer which may be invaginated at planar end

Viral Pre - Replication Latent Period

Viruses have an initial Latent Period before they begin replication that consists of the Adsorption Period + Eclipse Period

Bacterial Growth vs Viral Replication

Viruses replicate MUCH faster but eventually reach a plateau (not enough resources to replicate faster)

Viral Hemagglutination

Vruses attach to RBC surface molecules --> at certain concentrations, a viral suspension may bind together (agglutinate) the RBCs, preventing them from settling out of suspension - agglutination is rarely linked to infectivity Serial dilutions of virus suspension into with addition of standard amount of RBCs enables estimation of the number of virus particles can be made - less accurate than plaque assay, but cheaper and quicker May be modified to include the addition of an antiserum. By using a standard amount of virus, a standard amount of RBCs, and serially diluting the antiserum, one can identify the concentration of the antiserum [the greatest dilution (smallest amount) which inhibits hemagglutination]

Smallpox Virus

discussed later

Baltimore Classification Scheme Class I

dsDNA (herpesviruses, poxviruses)

Baltimore Classification Scheme Class VII

dsDNA, ssRNA intermediate produced during replication (hepadnaviruses)

Baltimore Classification Scheme Class III

dsRNA (rotaviruses)

Central Dogma Devation:

employ an RNA → DNA → RNA → protein sequence of information delivery

Insertional inactivation

inactivation of a gene in a plasmid or other cloning vector by insertion of foreign DNA at an endonuclease site - commonly used in vitro with lacZ

Eclipse Phase

lasts for 10-12 hrs, corresponds to the period during which the input virus becomes uncoated. As a result, no infectious virus can detected during this time (any infectious virus detected is simply virus that is still stuck on the cell membrane).

Central Dogma Devation: Rotaviruses and Picornaviruses

only the RNA → protein portion of the pathway

Relative Sizes and Shapes of Different Viruses

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