Intro To Virology PDF

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Warning: TT: undefined function: 32 Warning: TT: undefined function: 32INTRO TO VIROLOGYVirus – means poison (in Latin)Obligate Intracellular Organisms; Complicate Parasites - Cannot survive outside the host - Only becomes active inside the host - No metabolic systems of their own; highly dependent ...

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Ephesians 4:15

University of Santo Tomas

JRMV 2020

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INTRO TO VIROLOGY Virus – means poison (in Latin) Obligate Intracellular Organisms; Complicate Parasites • Cannot survive outside the host • Only becomes active inside the host • No metabolic systems of their own; highly dependent on its host cell • Very specific and has a limited number of hosts o Has target receptors o HIV → T Lymphocytes (T helper) ▪ With CD4 o The virus will not target a cell unless it has its specific receptor o Trophism – the virus will target a specific type of cell/organ • Filterable organisms o 1/10 the size of a bacterial cell; Very small o Even if you filter the specimen, the organism will pass through o Membrane Filter – 0.22 um filter used in Bacteriology lab o Hepa Filter 0.3 um • Taxonomy: o -viridae → family o Family, first letter is capitalized The Viral Particle • Virion – complete viral particle o If Genome + Capsid = Naked Virus o If all 3 components = Enveloped Virus • Composed of o Genome (DNA or RNA, not both) ▪ Classification based on genome: • DNA Virus • RNA Virus ▪ Each virus only has one copy of the genome (Haploid) except for Retroviruses (diploid) • Retrovirus → HIV ▪ Genetic material can be: • Single stranded of double stranded (ss or ds) • Linear, segmented (fragmented, separated into pieces), or circular • Segmented Genome o RNA Virus (easily mutated to form a new virus) o Capsid ▪ Protein coat ▪ Composed of repeating subunits of polypeptides (capsomeres)

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Function: • Protection • Shape; Structural • Determines specific viral antigenicity • Attachment (if the virus is naked) Structural Patterns • Helical • Icosahedral • Complex

Envelope ▪ Lipoprotein • Lipid rich ▪ Host-cell derived • From the cell membranes OR nuclear membranes ▪ Surrounds the capsid of certain viruses ▪ It is acquired during viral maturation by a budding process through the host’s membranes ▪ Other Structures • Glycoprotein Spikes → Peplomers (for attachment) • Matrix Protein → found in between the envelope and the capsid o Contain enzymes needed for replication • Tegument → found in between the envelope and the capsid o Contain regulatory proteins that are needed or required for replication, transcription, translation. o Example: HSV (herpes simplex virus) and CMV (cytomegalovirus) Defective Virus o Deficiency/Problem in replication of the virus o Cannot replicate on its own; (Hepatitis D needs Hepatitis B) o

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Classification of Viruses • Based on presence of Lipoprotein Envelope o Naked Virus ▪ More resistant to disinfectants, extreme temperatures, to pH, chemicals ▪ More stable ▪ Ether stable / Ether resistant • If you add ether, the virus does not dissolve (not destroyed by organic solvents) ▪ Mode of Transmission • Oral/Fecal

Ephesians 4:15

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University of Santo Tomas

JRMV 2020

Since the viruses can generally survive the acidic pH of the stomach

Enveloped Virus ▪ Less resistant; easily inactivated by disinfectants ▪ Ether labile viruses / Ether susceptible • Soluble in ether. If you add organic solvent the virus will be destroyed • The lipid envelope will be dissolved ▪ Mode of Transmission • Sexual Contact (HIV) • Blood borne • Direct Contact • Droplet Transmission ▪ Advantage: • Ability to hide from the immune system • Since the envelope is derived from the host Based on Genome o DNA Virus ▪ More stable, less prone to errors ▪ Cause latent, long-standing infections ▪ Usually double stranded, and linear or circular • Except: Parvoviridae, Circoviridae (ss) ▪ HAPPY?? ▪ The virus replicates by hijacking the cell ▪ In DNA Viruses, the virus hijacks the nucleus • Virus inserts DNA genome into the nucleus. • (+) Strand used for DNA replication o Should undergo transcription to convert DNA to mRNA. ▪ Requires polymerases o mRNA is needed for translation to form a protein (now viral in nature) ▪ Viral proteins and enzymes • (-) Strand is ignored • All DNA viruses replicate in the nucleus except Poxviridae (replicates in the cytoplasm) o RNA Virus ▪ Easily mutated as they are prone to error ▪ Usually single stranded and segmented • Except Reoviridae, and Picobirnaviridae (ds) ▪ Sense or (+) strand RNA virus (+ssRNA) → 5’ – 3’ • Viruses that function already as mRNA • Already infectious ▪ Anti-sense or minus strand virus (-ssRNA) → 3’ – 5’ o

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Polarity opposite of mRNA; needs to be transcribed first • Not the usual orientation • Needs to be transcribed using RNA dependent RNA Polymerase o Usually comes with the virus ▪ Retroviruses • Retroviridae • Replicate in the nucleus (also Orthomyxoviridae) • Contain reverse transcriptase o To produce DNA intermediate o Transcribed to form mRNA and is then translated to form viral proteins and enzymes. Based on Structural Pattern o Helical Viruses ▪ Cylindrical ▪ Shaped like hollow protein cylinders which may be either rigid or flexible • Most Helical are flexible except Rhabdoviridae ▪ Nucleic acid and protein are closely associated • Genome intertwined with protein • Nucleocapsid o Not the same as virion ▪ Most helical viruses are enveloped ▪ All helical viruses are RNA viruses • Tobacco Mosaic (virus of tobacco plants) • Ebola Virus o Icosahedral Virus ▪ Heat stable ▪ Polygonal, geometric shapes • 20 triangular sides (equilateral) • 12 vertices (point where the triangular sides meet) • 60 identical subunits ▪ Arranged in a cubical manner (appear spherical when viewed at low power in the EM) ▪ Naked icosahedral viruses → equipped with protein spikes (on the vertices) • Mediates attachment to the target host cells ▪ Can be crystallized and viewed using radiocrystallography ▪ Adenovirus ▪ More commonly DNA Viruses o Complex Viruses ▪ Capsid symmetry but not purely icosahedral or helical •

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Usually ether resistant (have a complex protein coat) Poxviridae • Contain layers (multilayered) • Dumbbell shaped core • Overall appearance is brick shaped • Largest virus Bacteriophages • Viruses of bacteria • Contain very unusual structures • Tail fiber and Pin aids in injecting the genome • The virus itself does not enter the host

Reaction of Viruses to Physical and Chemical Agents • Heat and Cold o Heat labile – enveloped viruses o Heat stable – icosahedral viruses o Generally, infectivity is destroyed at 50-60C for 30 minutes (except HBV, polyomavirus) o Hepatitis B → most common blood borne pathogen • Stable with 1mol/L of salts • pH: generally stable (5-9) • Ether susceptibility and detergents: Enveloped Viruses • Formalin inactivates viruses (especially single stranded viruses) • Penetrable by vital dyes • Radiation: inactivates viruses

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Viral Replication • Viral Growth Curve o Viral Adsorption and Entry ▪ Looks for the target cell with the target receptor. ▪ Attachment of the virus to the host cell receptor. • Specific binding with capsid proteins and carbohydrate receptor of the host cell • Tropism ▪ On the surface of the cells, (Glycocalyx) has the receptors. • Surface glycoproteins that correspond to the virus promoting attachment or adhesion. • If we do not have the receptor, we will not get infected ▪ Recognition of a suitable host cell o

Penetration (Viral Entry) ▪ Varied mechanisms of entry through: • Fusion with the cell membrane (Syncytia formation) o Utilized by enveloped viruses • Endocytosis o Virus enters via a cytoplasmic vacuole • Translocation o Usually the mechanism of naked viruses • Injection of Viral Material (genome) o Bacteriophages o Use of several tail fibers and lysozyme Uncoating ▪ Disassembly (once the virus is in the host cell) ▪ Gets rid of the envelope and capsid (loss of protein coat) ▪ Exposes the genome (nucleic acid) • Free genome (picornaviruses) • Nucleocapsid (reoviruses) o Double walled capsid Macromolecular Synthesis ▪ Replication stage of viral material (either in the nucleus or cytoplasm) • Expression of genetic material and protein polymers ▪ Coincides with the eclipse phase • Cannot detect the viral particles since they are disassembled • Sudden fall in viral particle ▪ Coincides with Latent period • No symptoms seen ▪ Formation of inclusion bodies • Tells us that there is a viral infection • Indicator of viral replication • Can be found in the nucleus (DNA virus) or in the cytoplasm (RNA virus) • These inclusions are diagnostic (clue) for the viral infection. Viral Release ▪ Lysis of the infected cell • Viral particles released are ready to infect other cells

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Exocytosis of viral particles • Released via vacuoles or vesicles (cytoplasmic vacuole) • Transports the produced viruses • Can either be used by enveloped or naked virus Budding off • Forms a bud (forms an envelope) Cell to cell transport • Transport via syncytia • Fusion of infected cells with other cells • Change in morphology of the cells (become multinucleated)

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Viral Growth Cycle

Pathogenesis and Life Cycle • Incubation period o Patients usually asymptomatic o Viral replication is taking place o Symptomatic phase starts when the viruses are released (viral release) o Best time to collect samples for viral specimens are during the acute phase • Spread o Infects nearby cells, migratory cells, disseminates via the blood (viremia), neuronal, via lymph ▪ Viruses usually replicate at the primary site of entry and may not spread to distant sites ▪ Primary Viremia → Initial spread of viruses to the blood ▪ Secondary Viremia → second spread of viruses ▪ Neuronal Spread → Rabies (target cell are CNS cells) • Initial target are peripheral nervous system. • Prodromal o Early nonspecific symptoms (fever, aches, pain, and nausea) • Active Disease o Once the virus is in the actual target cell o Has immediate or long-lived infection (chronic) ▪ Latent infections ▪ Sometimes, these viruses are triggered by triggering factors (stress, fatigue, and other reasons) – Reactivation o Specific symptoms o When the virus is in the lytic phase (destroys cells after viral replication) o Lytic Viruses – destroy host cell after replication ▪ Manifestation of acute infection o Lysogenic – members of Herpesviridae ▪ Integrates the virus into the genome (by forming a provirus) ▪ As the host cell replicates, the viral genome also replicates ▪ Usually DNA viruses

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▪ Subclinical infection o Pseudolysogenic or Episomal ▪ Viral genome is not incorporated into the host cell genome. ▪ Viral persistence without integration Autoimmune pathogenesis – immune reaction allowing cross-reaction with host tissue antigens Promotion of transformation or immortalization of host cells Oncogenesis – transformative viruses o Stimulate uncontrolled growth of host cells o Polyoma viruses Resolution of Disease o Innate IR and Adaptive IR o Host Recovery o Viral Shedding ▪ Usually where the virus enters ▪ Shedding of the virion into the environment ▪ As long as there are symptoms, that’s when we are shedding • Some viruses, when there are no symptoms that’s when we become infectious ▪ Rabies → no shedding Frequency of Virus Colonization/Infection o Seasons: Winter vs Spring o Age Group: Infants vs Adults ▪ Rotavirus (diarrhea among infants)

Effects of Viral Infection on the Host Cell • Diagnostic • Cytopathic Effects o Structural changes leading to necrosis o Seen in virally infected cell cultures (diagnostic purpose) o Can also be seen in tissue sections • Plaque Formations o Estimates the number of viruses by the number of plaque-forming unit o More plaques = More viruses o Plaque assay • Formation of multinuclear giant cells/ Cell fusion o Syncytia formation o Seen in Respiratory Syncytial Virus • Cytoplasmic Vacuoles • Inclusion bodies o Nuclear Inclusions → DNA Virus o Cytoplasmic Inclusions → RNA Virus o Negri Bodies in rabies (appear eosinophilic found in the neurons → usually pyramidal)

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▪ Cytoplasmic inclusion ▪ Hallmark Sign ▪ Pathognomonic Sign (Diagnostic) Cell Degeneration and necrosis Apoptosis o Defense mechanism of host cell (no inflammation will occur if there is apoptosis) o Programmed cell death Noncytocidal infection o No extensive restructuring of host cell o Virions are released by budding o Usually associated with enveloped virus Appearance of new antigenic determinants on the cell surface

Modes of Transmission • Direct Transmission o Droplet/Aerosols (Influenza viruses, coronaviruses) o Sexual Contact o Hand-mouth o Hand-eye o Mouth-mouth o Exchange of contaminated blood • Indirect Transmission o Fecal-oral o Fomites • Animal to Animal o Humans are accidental host (zoonosis) o Bite of infected animal o Droplet of Aerosol infection of rodent urine or feces • Via arthropod vectors Classification and Taxonomy of Viruses • Morphology • Physiochemical Properties • Genome • Macromolecules • Antigenic Properties • Biological Properties Taxonomy (International Committee on Taxonomy of Viruses) • Family names end in -viridae (Capitalized) • Subfamily names end in -virinae (Capitalized) • Genus end in -virus (capitalized)

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Viral species: group of viruses sharing the same genetic information and host. Common names of virus are used Subspecies are designated by a number

Baltimore Classification • Group I: dsDNA Viruses • Group II: ssDNA Viruses • Group III: dsRNA (Ambisense) • Group IV: (+) sense ssRNA • Group V: (-) sense ssRNA • Group VI: Reverse transcribing (+) sense ssRNA • Group VII: Reverse transcribing DNA

Ephesians 4:15

Baltimore Classification Genome Group I dsDNA Viruses

Enveloped Viruses Herpesviridae Poxviridae

University of Santo Tomas

JRMV 2020

Naked Viruses Adenoviridae Papillomaviridae Polyomaviridae

Notes Genomes are stable and able to persist in the host cell - not usually associated with mutations - latency is probable as the virus can persist in the host cell. (well known in Herpesviridae) Easily mutates and can be packed in small particles - able to mutate (not as stable) Mutagenic; must create its own polymerases (RNA dependent RNA polymerase) - Prone to error (mutations) Equivalent to mRNA; genomes are directly infectious; highly mutagenic - no need for the genome to be read before it can manifest symptoms

Group II

ssDNA Viruses

Parvoviridae

Group III

dsRNA (Ambisense)

Reoviridae

Group IV

(+) sense ssRNA

Togaviridae Flaviviridae Coronaviridae

Group V

(-) sense ssRNA

Common viral agents of disease; template for mRNA - RNA dependent RNA polymerase to undergo transcription by using the genome as the template

Group VI

RT (+) sense ssRNA

Orthomyxoviridae Arenaviridae Paramyxoviridae Bunyaviridae Rhabdoviridae Filoviridae Hantaviridae Nairoviridae Retroviridae

Group VII

RT DNA

Hepadnaviridae

Produce RNA intermediate that integrates itself to the host genome - DNA virus that replicates in the cytoplasm. -

Picornaviridae Calciviridae Hepeviridae Astroviridae

Produce DNA intermediates (that can integrate into the host genome) - needs the enzyme Reverse Transcriptase - Replicates in the nucleus, for them to replicate in the nucleus, RNA must be converted first to the DNA intermediate

Ephesians 4:15

University of Santo Tomas

Parvovirus B19

Prevention, Treatment, and Control • Immunization (Vaccination) • Antiviral agents

JRMV 2020

Enlarged Ground Glass Nuclei with chromatin precipitation in erythroid precursors

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Antiviral Susceptibility Testing • Phenotypic Susceptibility o Plaque Reduction Assay o Dye uptake Assay o DNA hybridization o EIA o Flow cytometry o NA Inhibition Assay • Genotypic Susceptibility Assays (Faster) o Pyrosequencing o Next Generation Sequencing

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Laboratory Diagnosis • Direct Detection (Detects the viral particle/ viral antigens) o Microscopy ▪ Use of light microscopy (CPE) • Detection of visual changes in virally infected cells (Like inclusion bodies and syncytia formation) • Smears are stained with Giemsa, Pap’s or H and E • Presence of CPE are pathognomonic for the presence of a viral infection. Virus Rabies Yellow Fever Molluscum Contagiosum HSV PoxVirus (Vaccinia, Variola) Adenovirus Measles CMV HPV HTLV-1 RSV BK Virus JC Virus

Comment (Inclusion bodies seen in tissue specimens) Negri Bodies Councilman Body (seen in bhepatocytes – hepatitis) Lipschultz Body/Granules Present, Henderson-Paterson Bodies (Intracytoplasmic – associated with Poxviruses) Cowdry Type A Guarnieri body (Smallpox)

Stain Seller’s H and E Giemsa

Smudge Cells (cells have large round nuclei) Multinucleated Giant Cell; Dawson Bodies (SSPE, Warthin-Finkeldey cells) Owl’s Eye Koiloetween cytes (squamous cells with wrinkled prune nucleus and perinuclear halo – basophilic cytoplasm) Flower Nuclei in T cells Syncytia Formation Decoy Cells (seen in urine speimens – Glassy nucleus) Bizarre Astrocytes, Oligodendrocyte Nuclei Enlarged Foci of Demyelination in White Matter of the Brain

H and E

H and E

H and E Pap’s Wright H and E Pap’s H and E Luxol Fast Blue

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Use of EM • Detection of Virions • Useful for nonculturable viruses and novel viruses (Norwalk, Astrovirus, Calicivirus, Coronavirus) • Disadvantages – laborious and expensive • Uses negative staining using Phosphotungstic acid, Uranyl acetate, Lead, and osmium tetroxide. • Viruses seen as light structures against a dark background • Preferably done if the viral titer is at least 106 to 107 particles/mL Detection of Viral Antigens (immunodiagnosis) • Immunohistochemistry o Direct IHC – 1 antibody (usually labeled IgG) o Indirect IHC – 2 antibodies (2nd AB is labeled – usually Anti-IgG) • Direct FAT – use of virus specific fluorescein labeled monoclonal or polyclonal antibody • Indirect immunofluorescence (Antigen Capture Techniques) • Latex Agglutination • EIA, RIA • CHemiliuminescence • Immunoperoxidase – staining tissue sections for viruses • Immunochromatography – lateral flow immunoassays Other Methods: ELVIS – Enzyme Linked Virus Inducible System (for HSV) Manner of Reporting for Fluorescence techniques:

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Nucleic Acid Base Detection o Molecular Methods of Detection of Viruses ▪ Quicker release of results ▪ More sensitive ▪ Quantitative ▪ Detects unculturable viruses ▪ Can detect multiple viruses ▪ Costly and needs more specialized training ▪ COVID-19 Gold Standard is still PCR o Specimens should be processed first prior to amplification ▪ Lysis of cells (mechanical disruption, addition of detergents, addition of proteinase K) ▪ Nucleic Acid Extraction • Manual – use of ethanol that precipitates the DNA, phenol and chloroform that removes other debris • Automated – use of magnetic beads to bind nucleic acids • Also needed to remove inhibitors o PCR ▪ Principle: Gene Amplification ▪ Reagents needed in PCR: • Template or Target • dNTP – nucleotides with deoxyribose • Primers • DNA polymerase (Taq polymerase) • Magnesium – cofactor for DNA polymerase • Buffer (at neutral pH) ▪ Steps in PCR: • Denaturation • Annealing • Extension ▪ RT-PCR ▪ Nested PCR ▪ Multiplex PCR o Genome Sequencing Isolation of Viruses Using Cell Cultures (Gold standard in diagnosing viruses) o Use of Cell or organ cultures ▪ Organ Cultures – Rarely used (mostly for research purposes only) ▪ Embryonated Egg...