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Viruses (FACTS)

- Most abundant microbes on earth
- Infectious particles either active or inactive (acellular entities)
- Viral replication inside a cell usually causes death or loss of function of that cell
- OIP: cannot multiply unless it invades a specific host cell and instructs its genetic and metabolic machinery to make and release quantities of new viruses (Obligate intracellular parasite)

Tulip Mosaic Virus

- "harmless virus" (can be used to treat genetic diseases)
- alters the development of the plant cells and causes varying patterns of colors in the petals

Gene therapy

normal gene is inserted into a virus vector and the patient is infected w/ this altered virus in order to correct defect

ONYX

- mutant adenoviruses
- induce apoptosis in cancer cells

Bacteriophages (About)

- FUTURE: treating bacterial infections; bacterial viruses can selectively attack and destroy their host bacteria w/o damaging human cells; prevent food infections (E. coli)
- have a polyhedral nucleocapsid along with a helical tail and attachment fibers
- often bacteria tend to infect humans as more pathogenic
- full completion of viral infection through lysis and release of virions.
- this type of virus can (occasionally) enter a reversible state of lysogeny and is incorporated into the host's genetic material

Sizes & opterational & viewing Viruses

- Size range: most <0.2 μm; requires electron microscope
- Virion: fully formed virus able to establish an infection (extracellular virus particle that is virulent)
- Shadowcasting: attaches a virus preparation to a surface and showers it w/ a dense metallic vapor directed from a certain angle
- Density gradient centrifugation (sucrose medium) separation

Properties of Virus

- Contain what they need to invade and control a host cell (external coating and a core containing one or more nucleic acid strands of either DNA or RNA
- look at slide

Common Viral Organization of Envelope

- All virus has capsid (shell that surrounds the nucleic acid in the central core)
- Capsid & nucleic acid = "nucleocapsid"
- Enveloped virus: composed of a nucleocapsid surrounded by a flexible membrane called "envelope" (usually has special receptor spikes inserted into it)
- Only containing a nucleocapsid = "naked virus"

Capsids

- All viruses have
- protein coats that enclose and protect their nucleic acid.
- Each is constructed from identical subunits called capsomers made of protein.
- Together with the nucleic acid are nucleoscapsid.
- Some viruses have an external covering called envelope virus; those lacking an envelope are naked virus
- Can spontaneously self-assemble into the finished capsid
- Depends on shape of capsomers: helical & icosahedral

Two external structural types of Envelope

Helical & Icosahedral

Helical

- continuous helix of capsomers forming a cylindrical nucleocapsid
- rod-shaped capsomers that form to together to create hollow discs resembling a bracelet
- Nuclei acid strand is coiled
- Steps: 1) Capsomers assemble into hollow discs
2) The nucleic acid is inserted into the center of the disc
3) Elongation of the nucleocapsid progresses from both ends, as the nucleic acid is wound "w/n" the lengthening helix
- Naked virus: tobacco mosaic virus: attacks tobacco leaves (loose)

Icosahedral

- 3-dimensional
- Single or multiple capsomers
- Envelop may or may not be absent
- Ex. herpes simplex (cold sores): enveloped
- Ex. Poliovirus crystals (crystalline is common nature)
- Geometric capsids of the
same virus (SE 150,000x)
- 20-sided with 12 corners
- Structure: a) Faced or "face" of capsid is composed of 21 identical capsomers arranged in a triangular shape. A vertex or "point" consists of five capsomers arranged w/ a single penton in the center. Subject to variation b) Assembled virus showing how the facets and vertices come together to form a shell around the nucleic acid c) 3-dimension showing fibers attached to the pentons (penton = base) d) Negative stain of this virus highlights its texture and fibers that have fallen off
- Vary in the number of capsomers
- Each capsomer may be made of 1 or several proteins
- Some are enveloped

Viral Envelope

- mostly animal viruses
- acquired when the virus leaves the host cell
- exposed proteins on the outside of the envelope, called spikes, essential for attachment of the virus to the host cell
- Comes from taking membrane from host cell
- spikes: peplomers that are essential for the attachment of viruses to the next host cell (glycoproteins exposed outside envelope)
- Shape can be pleomorphic and can be spherical to filamentous
- Can Exit by: bud off; nuclear envelope; ER

Functions of Capsid/Envelope

- Protects the nucleic acid when the virion is outside the host cell
- Helps to bind the virion to a cell surface and assists the penetration of the viral DNA or RNA into a suitable host cell
- Indispensable
- First bind to cell surface -> assisting in penetration of viral nucleic acid
- Can stimulate the immune system to produce antibodies that can neutralize viruses and protect the host's cells against future environments

Complex viruses

- atypical viruses
- Pox viruses & some bacteriophages
- More intricate structure than helical, icosahedral, naked or enveloped

Orthopoxvirus

- lack a typical capsid and are covered in outer layer by a dense layer of lipoproteins & coarse fibrils
- complex
- Large DNA virus
- pocks
- cells round up
- inclusions appear in cytoplasm
- bioterrorism
- smallpox & cowpox
- Pox virus

Paramyxovirus

- Enveloped virus
- Helical nucleocapsid
- RNA
- cold sores and genital herpes
- nuclear inclusions form
- cells fuse to form multinucleated giant cells
- Mumps

Herpes simplex virus: "simplexvirus"

- DNA
- Enveloped viruses
- Icosahedral nucleocapsid
- Type 1: fever blister, cold sores
- Type 2: genital herpes

Lentivirus

- RNA
- Enveloped viruses
- Icosahedral nucleocapsid
- Giant cells w/ numerous nuclei (multinucleate)
- HIV-AIDS

Mastadenovirus

- DNA
- Helical capsid
- Naked virus
- clumping of cells
- nuclear inclusions
- ONYX
- cold syndrome
- running nose
- coughing
- Adenovirus

Papillomavirus

- DNA
- Naked virus
- Icosahedral capsid
- cervical cancer
- Gardasil, 9-26 yr. old, 3/6m
- "HPV"
- Several types of warts: Genital warts

Bacteriophage (Structure)

- DNA
- Complex virus
- Flexible-tailed (Helical)

Nucleic acids

- Viral genome
- Carries genes necessary to invade host cell and redirect cell's activity to make new viruses
- Number of genes varies for each type of virus - few to hundreds
- "Genetic Parasites"
- DNA & RNA

Viral genome

- either DNA or RNA but never both
- sum total of the genetic information
- Small number genes due to tiny space

DNA (viruses)

- Nucleic acids
- usually double stranded (ds) but may be single stranded (ss)
- circular or linear

RNA (viruses)

- usually single stranded, may be double stranded, may be segmented into separate pieces with their own individual genes
- ssRNA genomes ready for immediate translation are positive-sense RNA.
- ssRNA genomes that must be converted into proper form are negative-sense RNA.
- Ex. Influenza virus
- Nucleic Acid

Retro virus: "Retroviridae"

- few virus types that converts its nucleic acid from RNA to DNA inside its host cell
- "Oncornarvirus": Human T-cell leukemia (HTLV)
- "Lentivirus": HIV (Type 1 & 2) cause AIDS

Viral Enzymes

- pre-formed can be present
- polymerases: synthesizes DNA or RNA (synthesizes a large molecule from smaller subunits; form of replication)
- replicases: copy DNA (or RNA)
- reverse transcriptase: synthesis of DNA from RNA (AIDS virus)
- General Structure

Viral Classification

- Main criteria presently used are structure, chemical composition, and genetic makeup.
- No taxa above Family (no kingdom, phylum, etc.)
- Currently recognized: 3 orders, 63 families, and 263 genera of viruses
- Family name ends in -viridae, i.e.Herpesviridae
Genus name ends in -virus, Simplexvirus,Herpes simplex virus I (HSV-I)
- animal, plant, or vacterial viruses
- Virus is placed in a species on the basis of a collection of properties such as host range, pathogenicity, or antigenicity (Consisting of members that have a number of properties in common but have some variations)
- Vernacular language used
- Family: capsid, nucleic acid strand number, presence and type of capsid, overal virus size, area of the host that virus mustiplies (Size & Shape)

Modes of Viral Multiplication of animal virus

1) absorption 2) penetration 3) uncoating 4) Synthesis 5) Assembly 6) Release
- nature profoundly affects pathogeicity, transmission, the responses of the immune defenses, and human measures to control viral infections

Absorption

- binding of virus to specific molecule on host cell
- Virus coincidentally collides with a susceptible host cell
- adsorbs specifically to receptor sites on the cell membrane (attachment)

Penetration

- genome enters host cell
- Flexible cell membrane is penetrated by the whole virus or its nucleic acid by: endocytosis or fusion

Uncoating

the viral nucleic acid is released from the capsid

Synthesis

viral components are produced

Assembly

new viral particles are constructed

Release

- assembled viruses are released by budding (exocytosis) or cell lysis
- Number of viruses released is variable: a) 3,000-4,000 released by poxvirus; b) >100,000 released by poliovirus

Host Range (Absorption Traits)

- Spectrum of cells a virus can infect (limitation)
- Receptor sites are usually glycoproteins for cell's normal functioning
- Virus have tissue specificities that can target cells called "tropisms" (having a specal affinity for an object or substance)
- Prominent spikes
or Naked nucleocapsids (molecules on capsid that adhere to cell membrane receptors)

Hepadnavirus

- human liver cells
- has host range (not large)
- Hepatitis B (HBV)
- Serum hepatitis

Enterovirus

- primate intestinal and nerve cells
- intermediate; affect only certain range
- cell lysis
- no inclusions
- go into vertebrae & paralyze
- 20% victims become crippled
- Poliomyelitis (Poliovirus)

Lyssavirus

- various cells of many mammals
- mammals nerve cells; affect many
- inclusions in cytoplasm in brain (Negri bodies)
- no change in cell shape
- Rabies Virus (Hydrophobia)
- Enveloped virus
- Helical nucleocapsid
- RNA

Endocytosis

- entire virus is engulfed and enclosed in a vacuole or vesicle
- when enzymes in the vacuole dissolve the envelope and capsid, the virus is said to be uncoated, a process that releases the viral nucleic acid into the cytoplasm

Fusion

- most cases / common
- Ex. influenza & mumps virus
- envelope merges directly with membrane resulting in nucleocapsid's entry into cytoplasm

Replication & Protein Production

- Varies depending on whether the virus is a DNA or RNA virus
- DNA viruses generally are replicated and assembled in the nucleus.
- RNA viruses generally are replicated and assembled in the cytoplasm.
^ Positive-sense RNA contain the message for translation.
^ Negative-sense RNA must be converted into positive-sense message.
- Synthesis
- Viral nucleic acid alters the genetic expression of the host and instructs it to synthesize the building blocks for new viruses
- Can produce viral components: new RNA, proteins for the capsid, spikes and viral enzymes

Budding

- exocytosis; nucleocapsid binds to membrane which pinches off and sheds the viruses gradually; cell is not immediately destroyed
- to membrane by the viral spikes; enveloped viruses
- simultaneously picks up envelopes & spikes
- comes off surface of cell

Lysis

- nonenveloped and complex viruses released when cell dies and ruptures

Cytopathic (disease) effects: virus-induced damage to cells

- Changes in size & shape
-Cytoplasmic inclusion bodies
- Nuclear inclusion bodies (compacted w/ massive amounts of viruses)
- Cells fuse to form multinucleated cells.
-Cell lysis
- Alter DNA
- Transform cells into cancerous cells
- Connection between viruses and chronic afflictions
- Most number of cases
- Most common are acute
Human cell product called "interferon" (vaccine type tool; limited)
- ** Syncytia: (common) fusion of multiple host cells into single large cells containing multiple nuclei
- Develop intracellular changes

Persistent Infections

- cell harbors the virus and is not immediately lysed
- Can last weeks or host's lifetime; several can periodically reactivate - chronic latent state
- Ex: Measles virus; Herpes simplex virus; Herpes zoster virus
- Inactive over long periods of time
- Both herpes viruses go into latency in nerve cells and later emerge under the influence of various stimuli to cause recurrent symptoms

Morbillivirus

- may remain hidden in brain cells for many years
- Measles virus
- red

Varicellovirus

- chickenpox and shingles
- Varicella zoster virus (VZV)

Transformations: Cancer

- Some animal viruses enter host cell and permanently alter its genetic material resulting in cancer
- Cells have increased rate of growth, alterations in chromosomes, and capacity to divide for indefinite time periods resulting in tumors.
- Mammalian viruses capable of initiating tumors are called oncoviruses. (Papillomavirus & Epstein-Barr virus)
- Enter host cell and their nucleic acid becomes integrated into the host DNA
-Generally have increased rate of growth, alterations in chromosomes, changes in the cell's surface molecules, and the capacity to divide for an indefinite period
- Oncogenic: animal viruses involved in cancer
- Animal virus can be acute infections or persist in host tissues as latent infections that can reactivate periodically throughout the host's life

Epstein-Barr virus

- Burkitt's lymphoma (Herpes virus)
- cause cancer

Multiplication Cycle in Bacteriophages

- Bacteriophages - bacterial viruses (phages)
- Most widely studied are those that infect Escherichia coli (complex structure, DNA; intestinal bacterium; T-even phages [T2 & T4])
- Multiplication goes through similar stages as animal viruses.
- Only the nucleic acid enters the cytoplasm - uncoating is not necessary.
- Release is a result of cell lysis induced by viral enzymes and accumulation of viruses - lytic cycle.

6 Steps in Phage Replication

Outside cell in Temperate phages

1. Adsorption - binding of virus to specific molecule on host cell
2. Penetration -genome enters host cell
3. Replication - viral components produced; Viral replication and synthesis of viral protein
4. Assembly - viral components assembled; New phage parts spontaneously assemble into bacteriophages
5. Maturation - completion of viral formation
6. Release - viruses leave cell to infect other cells; lysis

Lysogeny: Silent Virus Infection

- Not all phages complete the lytic cycle.
- Some DNA phages, called temperate phages, undergo adsorption and penetration but don't replicate.
- The viral genome inserts into bacterial genome and becomes an inactive prophage (the cell is not lysed)
- Prophage is retained and copied during normal cell division resulting in the transfer of temperate phage genome to all host cell progeny
- Induction can occur resulting in activation of prophage followed by viral replication and cell lysis.
- is a less deadly form of infection then the full lytic cycle and is thought to be an advancement that allows the virus to spread w/o killing the host
- Phages sometimes serve as transporters of bacterial genes called "Transduction" (genes for toxic and resistant to antibiotics can spread)
- results in the spread of the virus without killing the host cell
- Ex. Cornebacterium diphtheriae, Vibrio chlorea; Clostridium botulinum

lysogenic conversion

- Phage genes in the bacterial chromosome can cause the production of toxins or enzymes that cause pathology
- bacterium receives genes from temperate host
- Process of lysogeny

Corynebacterium diphtheriae

- "bacteriophage byproduct to produce severe disease"
- lysogeny

Vibrio cholerae

- infection of the small intestine that causes a large amount of watery diarrhea.
- Symptoms: Abdominal cramps; Dry mucus membranes or mouth; Dry skin; Excessive thirst; Glassy or sunken eyes; Lack of tears
- lysogeny
- - "chlorea"

Clostridium botulinum

- serious illness that causes flaccid paralysis of muscles.
- lysogeny
- "botulism" (botulin toxin)

Techniques in Cultivating and Identifying Animal Viruses

- Obligate intracellular parasites: that require appropriate cells to replicate
- Methods used:
a) cell (tissue) cultures
b) bird embryos
c) live animal inoculation
- Procedure used:
1) To isolate and identify viruses in clinical specimens
2) To prepare viruses for vaccines
3) Detailed research on viral structure, multiplication cycles, genetics and effect on host cells

Cell (tissue) cultures

- cultured cells grow in sheets that support viral replication and permit observation for cytopathic effect
- are cultures of host cells grown in special sterile chambers containing correct types and proportions of growth factors using aseptic techniques to exclude unwanted organisms
- in vitro cultivation systems (commonly used)

Bird Embryos

- incubating egg is an ideal system; virus is injected through the shell
- Perfect for viral propagation
- Common: chicken, duck, turkey
- Pocks: shows viral development (discrete, opaque spots) [Smallpox]
- Detected also by: agglutinate red blood cells; reaction w/ antibody that cause them to affix to corresponding virus
- Used for vaccines
- Inserting vaccine for highest levels of sterile and aseptic precautions
- Targets: allantoic cavity (embryonic waste removal); amniotic cavity (sac protects embryo); chorioallantoic membrane (gas-exchange membrane); yolk sac (nourishment of embryo)

live animal inoculation

- occasionally used when necessary
- Paws of armadillo & Monkey Kidney Cell Culture
- alternate method for viruses that do not readily grow in cultures (normal) or embryos
- Laboratory animals

In Vitro cultivation systems

- Form plaques (symptoms of CPE)
- Degeneration and lysis of infected cells in the monolayer of cells.
- Virus-infected cells have been destroyed show up as clear, well-defined patches in cell sheet
- Virus attacks cell by cell creating round, clear spaces from the lysed cells
- experiments test tubes or artificial environments

Plaques

- consist of open spaces where cells have been disrupted by viral infection
- "patch or spot"
- Symptom of CPE

In Vivo cultivation system

- experiments in living body

Types of Cytopathic Changes in Cells

- Multinucleate giant cells (Herpes simplix virus)
- Inclusion bodies in cytoplasm or nucleus (Cytomegalovirus [CMV])

Steps to Treatment

Diagnosis -> identification -> direct examination -> samples -> standard procedures -> definite diagnosis -> screening tests

Influenza virus

- cells round up
- no inclusions
- pain & tension
- "flu"
- Type A: Hong Kong, Asian, Swine

Viral Diagnosis

- Animal agents most difficult
- Consider overall picture such as signs & symptoms
- Cytopathic effects
- Cell & animal culture (no agar plate)
- SEM: microscope, discover virus (see spikes; very virus has specific shape); Scanning electron microscope; parts virus
- Virus antigen
- Serological screening: screen for immune response by testing for antibodies
- Antibodies Western Blot: HIV; test specific proteins
- Polymerase Chain Reaction (PCR): sample tissues blend it down get nucleus out and see nucleotides; 90 degrees separate, 70 degrees for enzyme; thermocycline (heat -> cool -> separate)
- Focus on practical aspect:
Fungus or parasite (worms [name, disease])
- Antiviral drugs can cause serious side effects

Reovirus

- cell enlargement
- vacuoles
- inclusions in cytoplasm

Types of Noncellular infections agents

- Satellite viruses: adeno-associated virus; delta agent
- Viroids

Satellite viruses

- dependent on other complete viruses for replication
- Adeno-associated virus & delta agent
- require larger viruses to cause diseases
- noncellular infectious agents

adeno-associated virus

- replicate only in cells infected with adenovirus
- up virus that cause co-symptoms (minor groups)
- noncellular infectious agents

delta agent

- naked strand of RNA expressed only in the presence of hepatitis B virus
- not in plants, but in animals, hijack (get in liver)
- noncellular infectious agents

Viroids

- noncellular infectious agents
- short pieces of RNA, no protein coat; only been identified in plants (so far)
- extremely small lengths of protein-coated nucleic acid

Prion proteins

- PrPc & PrPsc
- resistant to Proteinase K (enzyme that digest protein)
- Protein fibers that cause spongiform encephalopathies
- abnormal version of protein that can induce spontaneous abnormal folding in normal protein
- Extreme resistance to to disinfectants, radiation and sterilization techniques, high temperature, concentrated chemicals

PrPsc protein

- "Mad Cow"
- Beta protein (bad)
- nails can't
- Lose memory
- Confusion
- rapid deterioration & brain shrinkage
- Turns alpha into beta
- Comes spongy (suck up water)
- transmittable
- mutant
- Proteinase K resistant

PrPc

- normal protein
- alpha helix: alpha protein
- turn into protein when buried
- Proteinase K non-resistant

Creutzfeldt-Jakob Syndrome (CJS)

- chronic disease
- fatal familial insomnia, kuru, brain progressively deteriorates and loss of motor, sensory and cognitive coordination
- no treatment
- spongiform pathology
- Brian shrinkage
- Protein PrPssc
- Not same as mad cow

Bovine Spongiform Encephalopathy

- Develops a spongelike appearance due to loss of nerve and glial cells
- Buildup of tiny protein fibrils in the brain tissue
- Mad Cow Disease
- Protein PrPsc
- Symptoms: staggering gait, weakness, weight loss

Transmissible Spongiform Encephalopathies (TSE)

- Spread human to human by direct contact, contaminated foods, ect.
- Various effects on nervous tissue agent
- Develops spongelike appearance due to loss of nerve and glial cells
- buildup of tiny protein fibrils in brain tissue
- lose coordination, difficulty moving and eventually progress to Brian degeneration eventually leading to collapse and death

Cytomegalovirus

- CMV (HUMAN)
- CMV infections: inclusion bodies in cytoplasm or nucleus

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