Introduction!

This is a blog on General Virology. Virology is a branch of science that deals with viruses and viral diseases. All facts and data represented here have been summarised from the notes given to us by our helpful lecturers, and from the websites and books that we researched on.

This website was designed in a simple and concise manner, to provide you, the reader, with the knowledge you need to acquire.

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Wong Wei Sheng Wilson (082571G)
Liang Weisong Edwin (083818Q)
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Nur Nurulshahqairani Binte Azmi (082673C)

 

Prions, Viroids and Virusoids

In the 1920s, several cases of a slow but progressively dementing illness in humans were observed independently by Hans Gerhard Creutzfedlt and Alfons Maria-Jakob. It was characterized by mental degeneration, loss of motor function, and eventual death.

In 1982, Stanley Prusiner proposed that the infective causative agent is an exceedingly small proteinaceous infectious particle called a prion.

Introducing Prions

Prions are rogue proteins that transform other cellular protein (PrPC) to the prion form PrP^SC. Research indicates that prions are normal proteins that become folded incorrectly (possibly due to mutations).

Prions cause fatal, neurological degenerative diseases, including Creutzfeldt-Jakob disease (CJD), kuru, scrapie (Transmissible spongiform encephalopathy), mad cow disease (Bovine spongiform encephalopathy) and chronic wasting disease. The disease can be passed on to humans—new-variant CJD.

Important Characteristics

   ·      Prions are resistant to inactivation by heating to 90°C, which will inactivate viruses.

   ·       Prion infection is not sensitive to radiation treatment that damages virus genomes.

   ·       Prions are not destroyed by enzymes that digest DNA or RNA.

   ·       Prions are sensitive to protein denaturing agents such as phenol and urea.

   ·       Prions have direct pairing of amino acids.

Mechanism of Action

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Winter/clip_image012.jpg

   1.      Initial infection and eventual interaction with normal cellular PrP^C.

a.       PrP^Sc forms a heterodimer with normal PrP^C

b.      Template for altering the protein fold

c.       Tightly coiled alpha helix becomes converted to loose beta sheets.

   2.      PrP^C and PrP^Sc are internalized and interact within caveolae-like vesicles where PrP^C is converted to PrP^Sc.

   3.     PrP^Sc may be recycled to the cell surface(shedding) to interact with PrP^C from other cells and spread the infection, or remain and accumulate within the cell.

   4.      When PrP^Sc accumulates in the cell, they stick together inside cells, forming small fibrils. Because the fibrils cannot be organized in the plasma membrane correctly, such aggregations eventually kill the cell.

   5.      Cell death would release PrP^Sc and spread the infection; partially released proteolyzed PrP^Sc 27-30 may aggregate within plaques.

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Winter/clip_image010.jpg

In Step 1(c): Three-dimensional structure of PrP C (left) and proposed 3D structure of PrP Sc (right). Alpha helices are indicated in green while beta sheets are indicated in blue. PrP C is composed primarily of alpha helices while PrP Sc is composed primarily of Beta pleated sheets.

Note:

·       α-helical, cellular isoform of the prion protein is PrP^C. It is a membrane-anchored glycoprotein.

        ·       Insoluble, β-sheet, infectious, disease-causing isoform is PrP^Sc

PrP C	PrP Sc
primarily alpha helix structure	primarily beta sheet structure
protease susceptible	resistant to protease
monomeric species	forms multimeric aggregates
stable monomer	less stable monomer, self propagates for stability
normal resistance	extremely resistant to heat, chemicals, radiation and strong solvents
detergent soluble	detergent insoluble

 Evidence that PrP is the protein involved.

·         Mouse with knockout PrPC gene ·         No PrPC protein ·         Infect with prions ·         Nothing to convert ·         Mouse lives

Mouse with normal PrPC gene Produce PrPC protein Infect with prions Prions convert normal PrPC to rogue proteins Mouse dies

 

Pathogenesis

On the light-microscopic level spongiform changes in the brain, degeneration of neurons and astrocytosis are histopathological hallmarks of prion diseases.

Transmission:

Eating beef or mutton contaminated with prions.

Model of propagation of PrP^Sc

Viroids

   ·       Viroids are infectious agents composed exclusively of a single piece of circular single stranded RNA which has some double-stranded regions.

   ·       They mainly cause plant diseases (25 main sequences identified) by interfering with mRNA processing—they are catalytic ribozymes that cleave RNA to produce fragments containing a 5’-hydroxyl and a 2’, 3’-cyclic phosphate.

   ·       Only one sequence is known to infect man (Hepatitis D)

Hepatitis D

   ·       Only human disease known to be caused by a viroid.

   ·       Previously called delta agent

   ·       There is extensive sequence complementarity between the hepatitis D viroid RNA and human liver cell 7S RNA

o   The 7S RNA structure involved in the translocation of secretory and membrane-associated particles.

   ·         Hepatitis D viroid causes liver cell death via sequestering this 7S RNA and/or cleaving it.

Transmission

   ·       Co-infection with Hepatitis B virus

   ·       Bodily fluids

o   Unprotected sex

o   Sharing contaminated needles

o   Close proximity

http://www.thailabonline.com/blood/hepdslide_1.gif

Virusoid

Virusoids are infectious agents that infect plants in conjunction with an assistant virus.

They are not considered viruses but are subviral particles.

The size and structure is similar to viroids.

Its genome is a single molecule of single stranded circular RNA that is several hundred nucleotides long and codes for nothing but its own structure.

Emerging Viruses

Definition of emerging viruses:

Known viruses that have newly appeared in a human population and is rapidly increasing in disease incidence.

Source of emerging viruses:

    · Animals are usually the reservoirs of emerging viruses (zoonotic viruses).

    · Viruses were probably endemic at low levels in localized areas.

    · However, they could have “jumped” species, acquired a new host range and spread from there.

Examples of emerging viruses:

Dengue virus, Avian Influenza virus (e.g. H5N1 strain), SARS coronavirus, Nipah virus and Ebola virus.

http://bepast.org/docs/photos/nipah%20virus/nipah%20virus.jpg

Nipah Virus

http://m.blog.hu/li/lightscience/image/gpw-20050430a-fullsize-Ebola-virus-CDC-PHIL-ID-1181.jpg

Ebola Virus

http://ec.europa.eu/health/ph_threats/com/Influenza/images/influenza.jpg

Reasons for Emergence

Virus Factors

    · Spontaneous evolution of a new virus entity

    · Generation of a novel strain due to co-infection of different strains in an individual (random assortment)

Human Factors

    · Concentration of people with shared lifestyle

o E.g. the sharing of hypodermic needles, unsafe sex

    · Breakdown in public health

o Mainly in third world countries

    · Climate change

o E.g. increased wet seasons will lead to more stagnant water which might make it more conducive for mosquito breeding in a particular area. Aedes mosquito is a vector of dengue fever. Thus, the spread of the dengue flavivirus will be enhanced.

    · Man invading natural habitat of animal

o Deforestation for agricultural needs

o Closer to wild animals

o Increasing the possibility of zoonoses

General Viral Replication Cycle

Viral Growth Curve

· The amount of virus is plotted on a logarithmic scale as a function of time after infection.

· At the start, the virus disappears but viral nucleic acid continues to function and begins to accumulate within the cell as the virus is replicating in the cell. The time during which no virus is found inside the cell is known as the eclipse period. The eclipse period ends with the appearance of virus.

· The latent period is defined as the time from the onset of infection to the appearance of virus extracellularly.

· Alterations of cell morphology accompanied by marked derangement of cell function begin toward the end of the latent period.

· At the end of the latent period, new progeny viruses are assembled and released as the cell bursts.

· Infection begins with one virus particle and ends with several hundred virus particles having been produced. This is known as productive infection.

Viral Growth Cycle

6 main steps:

  1. Attachment

  2. Entry (penetration)

  3. Uncoating

  4. Replication of genome and gene expression

  5. Assembly

  6. Release

http://staff.vbi.vt.edu/pathport/pathinfo_images/CCHFV/rc.jpe

Attachment

    · The virus attachment proteins on the surface of the virion attach to specific receptor proteins on the host cell surface through weak, noncovalent bonding.

    · The specificity of attachment determines the host range of the virus.

    · Receptor on host cell mediates viral entry into cell.

Routes of Host Entry

    · Clathrin coated pits in endocytosis: Naked viruses such as the Hepatitis C virus.

    · Receptor-mediated endocytosis: Enveloped viruses such as the Epstein Barr Virus.

    · Fusion: Lipid bilayer of virus fuses with lipid bilayer of host cell.

    · Direct penetration: Translocation of entire virus particle mediated by receptor.

Uncoating

    · The viral genome must be separated from its protein coat once the virus enters the host cell’s cytoplasm. This process is called uncoating.

    · Naked viruses are uncoated by proteolytic enzymes from host cells or from the viruses themselves.

    · The uncoating of viruses such as the poxviruses is completed by a specific enzyme that is encoded by viral DNA and formed soon after infection.

    · Polioviruses begin uncoating even before penetration is complete.

Replication and Expression

    · After uncoating has taken place, genome replication and gene expression takes place—mRNA is synthesized, processed and translated to produce viral proteins.

    · At this point, viruses follow different pathways depending on the nature of their nucleic acid and the part of the cell in which they replicate.

Assembly

    · Assembly depends on the site of synthesis

    · Sites of protein synthesis and processing are the endoplasmic reticulum and Golgi body.

    · Sites for assembly are the nucleus, endoplasmic reticulum and Golgi body.

    · The progeny particles are assembled by packaging the viral nucleic acid within the capsid proteins.

Release

    · Virus buds off the plasma membrane of the host cell, and an envelope forms around the new viruses.

o Virus specific proteins enter the cell membrane at specific sites

o The viral nucelocapsid then interacts with the specific membrane site mediated by the matrix protein.

o The cell membrane evaginates at that site, and an enveloped particle buds off from the membrane.

o This usually occurs with enveloped viruses. An exception is the herpesviridae family, whose members acquire their envelopes from the nuclear membrane rather than the outer cell plasma membrane.

    · Alternatively, there may be an accumulation of particles in vesicles and release via exocytosis. This usually occurs with enveloped viruses.

    · Host cell lysis may follow.

Life Cycle of HIV virus

Methods of Study of Viruses

Isolation and Cultivation of Viruses

Animals and eggs can be used for virus cultivation. However, this way of cultivating viruses has been mainly replaced by cell culture due to the inconvenience and safety involved in handling animals. Animals and eggs still have their advantages for use as a culture “media”—this method is still used to cultivate viruses that have no known host in vitro (e.g. influenza virus in chick embryo)), and to study viral pathogenesis in a whole host (e.g. polo studies in chimpanzees). Plants may also be used to cultivate certain plant viruses. The tobacco mosaic virus may be used to determine virus numbers based on the number of virus plaques on its leaves.

Cell tissue culture comprises of cells being grown in vitro on continuous cell lines. Continuous cells lines are the preferred choice of viral culture because they can be sub-cultured indefinitely, theoretically. They are derived from polyploid or multiploid cancerous cells. Viruses that do not grow in vitro have to be grown in animals, plants or eggs.

Detection, identification and diagnosis (elaborated later)

  1. Tissue culture methods

a. Cytopathic effect

b. Plaque assay

  2. Physical methods

a. X-ray crystallography

b. Electron microscopy

c. Ultracentrifugation

  3. Serological methods

a. Haemagglutination (HA)

b. Haemagglutination Inhibition (HI)

c. Virus neutralisation

d. Complement Fixation

  4. Immunological methods

a. Immunofluorescence

b. Immunogold EM

c. Immunoprecipitation

d. Immunoblot

e. Enzyme Linked Immunosorbent Assay (ELISA)

  5. Molecular biology methods to analyse viral proteins

a. SDS PAGE

b. Western blot

c. Protein sequencing

d. X-ray crystallography

  6. Molecular biology methods to analyse viral genome

a. Restriction analysis

b. DNA sequencing

c. Southern blot

d. Northern blot

e. PCR/RT-PCR

Cytopathic Effect:

Cytopathic effect (CPE) is an alteration in cell morphology resulting from viral infection of a cell culture monolayer. CPE may be used as a presumptive identification of a virus. If the virus does not produce a CPE, its presence can be detected by several other techniques such as haemadsorption.

http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/cummings/baculovirus.JPG

Plaque Assay:

Plaque Assays are used for the quantitative measure of infectious centres by counting the number of infectious virus particles in a given sample in Plaque forming units.

The plaque assay is based simply on the ability of a single infectious virus particle to give rise to a macroscopic area of cytopathology (i.e. plaque, focus of infection) on an otherwise normal monolayer of cultured cells.

Specifically, if a single cell in a monolayer is infected with a single virus particle, new virus resulting from the initial infection can infect surrounding cells which, in turn, produce virus that infects additional surrounding cells. Multiple rounds of infection give rise to an area of infection called a plaque.

*The semisolid medium prevents the formation of secondary plaques through diffusion of virus from the original site of the infection to new sites, ensuring that each plaque that develops in the assay originated from a single infectious particle in the starting inoculum.

http://www.v-bio.com/pictures/p06_small.png

Advantages

  1. Simple, inexpensive, precisely quantitative method.

  2. Higher accuracy at lower concentration—useful for samples with very low virus counts (in food and water samples).

Disadvantages/Limitations

  1. Counts only viable virus (virus capable of multiplying)

  2. Only works for viruses that infect monolayer cells and viruses that cause cell lysis (or other cytopathic changes that can be observed)

  3. Must know culture conditions for the virus studied.

  4. Requires time for incubation, time consuming.

Calculation

Original Virus Concentration (in PFU/ml) = Final Virus Concentration * Dilution Factor * (1/volume pipetted in well (in ml))

X-ray crystallography:

Viruses are crystallized and by using X-ray diffraction techniques, the structure of the virus can be elucidated from the diffraction pattern produced.

Electron microscopy:

This includes Transmission EM, Scanning EM and STEM. Electron microscopy involves scattering electrons on a specimen to “illuminate” it and magnify it for viewing. The resolution of an electron microscope is much greater than a light microscope as the wavelength of an electron is much smaller than that of a light proton.

http://www.biologie.uni-hamburg.de/b-online/ge03/14.gif

Ultracentrifuation:

Ultracentrifugation has assumed growing importance in virus diagnosis as a technique by which to concentrate and purify viruses for immediacy diagnosis on the basis of electron microscopy as well as for purely virological and serological tests.

Ultracentrifugation has proved to be helpful for sizeable improvement of sensitivity for detection, which, in turn, has been conducive to time saving. The preparational ultracentrifuge enables also direct diagnosis by determination of isodensities and sedimentation coefficients of viruses and their components.

Haemagglutination:

Agglutination of red blood cells caused by certain antibodies, virus particles or high molecular weight polysaccharides. This can be used to test for influenza and other viruses with two spike proteins neuraminidase and haemagglutinin, since these two proteins bind specifically to red blood cells.

Haemagglutination Inhibition:

This is used to quantify the virus by haemagglutination. If the virus and antibody are homologous, the antibody bound to the surface of the virus blocks its entry into the cell. This neutralizes infectivity, because it prevents viral replication and subsequent CPE formation or animal infection. Agglutination is also i

nhibited.

http://www.umanitoba.ca/science/microbiology/staff/cameron/graphics/401lab3passivehemagglutination%20group506.jpg

Virus neutralization:

If the virus and antibody are homologous, the antibody bound to the surface of the virus blocks its entry into the cell. This neutralizes viral infectivity, because it prevents viral replication and subsequent CPE formation or animal infection. It is one of the methods employed for the detection of virus-specific neutralizing antibodies.

Complement fixation:

Complement fixation is used to detect the presence of either specific antibody or specific antigen in a patient's serum. If the antigen (the unknown virus in the culture fluid) and the known antibody are homologous, complement will be fixed (bound) to the antigen-antibody complex. This makes it unavailable to lyse the “indicator” system, which is composed of sensitized red blood cells.

Immunofluorescence:

Microscopic method of determining the presence or location of an antigen (or antibody) by demonstrating fluorescence when the preparation is exposed to a fluorescein-tagged antibody (or antigen) using ultraviolet radiation.

http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/cummings/1245-MERGED%2020x%20wide%20field.JPG

Above: Immunofluorescence for Herpes Simplex Virus Antibody

Immunogold EM:

Locate specific proteins or antigens by attaching nano-gold particles to antibodies.

Immunoprecipitation:

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.

Immunoblot:

The immunoblot (also called Western blot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed using antibodies specific to the target protein.

ELISA (Enzyme-linked Immunosorbent Assay):

The basic principle of an ELISA is to use an enzyme to detect the binding of antigen (Ag) and antibodies (Ab). Antibodies are bonded to enzymes; the enzymes remain able to catalyze a reaction that converts a colourless substrate to a coloured product, indicating the presence of Ag:Ab binding. Depending on the test, an ELISA can be used to detect the presence of either Abs or Ags.

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SDS PAGE:

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility.

Polymerase Chain reaction (PCR):

A method for expanding small discrete sections of DNA by binding DNA primers to sections at the ends of the DNA to be expanded and using cycles of heat (to create single-stranded DNA) and cooler temperatures (to allow a DNA polymerase enzyme to create new sections of DNA between the primer ends).

http://www.copernicusproject.ucr.edu/ssi/HighSchoolBioResources/Genetic%20Engin%20Hum%20Genome/pcr.jpg

Southern Blot:

Identification of specific genetic sequences by separating DNA fragments by gel electrophoresis and transferring them to membrane filters in situ. Labelled complementary DNA applied to the filter binds to homologous fragments, which can then be identified by detecting the presence of the labelled DNA in association with bands of certain molecular size. This test was named after its discoverer, E.M. Southern.

http://www.biogate.it/microarrays/images/Southern_blot.gif