Do APPLE get DISEASES??

The green apple in the picture taken above seemed to have suffered from some kind of "skin allergy"!! It looks just like those itchy rashes on our own skin and the colour of those spots is somehow simliar. However, it is not "skin allergy"!! This is a kind of apple disease, Apple Scar Skin. It is one of the most destructive diseases affecting apple which is caused by Apple Scar Skin viroid (ASSVd). Fruit dappling appeared on several cultivars in Korea and has been distributed to major cultivated areas since 2001. ASSVd was identified from infected fruits by using nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL). Affected fruit bears small circular spots near the calyx end of the fruit. As the fruit matures, the spots enlarge and the spots increasingly contrast with the darkening background color of the fruit.

The mastermind of the apple disease mentioned earlier is VIRIOD!!

Well well well what are viriods?? Have any idea?

They are the smallest known agents of infectious disease. Whereas conventional viruses are made up of nucleic acid encapsulated in protein (capsid), viriods are uniquely characterized by the absence of a capsid. In spite of their small size, viroid ribonucleic acids (RNAs) can replicate and produce characteristic disease syndromes when introduced into cells.

Potatoes that suffered from PSTVd

Another example of plant disease caused by Viriods is Potato Spindle Tuber Viriod (PSTVd). Potato is the main host of this disease! Infected tubers are small, elongated, characteristically spindle or dumb-bell shaped, often with prominent bud scales evenly distributed over the surface. Futhermore, growth cracks may also occur.

Recommendation of the day!!

Fatal Protein: The Story of CJD, BSE and Other Prion Diseases By Rosalind Ridley, Harry Baker

We would like to recommend this interesting book specially for those who are interested in knowing more about the history of scrapie in sheep, the spread of kuru through cannibalism in Papua New Guinea, and the BSE epidemic in Britain - "mad cow disease" - which claimed over 170,000 cattle and threatened the rest of Europe. The authors presented a detailed description of the human prion diseases, and also examined the evidence that the new variant of the Creutzfeld-Jakob disease results from eating infected by BSE. At the heart of this book lies a discussion of the still controversial idea that the infectious agent in prion diseases is a normal brain protein which takes on abnormal, mutant shape, the so-called "prion theory".

Symptoms of CJD, Mad Cow and Scrapie

Overview of transmission of PRIONS!!

SPONGE-LIKE Human Brain!!!

MRI scan of a CJD brain, showing its spongy appearance.

Look at the picture carefully!!

Are you able to spot those tiny winy holes in the brain??

Classic Creutzfeldt-Jakob disease (CJD) is a human prion disease and also a neurodegenerative disorder with characteristic clinical and diagnostic features. This disease is rapidly progressive and always deadly. Most importantly, we would like to state out a point that classic CJD is not related to "mad cow" disease. Infection with this disease leads to death usually within 1 year of onset of illness.

*Important Note: Classic CJD also is distinct from "variant CJD", another prion disease that is related to BSE.


Four types of CJD




Cause of CDJ
- a specific protein called a prion, which lacks nucleic acids, resists proteolytic digestion, and spontaneously aggregates in the brain.

The tissue slide above displays sponge-like lesions in the brain tissue of a classic CJD patient. This lesion is typical of many prion diseases. Most cases are sporadic; 5% to 15% are familial, with an autosomal dominant pattern of inheritance.

In the present, there is only treatment for relieving symptoms. Current clinical trials are testing promising antiviral drugs such as amantadine, which has proven helpful in treating Parkinson's disease. Some patients taking these antivirals have shown brief periods of improvement, with no harmful side effects. However, no treatment has been discovered that stops CJD completely. It is invariably fatal, often within a year of the onset of symptoms.

The SHEEPS’ and GOATS’ KILLER!!

This disease was known since the 18th century, but till the 60s that scientists began to suspect that it wasn’t a typical disease, self-replicating outside of the usual means of nucleic acids. This fatal and infectious disease is none other than scrapie, which cause horrible symptoms on animals it infect before death. If you are curious about how the name of scrapie come about, continue to read on….. The name of the disease came from the disturbing behaviour of infected sheep, to scrape off their fleece against rocks. It is quite saddening if you try to visualise yourself in the picture of this happening, witnessing the whole entire process.

Scrapie causes a degeneration of the central nervous system which eventually results in a variety of behavioral, locomotive changes and death. Sad to say that up till today, there is still neither cure nor treatment. In other words, the cruelty of this killing will continue to happen on any innocent sheep or goat.

Do you have any idea of what causes scrapie??

An agent that is smaller than a virus causes scrapie. It is either a protein called a prion or another substance closely associated with the prion protein. Scientists still have many to learning about the scrapie agent and exactly how it works. It is known that the scrapie agent is extremely resistant to heat as well to normal sterilization processes. Besides that, it does not evoke any detectable immune response or inflammatory reactions in host animals. The incubation period is usually two to five years. Ultimately, the scrapie agent attacks the brain, leaving holes in it much like swiss cheese or a sponge, hence the name spongiform encephalopathy.

Have you heard of MAD COW Disease??

MOO!! I bet almost everyone does! Not because it is COMMON, but because of its outbreak in countries such as Europe, Britain, U.S.A, Canada and Japan. In 1986, the first occurrence of mad cow disease took placed in England. The scientific name for Mad Cow Disease is bovine spongiform encephalopathy (BSE), a progressive neurological disorder of cattle which can be transmitted to other species, including human beings. The disease is so named as it causes the brain of the victim to degenerate into a sponge-like consistency.
The actual cause of BSE is still unknown, however, it is generally accepted by the scientific community that the likely cause is infectious forms of a type of protein, prions, normally found in animals cause BSE. In cattle with BSE, these abnormal prions initially occur in the small intestines and tonsils, after which are found in central nervous tissues, such as the brain and spinal cord as well as other tissues of infected animals experiencing later stages of the disease.

Say HELLO to PRIONS!!!

Prions also known as proteinaceous infectious particles are microscopic protein particle but lacking of nucleic acid, transform other normal cellular protein (PrPC) to the prion form PrPSC. Also, thought to be the infectious agent responsible for scrapie and certain other degenerative diseases of the nervous system.

Prion diseases are associated with the conversion of the α-helix rich prion protein (PrPC) into a β-structure-rich insoluble conformer (PrPSc) that is thought to be infectious. Increasingly, PrPSc gets transformed until they completely clogged brain cells. Cells fail to function properly or stop working. Eventually, cells die and release prions into bloodstream to re-infect other cells.

In the picture above, on the left hand side is a normal conformer whereas on the other side is a rogue conformer. PrPSC forms a heterodimer with normal PrPC, then presence of template will alter the protein fold. After which, tightly coiled α-helix will be converted to loose β-sheets.

Prion diseases are often called spongiform encephalopathies due to the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum. They are a family of rare progressive neurodegenerative disorders that affect both humans and animals.
Majority of mammalian species will tend to develop these diseases.

Below are a few examples:

• Scrapie – for sheep
  
• TME (transmissible mink encephalopathy) – for mink


• CWD (chronic wasting disease) – for mule deer, elk
  
• BSE (bovine spongiform encephalopathy) – for cows
  

Humans are also susceptible to several prion diseases:

• CJD - Creutzfeld-Jacob Disease
  
• GSS - Gerstmann-Straussler-Scheinker syndrome
  
• FFI - Fatal familial Insomnia
  
• Kuru


• Alpers Syndrome (in infants)

* More information will be covered on scrapie, BSE and CJD in the next part!!

Viral Entry and Spread

Viral Entry

3 requirements to be fulfilled to ensure successful infection in an individual host:

• Sufficient virus must be available to initiate infection


• Cells at the site of infection must be accessible, susceptible and permissive for the virus


• Local host anti-viral defense systems must be absent or initially ineffective
  

To infect its host, a virus must first enter cells at a body surface. Common sites of entry include:

- Mucosal linings of the respiratory
- Alimentary tract
- Urogenital tract
- Outer surface of the eye (conjunctival membranes or cornea)
- Skin
* Only the two points in purple will be covered.

Alimentary Tract - a common route of infection and dispersal.

Eating, drinking, and some social activities routinely place viruses in the alimentary tract. It is designed to mix, digest and absorb food, providing a good opportunity for viruses to encounter a susceptible cell and to interact with cells of the circulatory, lymphatic, and immune systems.

Urogenital Tract

Some viruses enter the urogenital tract as a result of sexual contact. The urogenital tract is well
protected by physical barriers, including mucus and low pH (in the case of the vagina). Normal sexual activity can result in minute tears or abrasions in the vaginal epithelium or the urethra, allowing viruses to enter. Some viruses infect the epithelium and produce local lesions (e.g., certain human papillomaviruses, which cause genital warts). Other viruses gain access to cells in the underlying tissues and infect cells of the immune system (e.g., HIV type 1), or sensory and autonomic neurons (in the case of herpes simplex viruses).

Viral Spread

* Click on the picture for enlargement!!

Systemic infection, which was not mentioned in the diagram, is one that affects the whole body, probably travelling in lymph or blood. This is in contrast to a local infection which only affects the area where the infection entered.

The HIV Life Cycle

The picture shown above is not an art piece!!
It is actually the coloured scanning electron micrograph of a T4 cell (green) infected with HIV (red).

Infection: Several different kinds of cells have proteins on their surface that are called CD4 receptors. HIV searches for cells that have CD4 surface receptors, because this particular protein enables the virus to bind to the cell. Although HIV infects a variety of cells, its main target is the T4-lymphocyte (also called the "T-helper cell"), a kind of white blood cell that has lots of CD4 receptors. The T4-cell is responsible for warning your immune system that there are invaders in the system.

Replication: Once HIV binds to a cell, it hides HIV DNA inside the cell's DNA: this turns the cell into a sort of HIV factory.

Step 1: Binding

A virus consists of an outer envelope of protein, fat and sugar wrapped around a set of genes and special enzymes. HIV has proteins on its envelope that are strongly attracted to the CD4+ surface receptor on the outside of the T4-cell. When HIV binds to a CD4+ surface receptor, it activates other proteins on the cell's surface, allowing the HIV envelope to fuse to the outside of the cell. Entry can be blocked by entry inhibitors.

Step 2 : Reverse Transcription

HIV's genes are carried in two strands of RNA, while the genetic material of human cells is found in DNA. In order for the virus to infect the cell, a process called "reverse transcription" makes a DNA copy of the virus's RNA.
After the binding process, the viral capsid is released into the host cell. A viral enzyme called reverse transcriptase makes a DNA copy of the RNA. This new DNA is called "proviral DNA". Reverse transcription can be blocked by: Nucleoside Reverse Transcriptase Inhibitors (NRTIs), and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs).

Step 3 : Integration
The HIV DNA is then carried to the cell's nucleus, where the cell's DNA is kept. Then, another viral enzyme called integrase hides the proviral DNA into the cell's DNA. Then, when the cell tries to make new proteins, it can accidentally make new HIVs.Integration can be blocked by integrase inhibitors.

Step 4 : Transcription
Once HIV's genetic material is inside the cell's nucleus, it directs the cell to produce new HIV.
The strands of viral DNA in the nucleus separate, and special enzymes create a complementary strand of genetic material called messenger RNA or mRNA. Transcription can be blocked by antisense antivirals or transcription inhibitors (TIs), new classes of drugs that are in the earliest stage of research.

Step 5 : Translation
The mRNA carries instructions for making new viral proteins from the nucleus to a kind of workshop in the cell. Each section of the mRNA corresponds to a protein building block for making a part of HIV. As each mRNA strand is processed, a corresponding string of proteins is made. This process continues until the mRNA strand has been transformed into new viral proteins needed to make a new virus.

Step 6 : Viral Assembly and Maturation

The final step begins with the assembly of new virus. Long strings of proteins are cut up by a viral enzyme called protease into smaller proteins. These proteins serve a variety of functions; some become structural elements of new HIV, while others become enzymes, such as reverse transcriptase.Once the new viral particles are assembled, they bud off the host cell, and create a new virus. The virus then enters the maturation stage, which involves the processing of viral proteins. Maturation is the final step in the process and is required for the virus to become infectious. With viral assembly and maturation completed, the virus is able to infect new cells. Each infected cell can produce a lot of new viruses.
Viral assembly can be blocked by Protease Inhibitors (PIs).

Curious about how virus replicates??

Everyone has their own curiosity towards certain things or maybe even EVERYTHING!!

The general virus replication cycle can be divided into the following stages:

1. Attachment - a specific binding between viral surface proteins and their receptors on the host cellular surface. This specificity determines the host range of a virus.

2. Penetration - After attachment, viruses may enter the host cell through receptor mediated endocytosis, clathrin endocytosis or other mechanisms.

3. Uncoating - a process that viral capsid is degraded by viral enzymes or host enzymes.

4. Replication - involves assembly of viral proteins and genetic materials produced in the host cell.

5. Assembly - capsid is assembled around the genome.

6. Release - Viruses may escape from the host cell by causing cell rupture (lysis). Enveloped viruses (e.g., HIV) typically "bud" from the host cell. During the budding process, a virus acquires the phospholipid envelope containing the embedded viral glycoproteins.

* CLICK HERE!! If you want to have a better understanding on the cycle!! An animation have been found specially for YOU(:


Definitions

1. Receptor mediated endocytosis is an endocytotic mechanism in which specific molecules are ingested into the cell. The specificity results from a receptor-ligand interaction. Receptors on the plasma membrane of the target tissue will specifically bind to ligands on the outside of the cell. An endocytotic process occurs and the ligand is ingested.

2. Clathrin-mediated endocytosis is a vesicular transport event involved in the internalization and recycling of receptors participating in signal transduction events and nutrient import as well as in the reformation of synaptic vesicles.

The Attack of Emerging Viruses!!

As the name suggests, emerging virus is an infectious disease that has newly appeared in a human population or that has been known for some time but is rapidly increasing in incidence or geographic range.

Examples of emerging virus include:

• Ebola Virus (first outbreak in 1976 and discovery of the virus in 1977)


• HIV/AIDS (virus first isolated in 1983)


• Hepatitis C (first identified in 1989, now known to be the most common cause of post-transfusion hepatitis worldwide),


• Influenza A (H5N1) virus (well known pathogen in birds but first isolated from humans in 1997),


• Legionella pneumophila (first outbreak in 1976 as Legionnaire disease and since associated with similar outbreaks linked to poorly maintained air conditioning systems)

* The 2 viruses that were highlighted in different colour will be touched on!!

Hepatitis C, commonly known as HCV is a viral infection of the liver. It is the most common liver disease worldwide. Also known as the 'silent epidemic', Hepatitis C could have infected a patient for years before actually being discovered. The virus attacks the liver. It keeps on multiplying, killing the surrounding tissue. The immune system fights back, usually causing reactions such as inflammation and fibrosis of the liver. If not detected and treated in time, it could lead to liver cancer or cirrhosis.

It is estimated that approximately 1.8% of the American population is infected by HCV which is a major cause of chronic liver diseases including liver cancer and cirrhosis. Almost 80% of those infected by HCV will develop chronic infection. Out of this approximately 20% will develop liver cirrhosis and 5% will develop liver cancer.

Ebola virus

Being the notoriously deadly virus that causes fearsome symptoms, the most prominent being high fever and massive internal bleeding! Ebola virus kills as many as 90% of the people it infects. It is one of the viruses that is capable of causing hemorrhagic (bloody) fever.

Ebola hemorrhagic fever (Ebola HF) is a severe, often-fatal disease in humans and nonhuman primates (monkeys, gorillas, and chimpanzees) that has appeared sporadically since its initial recognition in 1976.

Reasons for emergence??

With increasing international travel and globalization of the world's economies, breakdown in public health in remote areas, changing climates, and altered human behaviour (includes man invading natural habitat of animals) and demographics, multiple viruses have emerged to occupy expanded ecologic niches, producing disease syndromes in parts of the world where they had never before existed. Most emerging viral diseases in humans in the 21st century have been zoonotic.

Methods of study of viruses

For isolation and cultivation:

• Embryonated eggs
  
• Animals and Plants
  
• Tissue culture

For detection, identification and diagnosis:

a) Tissue culture methods

b) Physical methods

c) Serological methods

d) Immunological methods

e) Others and molecular biology

1. Eggs

• First method used for virus cultivation
  
• Inconvenient
  
• Safety in handling animals
  
• Mainly replaced by cell culture except when:
  
• Virus has no known host in vitro
  
• Study of viral pathogenesis in a whole host
  

Some viruses will replicate in the living tissues of developing embryonated hens eggs, such as influenza virus. Egg-adapted strains of influenza virus replicate well in eggs & very high virus titres can be obtained. This method has proved to be highly effective for the isolation & culture of many (but not all) viruses.

http://chickscope.beckman.uiuc.edu/resources/egg_to_chick/figures/figure11.gif

2. Animals

Creation of transgenic animals & plants by means of the insertion into the DNA of the experimental organism of all or part of the virus genome, resulting in expression in the somatic cells (and sometimes in the cells of the germ line) of virus mRNA & proteins.

Some animal s that are used:

http://www.scanbur.eu/images/products/Lab_animals_sprague_dawley_.jpg

http://wwwimage.cbsnews.com/images/2004/05/31/image620404x.jpg

3. Plants

Tobacco Mosaic virus

Virus plaque leaves to determine virus number

http://www.apsnet.org/online/feature/tobacco/image/tobacco1.jpg

4. Tissue culture

- Cell tissue culture

- Cells grown in vitro

- Primary cell culture

- Continuous cell lines:

• derived from primary cell lines
  
• transformed/ cancerous cells
  
• polyploid or multiploid
  
• can be sub cultured indefinitely- theoretically
  
• method of choice to cultivate virus

Some viruses do not grow in vitro

http://www.nordiqc.org/Run-17-B1/Assessment/Image/NQCB1_HER-2/NQCB1-HER2

(A) Tissue culture method for detection

• Cytopathic Effect (CPE)
  
• Plaque Assay
  

Cytopathic Effect (CPE)

The presence of the virus often gives rise to morphological changes in the host cell. Any detectable changes in the host cell due to infection are known as a cytopathic effect. Cytopathic effects (CPE) may consist of cell rounding, disorientation, swelling or shrinking, death, detachment from the surface, etc.

http://www.medscape.com/content/2004/00/47/13/471342/art-eid471342.fig1.gif

Plaque Assay

What?

• Observing cell death in infected cell culture
  
• One virus infects one cell and spreads to surrounding cells
  
• Higher accuracy at lower concentration
  
• Features
  
• Very time-consuming
  
• Very simple method
  
• Only works for viruses that affect monolayer
  
• Only works for viruses that cause cell lysis
  
• Uses principle of one virus on the monolayer produces one plaque
  

http://www.sigmaaldrich.com

Virus count features

• Counts only viable virus
  
• Must know culture conditions for the virus studied
  
• Useful for samples with very low virus counts
  
• Requires time for incubation
  

(B)Physical methods

• X-ray crystallography
  
• Electron microscopy
  
• Ultracentrifugation
  

(C)Serological / immunological methods

• Haemagglutination(HA)
  
• Haemagglutination Inhibition(HI)
  
• Virus neutralisation
  
• Complement fixation
  
• Immunostaining
  
• Immunoprecipitation/immunoblot
  
• ELISA
  

Haemagglutination

http://www.microbiologybytes.com/introduction/graphics/haem.jpg

Influenza and other viruses

Two spike proteins( neuraminidase and haemagglutinin)

Complement fixation

• Mediated by antibody
  
• Antibody binds to antigen
  
• Complement cascade of molecules in blood serum initiated, causing lysis of infected cell or pathogen
  

http://www.dshs.state.tx.us/LAB/images/cf_test2.gif

(D)Immunofluorescence

• Antibody tagged with fluorescent dye
  
• Antibody attached specifically to antigen
  
• View specimen under exciting light
  
• Fluorescent microscope
  

Immunoprecipitation

http://www.molecularstation.com/images/immunoprecipitation.gif

Immunoblot

http://www.teagasc.ie/research/reports/dairyproduction/5067/image01.gif

ELISA

What?

• Enzyme –linked immunosorbant assay
  
• Antibody to detect antigen
  
• Antibody labelled with indicator
  
• Colour reaction
  
• System requires one molecule to be attached to solid surface
  

http://microvet.arizona.edu/Courses/MIC419/ToolBox/ELISA2.gif

molecular virology

• study genome organisation
  
• expression of viral genome
  
• replication of genome and progeny virus
  
• molecular basis of viral replication cycle
  

(E)Molecular biology & Others

Analysis of viral proteins

• PAGE/SDS PAGE
  
• Western blot
  
• Protein sequencing
  
• x- ray crystallography
  

Analysis of viral genome

• Agarose gels
  
• Restriction analysis
  
• Sequencing
  
• Southern blot
  
• Northern blot
  
• PCR/RT-PCR

How does PCR work?

Cycles of 3 steps repeated again and again

Step1: heatingà separate double stranded DNA

Step 2: coolingà primers bind

Step 3: increase temperatureàpolymerase work

Retroviruses

RETROVIRUSES

Most of the retroviruses we currently know (many!) infect vertebrates, but as a group, they have been identified in virtually all organisms including invertebrates - an evolutionarily successful design!

Legend: Process by which retroviruses use a host cell to replicate.

Retroviruses contain viral RNA and several copies of reverse transcriptase (DNA polymerase). After infecting a cell, the reverse transcriptase is used to make the initial copies of viral DNA from viral RNA. Once a DNA strand has been synthesized, a complementary viral DNA strand is made. These double strand copies of viral DNA are inserted into the host-cell chromosome and host-cell RNA polymerase is used to make virus-related RNA. These RNA strands serve as templates for making new copies of the viral chromosomal RNA and serve also as mRNA. mRNA is translated into viral proteins that are used to make the virus envelope. New viral particles are assembled, bud from the plasma membrane, and are released. An example of this process is illustrated in the replication of the retrovirus, HIV (human immunodeficiency virus).

Legend: Illustration of the structure of a retrovirus.

Retroviruses are infectious particles consisting of an RNA genome packaged in a protein capsid, surrounded by a lipid envelope. This lipid envelope contains polypeptide chains including receptor binding proteins which link to the membrane receptors of the host cell, initiating the process of infection.
Retroviruses contain RNA as the hereditary material in place of the more common DNA. In addition to RNA, retrovirus particles also contain the enzyme reverse transcriptase (or RTase), which causes synthesis of a complementary DNA molecule (cDNA) using virus RNA as a template. When a retrovirus infects a cell, it injects its RNA into the cytoplasm of that cell along with the reverse transcriptase enzyme. The cDNA produced from the RNA template contains the virally derived genetic instructions and allows infection of the host cell to proceed.
The virus that causes AIDS (acquired immune deficiency syndrome) is a retrovirus. It is called HIV for human immunodeficiency virus.

Taxonomy:
Group VI: RNA Reverse Transcribing Viruses

Genome:All retrovirus genomes consist of two molecules of RNA, which are s/s, (+)sense and have 5' cap and 3' poly-(A) (equivalent to mRNA). These vary in size from ~8-11kb. Retrovirus genomes have 4 unique features:

They are the only viruses which are truly diploid.
They are the only RNA viruses whose genome is produced by cellular transcriptional machinery (without any participation by a virus-encoded polymerase).
They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication.
They are the only (+)sense RNA viruses whose genome does not serve directly as mRNA immediately after infection. These two molecules are physically linked as a dimer by hydrogen bonds (co-sediment). In addition, there is a 3rd type of nucleic acid present in all particles, a specific type of tRNA (usually trp, pro or lys) - required for replication (below).Gene order in all retroviruses is invariant:

5' - gag - pol - env - 3'
Some retroviruses have additional genes: