Tuesday, February 3, 2009
6:19 PM
Herpesviridae Are a large family of DNA viruses which cause disease in animals and human. The family name, herpesviruses is derived from the Greek word herpein (to creep) whereby the viruses causes primary infection and the virus then remain latent until reactivation. The reactivation can be trigger due to stress, long exposure to sunlight, during menstrual period. The virus hides in the nerve tissue. It causes viral encephalitis. If the virus is passed from the mother to the infant during birth, it may cause brain damage to the child.
Herpes virus genome (linear ds DNA)
Consist of a spherical virion that have the DNA packaged in a liquid crystalline array in a central core surrounded by an icosahedral capsid, an amorphous layer of proteins and an envelope consisting of a lipid bilayer closely associated with amorphous protein.
Pathogenesis
·
Herpes simplex viruses(HSV), enter and hide in human nervous system and remain latent until reactivation. HSV1 is commonly related to herpes outbreak of the oral cavity such as cold sores or fever blisters however HSV2 is more usually related with genital herpes.
· Shingles and chicken pox were once thought to be separate illness however researchers now know that they are both caused by a virus of the herpes family known as Varicella zoster virus. Varicella: primary infection that causes chicken pox. Herpes zoster: reactivation of the varicelle zoster virus that causes shingles. Shingles often occurs many years after the initial chicken pox infection in people.
· Most healthy people who are infected by Cytomegalovirus after birth have no symptom however some may go on to develop an infectious mononucleosis (aka glandular fever) with prolonged fever and a mild hepatitis. Cytomegalovirus can be found in bodily fluids of any infected person and can be found in urine, saliva, blood, tears, semen and breast.
· Epstein - Barr virus (EBV): people who are infected with EBV is often asymptomatic but infection usually cause infectious mononucleosis. EBV can found in nasopharynx and in salivary gland.
Clinical Features- Herpes simplex
The symptoms vary based on the stage of the virus, the primary outbreak and recurrence. Both herpes simplex viruses 1 and 2 produce similar symptoms however they can differ in severity depending on the site of infection.
· General symptoms of primary herpes simplex infection: skin eruptions appear 2 to 12 days after the initial exposure to the virus. (skin lesion)
· Asymptomatic stages: after an outbreak, the herpes simplex virus becomes latent where HSV produces no symptoms at all and the virus is not transmissible.
· Symptoms of recurrence: the outbreak of infection is often preceded by an early group of symptoms such as itching skin, pain or abnormal tingling sensation at site of infection.
· Oral herpes such as cold sores is usually caused by HSV1 but can also be caused by HSV2.
It usually affects the lips, blister form on the lips but may also erupt on the tongue.
· Genital herpes typically affects the penis, vulva or rectum and is usually caused by HSV2 although the stastistic of HSV1-genital infection is increasing. Symptoms include blister, burning sensation and discharge for 1 to 3 weeks.
Clinical features- Varicella zoster· Symptoms of varicella (chicken pox): fever, skin lesion all over body, scratching the lesion can lead to secondary infection and causes scarring. Chicken pox is dangerous in pregnant woman as the virus can be passed onto the baby and causes problem include birth defects such as scars, eye problems and shortened limbs, etc.
· Shingles: after the chicken pox virus has been contracted, it travels from the skin along the nerve paths to the roots of the nerves where is becomes latent. If it reactivates, there is a burning sensation on the nerve paths along which the virus is travelling, follow by rash which is accompanied by fever and enlarged lymph nodes.
Lab diagnosis· The herpesvirus can be identify by culturing the virus where cells or fluids from a fresh sore are collected. A viral culture is the best method of identifying a genital herpes infection.
· ELISA (Enzyme-Linked ImmunoSorbent Assay) is a technique used mainly to detect the presence of antigen in a sample.
· Antibody test: blood tests can find antibodies that are made by the immune system to fight a herpes infection.
Epidemiology and control of Herpes simplex viruses· Almost 100% of the adult population is at risk of HSV1 due to kissing and close proximity.
· Up to 20% of the US population are infected with HSV2 mainly by sexual contact.
· To prevent risk of transmission: don’t kiss, practice safe sex and refrain from risky behaviour.
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Epidemiology and control of Varicella zoster
· Chicken pox can be prevented by vaccination.
· Avoid people who are infected by chicken pox.
By: man hua.
Monday, February 2, 2009
10:59 PM
Virus host interaction
Stages in virus life cycle
1. Attachment with the host receptor protein.
2. Entry of the viral nucleic acid into the host cytoplasm.
3. Replication and expression of the viral components.
4. Assembly (maturation) of the viral components into complete viral units.
5. Release (exit) of the completed virus from the host cell.
Introduction
The basic principle of viral infection of a given cell is that the cell’s outer surface contains the host cell receptor in which the virus attachment protein (VAP) can bind, the receptor on the host cell will then mediates viral entry into cell and the virus will then grow in the cell. However, this is only applicable within related species or to different types of cells within a host. For example, the common cold virus (Picornaviridae) are specific for the human upper respiratory track as they contain the appropriate VAP for binding to the receptors on the cell membrane of the specialized cells in the upper respiratory tract. There are other viruses that cause intestinal diseases only.
Attachment and entry: the virus blinding to the host cell receptor sites (small black spots) through its attaching proteins at the end of the tail fibres. Once firmly attached to the cell, a viral enzyme in the tail punches a hole in the host‘s cell and the core region of the tail is thrust through and the genome is injected into the host’s cytoplasm.
Replication and expression: once inside the host, the viral genome takes over the metabolism of the host and converting it to its own ends. The viral nucleic acid is then transcribed and translated and the various viral proteins proceed to create everything required to make new viruses.
Maturation and release: the various components accumulate inside the cell and start to SPOTANEOUSLY ASSEMBLY into new virus. However, this process is an orderly one so that each component is added in the proper sequence. Enzymes are produced causing the cell to burst or die thus a crop of new viruses are release into the environment.
Routes of host entry
->Endocytosis:
-non-specific endocytosis
-phagocytosis
-receptor-mediated endocytosis
-> Fusion
-> Direct penetration
1. Naked virus enters the host cell through translocation whereby particle crosses cell membrane intact.
2. Naked virus also enter the host cell through genome injection in which virus attaches to cell surface and release its genome that penetrates the cytoplasm by pore that has been created in the plasma membrane. E.g. bacteriophages.
3. Naked virus-Endocytosis: the virus attaches to the host cell receptor molecules and sinks into a clathrin coated pit. The pit invaginates and lastly closes off producing a clathrin coated vesicle and the contained virus is absorbed into the cytoplasm. E.g. adenovirus
4. Enveloped virus- virus enters cell by receptor mediated endocytosis whereby it depends on the interaction of the virus with a specific binding protein in the cell membrane called a receptor. E.g. influenza virus.
5. Enveloped virus-membrane fusion whereby the virus enters the cell by fusing its outer membrane with the plasma membrane of the host cell. The viral contents are then spilled into the cytoplasm of the cell. E.g. HIV virus.
Naked viruses have an exposed protein capsid while enveloped viruses are cloaked in cell membrane, added during budding from the host cell.
By man hua.
Sunday, February 1, 2009
10:03 PM
Detection, identification and diagnosis of virus
BASIC IMMUNOLOGY
Antigens are any foreign particle or molecule not recognised by the body and it causes an immune response. An antigen can be harmless such as grass pollen or harmful such as influenza virus. Antigen can be virus, bacteria, toxins, foreign proteins, etc. Disease causing antigens are called pathogens. The immune system is design for protecting against disease by identifying and killing the pathogens and tumour cells. Antibodies also known as immunoglobulin (Ig) are gamma globulin proteins that are found in blood or other bodily fluids and are used by the immune system to identify and neutralize foreign particles.
By Serological /Immunological methods· Haemagglutination assay (HA)
· Haemagglutination inhibition (HI)
· Virus neutralisation
· Complement fixation
The
haemagglutination assay is a method used to measure virus particle. This assay takes advantage of the fact that many viruses contain the two spike proteins: Neuraminidase and Haemagglutinin that blinds specifically to the red blood cells. Examples of viral protein with ability to bind to red blood cells are influenza virus and other virus.
Haemagglutination inhibition is whereby one measures the ability of soluble antigen to inhibit the aggulutination of antigen coated red blood cells by antibodies. The neutralisation of virus inhibits agglutination. Virus neutralisation is a method where antibodies are added to a virus preparation and the infectivity of this preparation is then measured using indicator cells. A subset of antibodies produced against any given virus will have the ability to neutralize the infectivity of the virus. These antibodies have the ability to neutralize the infectivity of virus; it prevents or lowers the virus infectivity.
Complement fixation is a test that is used to detect the presence of either specific antibody or specific antigen in a patient’s serum. If the antibody is present in the patient’s serum, it binds to the antigen and complement reagent is completely consumed in the reaction. The antibody bound to cellà complement bindingàcomplement cascadeà cell lysis. The complement of cascade of molecules in blood serum initiated causing lysis of infected cell or pathogen.
Immunofluorescence is where antibodies or antigen are tagged with fluorescent dyes.
This method usually uses two sets of antibodies whereby a primary antibody is used against the antigen of interest and follow by a secondary, dye-coupled antibody is introduced which recognizes the primary antibody. Under immunofluorescene, there are direct fluorescent-antibody (FA) technique and indirect fluorescent-antibody (IFA) technique.
-The antibody attached specifically to antigen.
-The specimen is viewed under exciting light using fluorescence microscope.
Immunogold electron microscopy has the same principle as immunoflourscence. However, instead of staining the antibodies with fluorescent dyes, gold particles (nano sized) are attached to the antibodies. The specimen is then viewed under electron microscope to localise specific proteins or antigens.
Immunoblot and immunoprecipitation are methods that allow one to detect specific viral proteins in lysates from infected tissues or cells.
In immunoprecipitation, the antigen is radioactively labelled. The cells are then lysed and the labelled antigne are precipitated out of solution using a specific antibody. The antibody-antigen complex is then isolated for analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It is then detected through x ray film.
In immunoblot also known as western blog analysis, the proteins are not prelabeled with radioactivity instead the whole protein sample is subjected to SDS-PAGE, so as to separate out the proteins on the basis of their size and other physical properties. The proteins are then transfer to nitrocellulose membrane, the membrane can then be probed with specific antibodies follow up, the antibodies is labelled with sensitive indicator (e.g. horse radish peroxidise).
Enzyme-linked immunosorbent assay also known as ELISA is biochemical technique used mainly in immunology that used an enzyme to detect the building of antigen (Ag) and antibodies (Ab). The colourless substrate was converted to a coloured product by the enzyme and this indicates the presence of Ag: Ab binding.
Generally, there are 5 types of ELISA:
-Direct ELISA
-Indirect ELISA
-Sandwich ELISA
-Competitive ELISA
-Multiplex ELISA
By man hua.
8:05 PM
Hey guys,
We all know that DENGUE is a serious problem during the rainy seasons in many countries. We also know the culprit who is the DEADLY AEDES MOSQUITOES. But, how many of us know that birds are the natural host of dengue. I know guys.. I think we should inoculated all these birds first! They are such a delicacy and a pest at the same time. Anyways, dengue as well as yellow fever and West Nile fever are flaviviruses which fall in the virus family Flaviviridae.
The Flaviviridae family consists of enveloped viruses and they have a single linear positive sense RNA genome. The complete genome is about 9500-12500 nucleotides long. Many species of Flaviviridae can replicate in both mammalian and insect cells. They also can survive for long periods in hosts by replicating in the host itself.
Many people have been victims of this dengue virus and it is believed that approximately 40% of the world’s population (about 2.5 billion people) is at risk for infection; either because they live in endemic areas or in regions where the mosquito vector is present. Statistics by World Health Organisation (WHO) show that the number of people infected each year range from 50 million to 100 million. It is worrying situation as a vaccine is not found yet.
Fatal dengue includes dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS).
Dengue fever is a severe flu-like illness. Its symptoms include rash, very high fever (104 - 105 degrees Fahrenheit), severe headache, extreme muscle and joint pain, nausea, vomiting, and enlarged lymph nodes.
Dengue hemorrhagic fever is even more severe and a life-threatening disease which causes early symptoms that are very similar to those of dengue fever. They would be very high fever, headache, muscle and joint aches, malaise, decreased appetite, bleeding, hypotension and vomiting. After several days, the patient may undergo symptoms of irritability, restlessness, and excess perspiration. Further complications include liver enlargement and circulatory failure.
Dengue shock syndrome usually occurs after 2 to 6 days. Patients usually exhibit the symptoms of Dengue fever and Dengue haemorrhagic fever first. Later, they would suffer from sudden collapse, “clammy” extremities, decrease in pulse, and circumoral cyanosis (blueness around mouth). At this stage, mortality rate can reach as high as 30%.
There are four serotypes of dengue virus. Infection by a serotype provides future infections by the same serotype, but does not offer cross protection for infection from other serotypes. In fact, a second infection by a different serotype can cause a phenomenon known as “immune enhancement” which may lead to a more severe form of the disease from the second infection.
Control mesures would include putting up a mosquito screen, removing stagnant water and also spraying insecticides.
lOShiNi
10:33 AM
Cell culture is whereby animal or plant cells are removed from tissues, continue to grow if provided with the appropriate nutrients and conditions and this process is carried out in a laboratory.
Types of cells grown in culture
Cell cultures are derived from either primary tissue explants of cell suspensions. Primary cells are cells that are cultured directly from a subject. Most primary cell cultures have a finite life span in cultures. However, if after several sub culturing onto a fresh media, the cell line did not die and transform into a continuous cell line. Such transformed cell lines display many alterations from the primary cultures which include changes in morphology, chromosomal variation (e.g. polyploidy where there are more than two homologous sets of chromosomes),etc.
Trypsinisation: cells are cultivated in glass or treated plastic surface flask in a suitable growth medium. Such adherent cells multiply to form a confluent monolayer of cells on the growth surface. Trypsin, a proteolytic enzyme can be used to detached the cells from the surface and from one another. The detached cells are then transferred to new flasks for sub culturing of these cells.
Tissue culture methods for detection
Virus, by subverting its host to make more copies of itself and next, replicating itself into large numbers. In doing that, virus usually cause some changes to its infected host such as morphology of the cell changes, cells swelling or shrinking, etc. These changes are known as cytopathic effects (CPE).
Plaque assay is a technique based on one virus infects one cell and spreads to surrounding cells; the virus then killed the cells and thus forming plaques. This method requires that the virus infects a cell line that grows as a monolayer. There is higher accuracy when the concentration of virus sample is low therefore serial dilution of the virus sample is done.
2. If the concentration of virus is low enough, then one virus will infect one cell.
3. If this infected monolayer is grown under a gel, only neighbouring cells can be infected after a virus burst.
4. This will result in the formation of a visible circular plaque as more cells are infected.
By man hua.
Friday, January 30, 2009
6:41 PM
Methods of studying viruses Isolation and cultivation (I) - In vivo
Animals and eggs are the first method used for virus cultivation. Suitable living mammals are chosen for cultivation of viruses. Only healthy animal that are free from any communicable disease are selected. The specific virus then can be introduced into the healthy animals. Examples of animal selected for virus cultivation are chimpanzees, mouse, sheep and rabbits. The animals are then slaughtered at the end of incubation period and are washed thoroughly and viruses are obtained from them. However, this method are inconvenient, it also concerns the safety in handling animals and animal rights.
Animal viruses can be cultivated using chick embryo technique whereby fertile hen eggs are selected. The eggs must not be more than 12 days old.
Preparation of egg for virus cultivation:
1. The shell surface is disinfected with iodine
2. Shell surface penetrated with a small sterile drill
3. After inoculation, the drill hole is sealed with gelatine & the egg is incubated
4. Virus may be able to region.
Cultivation of plant viruses
The leaves of the plant are mechanically inoculated by rubbing with a mixture of viruses and an abrasive. The virus would directly ntact the plasma membrane and infect the exposed host cells when the cell walls are broken by the abrasive. The role of the abrasive is often done by insects that suck of crush plant leaves and thus transmit viruses.
Due to rapid death of cells in the infected area, a localized necrotic lesion would develop and even if lesions does not occurs, the infected plant may show symptoms like change in pigmentation, etc. A picture of a plant infected with tobacco mosaic virus is shown.
Thursday, January 29, 2009
2:35 PM
Just like how we go through different stages in life, viruses too undergo various stages in their life cycle. I am just wondering whether it is as challenging as a human’s life.
Stages of Virus Life Cycle
There are
6 stages in a virus life cycle.
1. Attachment
Attachment is a specific binding between viral surface proteins and their receptors on the host cellular surface. This specificity determines the host range of a virus. This means that the virus particle is only able to attach to the host cell if it has the appropriate attachment protein. If not, attachment would not take place. For instance, HIV attacks only human's immune cells (mainly T cells), because its surface protein can interact with ‘CD4’ and chemokine receptors on the T cell's surface.
2. Penetration / EntryFollowing attachment, viruses may enter the host cell through receptor mediated endocytosis or other mechanisms.
Enveloped viruses·
Entry by fusing with the plasma membraneSome enveloped viruses fuse directly into the plasma membrane. Thus, the internal components of the virion such as the genome are immediately delivered to the cytoplasm of the cell.
·
Entry via endosomes at the cell surface 
Some enveloped viruses require an acid pH for the fusion to occur as they are unable to fuse directly with the plasma membrane. These viruses are taken up by invagination of the membrane into endosomes. As the endosomes become acidified, the latent fusion activity of the virus proteins becomes activated by the fall in pH and the virion membrane fuses with the endosome membrane. This results in delivery of the internal components of the virus to the cytoplasm of the cell.
Non-enveloped viruses Non-enveloped viruses may cross the plasma membrane directly or may be taken up into endosomes. They then cross (or destroy) the endosomal membrane.
3. Uncoating
Uncoating is a process that viral capsid is degraded by viral enzymes or host enzymes. The viral genome then enters the cell.
4. Replication and Expression
A single virus particle is in and of itself essentially inert. It lacks the components needed to reproduce. Viruses are intracellular obligate parasites which mean that they cannot reproduce or express their genes without the help of a living cell.
Once a virus has infected a cell, it will "marshal" the cell's ribosomes, enzymes and much of the cellular machinery to reproduce. Viral reproduction produces many, many progeny that when complete, leave the host cell to infect other cells in the organism.
The exact nature of what happens after the host is infected varies depending on the nature of the virus. In most cases, the process depends on the form of the genome. The process for double-stranded DNA, single-stranded DNA, double-stranded RNA and single-stranded RNA will differ.
Virus binding to the cell wall
Virus injecting its genetic material
Virus genome replicates
5. Assembly
Assembly of virus particles occurs in the nucleus. DNA enters the particles after immature capsids are formed. The capsids then undergo a maturation process, after which the cells die and virus leaks out.
Virus components and enzymes continue to be produced
Components of the virus assemble
Virus enzyme breaks down the bacterial cell wall causing the bacterium to split open
6.Release
Viruses may escape from the host cell by causing cell lysis (death). Enveloped viruses (e.g., HIV) typically "bud" off the host cell. Budding viruses do not necessarily kill the cells. During the budding process, a virus acquires the phospholipid envelope containing the embedded viral glycoproteins. Also, not all released viral particles are infectious. The ratio of non-infectious to infectious particles varies with the virus growth conditions.
lOSHiNi
It usually affects the lips, blister form on the lips but may also erupt on the tongue.
Clinical features- Varicella zoster.jpg)
Replication and expression: once inside the host, the viral genome takes over the metabolism of the host and converting it to its own ends. The viral nucleic acid is then transcribed and translated and the various viral proteins proceed to create everything required to make new viruses.
Maturation and release: the various components accumulate inside the cell and start to SPOTANEOUSLY ASSEMBLY into new virus. However, this process is an orderly one so that each component is added in the proper sequence. Enzymes are produced causing the cell to burst or die thus a crop of new viruses are release into the environment.
Detection, identification and diagnosis of virus
The haemagglutination assay is a method used to measure virus particle. This assay takes advantage of the fact that many viruses contain the two spike proteins: Neuraminidase and Haemagglutinin that blinds specifically to the red blood cells. Examples of viral protein with ability to bind to red blood cells are influenza virus and other virus. 
The Flaviviridae family consists of enveloped viruses and they have a single linear positive sense RNA genome. The complete genome is about 9500-12500 nucleotides long. Many species of Flaviviridae can replicate in both mammalian and insect cells. They also can survive for long periods in hosts by replicating in the host itself.
Trypsinisation: cells are cultivated in glass or treated plastic surface flask in a suitable growth medium. Such adherent cells multiply to form a confluent monolayer of cells on the growth surface. Trypsin, a proteolytic enzyme can be used to detached the cells from the surface and from one another. The detached cells are then transferred to new flasks for sub culturing of these cells.
