In a typical infectious disease, the three main factors are host, agent and environment. The vector acts like a transporter in between all the three factors.
The general virus replication cycle for animal viruses involves 6 stages. The attachment, penetration, uncoating, replication and expression, maturation and release.
http://www.jbc.org/content/281/13/8305.full.pdf
Viruses have two types of replication cycle. The lytic and lysogenic cycles. Viruses that undergo lytic cycle will replicate and released out of the infected cell and infect other healthy cells. However, viruses that undergoes the lysogenic cycle will replicates but may or may not be released out of the infected cell.
Taken from Mr William How lecture notes: Virus-Host Interaction
In the attachment stage for the animal virus, the attachment sites of the virus, which are typically made up of proteins, bind to the receptor on the host cells. The receptor of the host cells is typically made of maybe protein, glycoprotein or glycolipid and they are specific during attachment.
In the second stage of the replication cycle is the penetration stage. There are three ways of penetration that occurs in the eukaryotic cells. They are the receptor-mediated endocytosis, clathrin-mediated endocytosis and fusion. Receptor-mediated endocytosis is that the virus will binds with a receptor before entering the host cell. The clathrin-mediated endocytosis is also known as the naked virus endocytosis is that the virus without its envelope will enter the host cell by endocytosis. Fusion is the direct penetration of the enveloped virus into the host cell.
The third stage is the uncoating. Uncoating is the separation of the nucleic acid from its protein coat. It is the uncoating of the genetic material of the virus. In the fourth stage, the virus will express its genetic material (i.e. translation) and replicate the genetic material.
The fifth stage of the replication cycle is the maturation. In maturation, the protein caspid is assembled.
The last stage is the release of the virus. For enveloped viruses, the release of the virus is via budding. (i.e. a lipid envelope is formed around the caspid.) For non-enveloped virus, the release of the virus is via membrane rupture. (i.e. the membrane of the host cell burst)
In the virus growth curve, in particular to the latent stage, there is a fall of concentration of virus as there are no virus particles during replication of the virus. Also the virus is unable to be detected in the external medium unless the virus is released. The new replicated virus is assembled and released out of the host cell to infect other cells when the host cell burst.
Normal microflora (i.e. microbes) can be found in the surface tissues such as the skin, eyes and mouth, of a healthy individual but not in the internal tissues such as blood, brain and muscle.
Between the virus and the human, when one organism benefit while the other is unaffected, it is called commensalism. When both organisms benefit, it is called mutualism. When one organism benefit while the other is harmed, it is called parasitism.
Taken from Mr William How lecture notes: Virus-Host Interaction
There are 4 major portals of entry for virus to infect humans. They are the skin, mucous membrane, placenta and the parenteral route. Although the skin is a barrier to most pathogen, some can still enter via hair follicles, sweat glands, cuts and bruises.
The mucous membranes are the respiratory, gastrointestinal, urinary, reproductive and the eyes. They are thin, moist, warm and cells are living. Pathogens can cross the placenta and infect the foetus. The pathogens enters the host via parenteral route such as nail and needle punctures, bites, cuts, stab wounds and surgery.
The virus that enters into a host will also leave the host to infect other people. The portals of exit are generally same as the portal of entry. The pathogens either leave the host via secretion or excretion.
The pathogenicity is the ability of microbes to cause disease. The degree of the pathogenicity is the virulence. Some of the virulence factors are the adhesion, host evasion, latency and high mutability.
There are a few stages involved in the infectious disease in a host. They are the incubation, prodormal, illness, decline and covalescence. In the iceberg concept of infection, severe symptoms of an infection are surfaced. Situations such as mild symptoms, infection with no symptoms and exposure with no infection, are not surfaced out.
Viruses that leaves the human body (host) cannot survive long, thus the places where the viruses are maintained is called the reservoirs of infection. The three types of reservoirs are animal, human and non-living.
Diseases that are spread from animal hosts to human host are zoonotic diseases. Human host can be infected from direct contact with animals and its faeces, consuming animals and via vectors such as mosquitoes and fleas.
Human host can be infected via the human reservoirs such as infectious diseases that are contagious or via sharing of food (i.e. saliva) or sneezing and coughing.
Examples of non-living reservoirs are the soil, water and food. Virus that is in the food or water (contaminated food and water) is ingested into the human host. Thus, humans are infected with the infectious diseases.
In order for us to study viruses, we will have to know the methods of studying viruses. There are different types and ways of studying the viruses.
Embryonated eggs, Animals, Cell Culture and Plants are some ways isolation and cultivation of viruses.
Physical methods, Serological and Molecular Biology methods are some ways for detection, identification and diagnosis of virus cells.
For isolation and cultivation of viruses,
The first way is to use the Embryonated eggs. Embryonated eggs are also called chick embryos. They are
(i) Convenient
(ii) Inexpensive for many animal viruses.
Some observations of the viral growth in the embryos are
(i) Death of embryos
(ii) Embryo cell damage
(iii) Formation of pocks (or lesions) on egg membranes.
Embryonated eggs are still used for growing viruses for vaccines. There are 4 different places in the embryonated eggs that the virus can be injected. They are the chorioallantonic membrane, aminotic, allantonic and the yolk sac.
The second way of isolation and cultivation is using animals. Although using animals for isolation and cultivation is expensive (cost-ineffective), it is still used of rare viruses. Infected live animals are used for studying the human immune system’s response to the virus. The live animals must be healthy, free from diseases and slaughtered to harvest virus. Infection in live animals is specific to each animal.
Cell culture is the third way of isolation and cultivation. It is a preferred method compared to the embryonated eggs as it is more convenient to handle. Cells are grown in-vitro (in a stimulated environment – in culture medium in flask). Primary and continuous cell lines can be used for the isolation and cultivation of virus. Some viruses cannot grow in-vitro.
Vaccine actually contain weaken form of virus. Which help you become immune to that species of virus.
However the weaken virus can cause original diseases to people with weak immunity.
The only way to get rid of viruses is antiviral drugs and your immunity system. However antiviral drugss have side effect. Viruses are immune to antibiotics.
That is why doctors give you medicine to cure the symptoms which is cause by the virus and say that the only cure is to let nature take it's course.
The treatment of virus such as influenza will usually involve:
* Drinking plenty of water.
* Staying at home. People who go to work or school in this condition not only risk spreading the virus to their colleagues, but also run a higher risk of catching a bacterial infection.
* Taking a painkiller such as paracetamol (eg Panadol) or ibuprofen (eg Nurofen) to bring your temperature down.
* Vaccines have been developed against most viral diseases.The vaccine gives the body some help in quickly and effectively fighting the virus.
Did you know that?
There are about 1 million viruses in a teaspoon of sea water. Microorganisms constitute more than 90% of the biomass in the sea.
Viruses kills about 20% of this biomass each day and there are 15 times as many viruses in the ocean as there are bacteria and achaea.
Viruses are indirectly responsible for reducing the amount of carbon dioxide in the atmosphere by approximately 3 gigatons of carbon per year.
Role in Evolution.
Viruse are an important natural means of transporting genes between different species, which increases general diversity and drives evolution.
Viruses can be used as weapons.
An example is:
Successful recreation of the infamous 1918 influenza virus in a laboratory.
Primary and Secondary Cultures
Primary cultures are maintained by changing the fluid 2 or 3 times a week. When the cultures become too crowded, the cells are detached from the vessel wall by either trypsin or EDTA, and portions are used to initiate secondary cultures. In both primary and secondary cultures, the cells retain some of the characteristics of the tissue from which they are derived.
Cell Cultures
Cell cultures are separated into 3 types:-
1. Primary cells - prepared directly from animal or human tissues and can be subcultured only once or twice e.g. primary monkey or baboon kidney
2. Semi-continuous diploid cells - which are derived from human fetal tissue and can be subcultured 20 to 50 times e.g. human diploid fibroblasts such as MRC-5
3. Continuous cells - derived from tumours of human or animal tissue e.g. Vero, Hep2
Cell cultures vary greatly in their susceptibility to different viruses. It is of utmost importance that the most sensitive cell cultures are used for a particular suspected virus. Specimens for cell culture should be transported to the laboratory as soon as possible upon being taken. Swabs should be put in a vial containing virus transport medium. Bodily fluids and tissues should be placed in a sterile container.
Upon receipt, the specimen is inoculated into several different types of cell culture depending on the nature of the specimen and the clinical presentation. The maintenance media should be changed after 1 hour or if that is not practicable, the next morning. The inoculated tubes should be incubated at 35-37°C in a rotating drum. Rotation is optimal for the isolation of respiratory viruses and result in an earlier appearance of the CPE for many viruses. If stationary tubes are used, it is critical that the culture tubes be positioned so that the cell monolayer is bathed in nutrient medium.
The inoculated tubes should be read at least every other day for the presence of cytopathic effect. Certain specimens, such as urine and faeces, may be toxic to cell cultures that may produce a CPE-like effect. If toxic effects are extensive, it may be necessary to passage the inoculated cells. Cell cultures that are contaminated by bacteria should either be put up again or passed through a bacterial filter. Cell cultures should be kept for at least one to two weeks (longer in the case of CMV). Cell cultures should be refed with fresh maintenance medium at regular intervals or if required should the culture medium become too acidic or alkaline. When CPE is seen, it may be advisable to passage infected culture fluid into a fresh culture of the same cell type. For cell-associated viruses such as CMV and VZV, it is necessary to trypsinize and passage intact infected cells. Other viruses such as adenovirus can be subcultured after freezing and thawing infected cells.
http://virology-online.com/viruses/CPE.jpg
http://virology-online.com/viruses/vzv-cpe.gif
Cytopathic effects of enterovirus 71, HSV, and CMV in cell culture: note the ballooning of cells. (Linda Stannard, University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)
Cytopathic effects of mumps and measles viruses in cell culture: note the formation of syncytia. (Courtesy of Linda Stannard, University of Cape Town, S.A.)
Influenza and parainfluenza viruses do not ordinarily induce CPE, however they possess haemagglutinins and thus the ability to absorb guinea pig RBCs as they bud from the cell. This phenomenon is known as haemadsorption. Commonly employed cell cultures include primary monkey kidney, LLC-MK2 and MDCK cells. The cell cultures are incubated with a suspension of guinea pig RBCs at 4oC or RT for 30 minutes. The unabsorbed RBCs are then removed and the cell sheet observed microscopically for the presence of haemadsorption. Presumptive identification of virus isolates can usually be made on the basis of the type of CPE, haemadsorption, and selective cell culture susceptibility. For final identification, immunofluorescence, neutralization, haemadsorption inhibition, electron microscopy, or molecular tests are normally carried out.
Advantages of cell culture for virus diagnosis include relative ease, broad spectrum and sensitivity. It is limited by the difficulty in maintaining cell cultures, variability of cell cultures. Contamination by endogenous viral agents such as SV40, mycoplasma and bacteria may occur. Another problem in isolating certain viruses, especially myxo and paramyxo viruses is the presence of inhibitory substances or antibodies in the calf serum used in the cell culture media. Using fetal calf serum reduces this problem but adds to the expense.
Reference from http://virology-online.com/general/Test1.htm#VIRUS_ISOLATION
The cytopathic effect (CPE) is the cell deterioration. The difference in appearance of the cells will depend on the particular virus and type of culture cells.
The other way of detecting the viral growth is via the plaque assay. Cell death is observed in infected cell culture in a form of plaque. Plaque assay is useful samples with very low virus counts (such as food and water samples). One virus on the monolayer will infect one cell and produce one plaque. However, only viable virus is counted (live cells that are capable of multiplying). Also, plaque assay only works for viruses that infect monolayer cells and works for viruses that cause cell lysis.
The last method of isolation and cultivation of virus is using plants. However, using plants is an expensive method. There will be a reduction of quality and quantity of crops. The plaques formed on the leaves will determine the virus titers / numbers (concentration).
For detection, identification and diagnosis of viruses,
The first way is the physical methods. They are the X-ray crystallography, electron microscopy and ultracentrifugation.
The second way is the serological / immunological methods. There are 4 serological methods. They are the haemagglutination (HA), haemagglutination inhibition (HI), Immunoblot (also known as the Western Blot) and the Enzyme Linked ImmunoSorbent Assay (ELISA).
The detection of viruses using the first serological method, haemagglutination, is that the virus binds specifically to the red blood cells and causing the agglutination or the clumping of cells.
The detection of viruses using the second serological method, haemagglutination inhibition, is that the antibody produced in the body’s immune system neutralizes the virus and inhibits agglutination.
The detection of viruses using the third serological method is the Western Blot. A whole protein sample run through sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the sample is transfer to nitrocellulose membrane. The antibody in the protein sample is labelled with the sensitive indicator and this indicator converts the substrate to a colour and the results is captured on X-ray film.
The detection of viruses using the fourth serological method is the ELISA. ELISA is an antibody – antigen reaction which detects the presence of antibody and antigen. The antibody in the protein sample is labelled with the sensitive indicator and this indicator converts the substrate to a colour. In order for detection, it requires an antibody or an antigen to be attached to a solid surface.
The third way is the analysis of the viral genome using the polymerase chain reaction (PCR), northern and southern blot. The SDS-PAGE and western blot, protein sequencing and the X-ray crystallography are used for the analysis of viral proteins.
The PCR generates copies of specific DNA. The DNA strand is heated to 95C, breaking apart the two strands of the DNA double helix. The temperature is cooled to 55C, and primers are added to the mixture. The primers designate the boundaries of the DNA strand being duplicated. Polymerase is added to the mixture and the temperature is increased. Polymerase functions to add the corresponding nucleotide bases to the DNA strand. The result is two identical copies of the DNA strand. The process is repeated, each time doubling the number of copies of the DNA strand in the mixture.
The Northern Blot is used for analysis of RNA and the Southern Blot is used for analysis of DNA. Both Northern and Southern Blot has the similar procedure. The DNA that is prepared is cut with restriction enzymes and loaded onto the gel. The DNA undergoes gel electrophoresis, and DNA appears like a smear after the gel electrophoresis. The gel is then transferred to a nylon by capillary action for blotting. The nylon with the transferred DNA will undergo autoradiography to identify specific fragments of DNA.
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Eugene W.Nester
Denise G. Anderson
C.Evans Roberts,Jr
Martha T.Nester
McGraw Hill Companies
Principle of Virology: Molecularbiology, Pathogenesis, and Control
by S.J. FLint
L.W Enquist
R.M Krug
V.R. Racaniello
A.M. Skalka
American Society for Microbiology (ASM) Press
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Gerard J.Tortora
Berdell R. Funke
Christine L.Case
Daryl Fox Publisher.
