Sally E. Drury
Avian Virology, VLA Weybridge, Woodham Lane, New Haw. Addlestone. Surrey. KT15 3NB
Presented at the Northern Symposium
October 2nd 1999
WHAT IS A VIRUS AND HOW IS IT STRUCTURED?
Viruses are the smallest of the parasites living within cells (intracellular). Viruses do not replicate by dividing their component parts and then splitting into two (binary fission) as bacteria do. They have no energy generating system and do not possess many of the enzymes necessary for making nucleic acid. Furthermore, they do not have the means to translate genetic information into protein. Viruses can therefore only replicate by using the material and machinery provided by their host cell.
The simplest viruses consist of just nucleic acid and protein. The larger viruses have a more complex structure and consist of carbohydrate and lipid material as well.
A virus only ever has one type of nucleic acid; either RNA or DNA, but never both. It is here that the genetic information of the virus is contained.
The nucleic acid, or genome of a virus is large enough to code for between 5 to 100 proteins. These are structural proteins and viral enzymes. The structural proteins are arranged geometrically around the genome, forming the capsid. The capsid protects the genome. It is also these capsid proteins which enable the virus to attach to host cells. Some viruses have an additional envelope which surrounds the entire nucleocapsid.
Viruses are so small that they cannot be seen under the ordinary light microscope. The smallest viruses are about 20 nanometers (e.g. circovirus and parvovirus) and the largest about 250 nanometers (e.g. poxvirus) in diameter. The lower limit of the light microscope is 1micron, which is 1000 nm. Therefore the light microscope is not powerful enough to make virus particles visible.
So the electron microscope is necessary to visualise virus particles. This microscope is powerful enough to actually show the morphology, or structure of individual particles. It is used as a rapid and simple diagnostic tool for virus detection in many different sample materials. It is also frequently used to confirm the identity of a virus which has been isolated in cell culture. In order to see a virus particle clearly enough to identify it, a magnification of at least 21,000 times will be necessary.
Because viruses can only replicate in living cells, the development of cell culture techniques has been an enormous step forward in enabling viruses to be grown outside of the host animal. This means that studies and tests can be carried out without the need to use laboratory animals.
Cell cultures are used routinely as a viral diagnostic tool. By looking for a cell damaging (cytopathic) effect in cell cultures, which have been deliberately infected with sample material, the presence or absence of virus can be established. Different viruses cause different and typical cytopathic effects and so their presence can be recognised.
The cells of all organisms can be infected by viruses:- mammals, birds, reptiles, amphibians, fish, insects, plants, fungi, and even bacteria. Viruses infect living cells and then use the host's cellular machinery for their own replication. This often takes place at the expense of the host cell's metabolism. One single virus particle can initiate a thousand (103) to a hundred thousand (105) progeny virus particles in the same cell. Following viral replication, particles are released from the cell in a variety of ways. Virulence is the deciding factor in the ability of a virus to cause disease. The more virulent a particular virus, the more able it is to cause widespread cellular damage and thus symptoms of disease or even death in the affected animal. Symptoms may also be caused by the animal's immune response to invasion by the virus or by the formation of tumours following cell transformation.
HOW DO VIRUSES REPLICATE?
It is useful to understand how viruses get into cells and how they are released from them following replication. Sometimes the cells may be killed as a result. Some viruses may cause cells to be transformed and become malignant. Knowing the principles of the replication cycle allows us to gain insight into stages at which viruses might be vulnerable to attack by chemotherapeutic agents.
The first stage of development is attachment of the virus to the cell surface. This requires surface capsid proteins, or envelope spikes in the case of the more complex enveloped viruses. Infection follows viral attachment to a specific receptor site on the cell surface. This explains the specificity of viruses to a particular host range only. Some viruses infect only one species, because the specific receptor sites to which they attach are only found on the cells of one species. An example of this type of virus would be feline panleukopenia virus, because receptors only occur on feline cells. The opposite of this would be a virus such as rabies, which can attach to the nerve cells of all mammals, because the specific receptor site is found on the neural cells of all mammals.
The next stage is penetration of the virus into the cell. This happens by adsorption and the virus is incorporated into vesicles in the cell's cytoplasm. There are some viruses, and herpesvirus is one of them, that are able to fuse their envelope with the cell membrane and then release nucleocapsid into the cytoplasm that way.
Once inside the cell, the next process, known as uncoating, involves the viral nucleic acid being released from protecting capsid protein into the cell's cytoplasm. At this stage the genetic material of the virus lies unprotected (and vulnerable to attack!) within the cell. Viral synthesis begins as the virus now takes over the cell's machinery and instructs it to make virus protein and new viral nucleic acid. The new nucleic acid associates with the capsid protein and new nucleocapsid is formed. Enveloped viruses (like herpesvirus) additionally cause viral envelope glycoprotein to be inserted into the host cell membrane. Assembly of the nucleocapsid takes place at the same site where the new nucleic acid has been formed. For most of the DNA viruses (like herpesvirus) this is the cell nucleus. For most RNA viruses it will be the cell cytoplasm. Enveloped viruses release their particles by a process known as budding. It is during this budding outwards through the cell nuclear membrane or plasma membrane that the nucleocapsid becomes enveloped in the viral glycoprotein inserted there during the synthesis stage.
HOW DO VIRUSES DAMAGE CELLS?
There are three main ways in which cells of the host animal may be affected by a virus infection.
- Cytopathic effect (CPE)
As the virus multiplies in the host cells, the morphology, or shape of these cells is altered. Finally, this rapid multiplication of virus leads to the death of the affected cells. Some common visible effects are:
- Rounding-up Of cells and formation of cell aggregates e.g. adenovirus.
- Rounding-up followed by shrinking of cells e.g. picornavirus.
- Fusing of cells into syncytia (giant, multinuclear cells) e.g. herpes-, or paramyxovirus.
The changes occur due to the effect of viruses either on the cellular macromolecules or on the cell membranes.
- Cell transformation.
Oncogenic (cancer causing) DNA viruses produce a transforming early protein which binds to the host cell DNA and causes cell division to occur in an uncontrolled way. Other oncogenic viruses either contain an oncogene in their own genome or are responsible for activating a cellular oncogene once they have become integrated into the cell. Either way, as with the DNA viruses, a transforming protein is produced and this initiates an enzyme cascade towards uncontrolled clonal cell division.
- No obvious effect.
In some cases the virus replicates without damaging or altering the cell overtly. All the cells are infected but continue to divide. In this way the virus is continually replicated as well. (e.g. leukaemia viruses) This is known as a steady state infection. This type of virus replication causes a confusing picture of healthy looking cells which are, nevertheless, infected.
Sometimes a minority of cells is infected and so any obvious cpe is transient. This occurs in the case of feline panleukopaenia virus which only replicates when host cells are in the DNA synthesis phase. In other words, only when the cells are in a particular phase of their growth will the virus be able to replicate. Cell cultures supporting this kind of infection are known as carrier cultures. So-called latent infections also occur, when viral nucleic acid is integrated into the host cell DNA but there is no synthesis of virus. (e.g. herpesviruses and retroviruses) When such cells are cultured in the laboratory, the virus may be re-expressed after several *passages and signs of the infection will then be noted. (Stress conditions in an infected animal will also cause reactivation of the virus. No lesions will be caused, but virus will be excreted.)
*One passage describes the serial transfer of virus from one cell culture in which it has been replicating to a new cell culture. Each passage will have a standard number of days duration.
The herpesviruses are enveloped, icosahedral viruses with a double stranded DNA nucleoid.
The capsid surrounding the nucleoid, is made up of 162 capsomeres. and is closely surrounded by another layer of protein known as tegument.
Enveloped, the herpesviruses measure 180 - 200nm in diameter. The envelope contains essential antigenic glycoproteins.
An unenveloped particle can measure between 70 - 100nm.
Only virions with both nucleoid and envelope are considered infectious.
They are divided into 3 sub-families:
|Alpha||these grow in a wide range of cells and are characterised by their rapid growth. They most commonly infect and produce lesions in the respiratory tract.|
|Beta||these are species specific, slow growing and usually cell associated. They can be characterised by the enlargement of infected cells (syncytia), giving them their other name of cytomegaloviruses.|
|Gamma||these are known to cause lymphoproliferative changes, being usually highly cell associated. (Their association is predominantly with lymphocytes.)|
Many different species are subject to herpesvirus infections, including horse cattle, pig, sheep and goats, various bird species, reptiles, dogs, cats and man.
Cultivation and cpe of the herpesviruses.
The alpha and gamma viruses can be cultivated in cells of more than one species, but the beta sub-family are species specific and will only produce infectious virus in cells of their own species.
Because viral capsid production takes place in the nucleus of the infected cell, this leads in time to the production of intra-nuclear inclusion bodies. Hence the typical cpe of rounded up cells with intra-nuclear inclusion bodies.
The use of some cell culture systems will also lead to syncytial (giant) cell formation.
HERPESVIRUSES DETECTED IN TORTOISES AT VLA WEYBRIDGE.
In October 1996, tortoise tissue samples were being received from the owner of a breeding colony of Leopard and Hermann's tortoises in South Yorkshire. The colony was experiencing an outbreak of lymphoproliferative disease. This was associated with clinical signs of nasal and ocular discharge. Pancreatitis and tracheitis were also evident. The eventual outcome was 100% mortality.
Using electron microscopy, herpesviru s particles were detected in both liver and spleen samples.
The average diameter of the virus particles seen was 105nm. The typical surface structure, morphology and icosahedral symmetry of herpesviruses were all clearly demonstrated.
The next challenge was to attempt isolation in order to be able to study and characterise the virus.
It is important to discover as much as possible about individual isolates of virus and to compare these with each other in order to understand more about the origin and spread of disease. (Epidemiology) Preventative measures can then be taken, thus limiting spread of the disease and maintaining healthy, disease-free breeding colonies.
We investigated several different types of cell culture in order to find the best system for isolation of the tortoise herpesvirus. Herpesviruses generally replicate better in epithelial cells rather than fibroblastic cells. We tried both types however, from a variety of sources:- three different species of tortoise embryo fibroblasts, chick embryo fibroblasts, chick embryo liver, and a commercial line of fat head minnow cells. The inoculated cells were incubated for 7 days for each of 3 passages. Incubation took place initially at 31°C
No visible cytopathic effects were noted and no viruses were isolated from any of the cells used.
SUCCESS AT LAST.
In 1998, tissues from a case of necrotic stomatitis in a Spur-thighed tortoise were received. The tortoise was about 70 years old, was weak and depressed and gasping for air. There was a bilateral mucoid discharge and widespread oedema of the subcutaneous tissue of the head and neck. This led to the neck being out-stretched and the eyelids closed. In addition there was a yellow necrotic plaque visible at the back of the throat on one side. Impression smears taken from the oral mucosa were stained and examined. These revealed a large number of intranuclear inclusion bodies consistent with a possible herpesvirus infection. The tortoise was euthanased following a rapid deterioration of the necrotic stomatitis. Tissue samples were sent to us for examination by electron microscopy and attempted virus isolation.
Using direct electron microscopy, herpesvirus particles were detected in liver and spleen tissues.
We obtained a turtle heart cell line, known as TH- 1, from the European Cell Culture Collection. This provided us with a culture of epithelial cells, which had already proven useful for the isolation of herpesviruses of tortoises in other laboratories.
We increased the time allowed for virus adsorption onto the cell sheet from 60 minutes to 2.5 hours and we increased the total incubation period from 7 to 12 or 14 days for each of the three passages. The incubation temperature was decreased from 31°C to 28’C.
Observation on a daily basis revealed cpe at early passage levels with all the tissues from the Spur-thighed tortoise. The cpe presented as discrete foci of refractile cells which later degenerated forming holes in the cell sheet. This is typical of the cpe seen with many herpesviruses.
We were able to confirm the identity of the cytopathic agent by examining the culture material by electron microscopy.
By a series of passages of the infective material, we were also able to adapt the agent to growth on our own cultures of spur thighed tortoise fibroblast cells.
CURRENT WORK AND THE FUTURE.
We are continuing to refine our cell culture techniques and looking for the ideal system to maximise our chances of isolating virus. Currently we are looking to purchase equipment to enable open systems of culture to be utilised. Both cell cultures and viruses require a certain pH level for growth and maintaining that level in open systems is a problem unless change of pH can be compensated for. This means a specially adapted incubator.
There are 4 new virus isolates, all from leopard tortoises, which have yet to be confirmed in their identity. It can be the case that virus replicates in cell culture, causing cell death so rapidly that very few particles are actually present within that culture. When we come to look on the EM for particles, there are insufficient. The identity of the cytopathic agent remains a mystery until we are able to increase the titre (or level) of that virus in the cell culture. The method for doing this is by passaging the virus and this takes time since each passage is approximately 14 days long!
Chickens have been inoculated intravenously with our first herpesvirus isolate in an attempt to produce antibody to this isolate. Six 10 week old chicks were given 0. 1 ml each of infected cell culture fluid. These will be boosted with a second dose after 2 weeks and a test blood sample taken 4 weeks after inoculation to see whether antibodies are present. We hope by this means to develop a serum neutralisation test which we will be able to offer as a useful diagnostic test for the future. With this test in place, it will be possible to screen tortoises prior to sale or introduction to an established colony for example, simply by taking a blood sample and testing it for antibodies to herpesvirus. In this way we hope to be able to prevent the spread of disease.
Testudo Volume Five Number Two 1999