|MadSci Network: Virology|
Your question is very timely. Yesterday I attended a symposium at my institute about the West Nile Fever outbreak that happened in New York City last summer and it was refreshing to hear the honest discussion of how the discovery of this pathogen actually took place. It normally takes some time to even realize that what you are seeing happening around you is an epidemic caused by a single pathogen. The West Nile Fever outbreak happened in people, crows and certain Zoo animals and many incorrect theories were created before the real viral pathogen was found. However, I will try to stay on task and deal with "an unknown pathogen in Brazil."
Ok, where do we start. Your phrase gives two important clues to a virologist. First the word pathogen means that animal(s) or human(s) were made sick and second that this happened in Brazil. The location and time of year where the disease breakes out can provide clues to the kind of virus and whether it uses a vector [insect or other animal] to spread, or whether it can spread from animal to man or man to man on its own. Those clues can be found in books on "geographic virology." You would not get a mosquito borne disease in winter, for example. The actual isolation and analysis of the possible kinds of virus depends on your getting a sample from a sick animal species.
While viral illnesses produce some signs and symptoms that are common to all viruses any unusual findings may help categorize what type of virus we are looking at. Viral diseases are also frequently "species specific" so knowing what animal species were affected could help in narrowing down the possibilities. When a pattern of disease occurrence and type of disease has developed during an epidemic it would help to involve a pathologist. He/she will dissect the animal and find what organs were harmed by the virus and may see microscopic changes in tissues that are sampled and made into slides. At the same time that the pathologist looks at tissues from animals or people who died from the disease caused by the pathogen, the virologist can grind up samples of these diseased tissues and use methods to isolate viruses from the tissues. Since viruses are very small, putting the slurry of tissue and media through a filter that removes particles larger than viruses and smaller than bacteria. The fluid that comes out can then be injected into experimental animals to see if they become ill, and fluid can be added to tissue culture cells that have grown on a surface to form a membrane.
If a virus is present, it may make the animals sick and reproduce the pattern of disease seen during the epidemic but that might not necessarily occur. Also, if a virus is present, it may cause invidual tissue culture cells to die and create holes in the confluent membrane of cells. These holes are calle plaques. Finding that disease is caused by a "filterable" agent, and/or causes plaques in tissue culture are classical methods for proving that a virus is involved but it does not say what kind of virus it is.
Viruses are of various sizes and shapes when examined by electron microscopy. Some of these sizes and shapes are useful in classifying the virus into groups. For example, ebola virus is a "filovirus" because it is long and thin and may look to some people like a strand of hair or a filament. The arrangement of proteins in a viruse's envelope creates certain symetrical shapes that an electron microscopist can use to further characterize it into groups.
These two methods are very dependent on the ability to grow the virus in small animals or in tissue culture cells. Sometimes virologists have to repeatedly inoculate the sample into animals or tissue culture cells to "adapt" the virus to grow in these respecive environments so he/she can have enough virus to analyze but electron microscopy.
Supposing the epidemic didn't kill anyone or any animals but just made them sick. If the animal has just gotten sick, it might be possible to isolate a virus out of the blood or secretions of the animal. Or, if the animal is sick in a particular way such as circling around, dragging his feet and staring peculiarly, you might want to humanely kill the animal and try to isolate the virus from the brain and spinal cord. This is exactly what is done if someone suspects an animal has the virus that causes Rabies.
Going back to the idea that the pathogen might let some animals live, allows yet another powerful method to be used. Most viruses are antigenic and will cause the immune system to produce antibodies against it. This might even occur when the virus is so pathogenic that it kills the host but there is enough time for antibodies to begin to appear just before death. It takes between 3 and 7 days for antibodies to start to appear.
If you take serum from infected animals and test whether the animals serum can prevent plaque formation in tissue culture cells that you have infected with a selection of "likely" viral pathogens that are known to be active in Brazil, you might find one that is neutralized by the animal's serum (you would say the serum neutralized the virus if less than the expected number of plaques form in the presence of antibody.). If you successfully isolated an "unknown" virus that causes plaques in tissue culture cells, you can take a collection of known convalescent serums for viruses you think might be involved and see if one of those sera neutralize plaques caused by the test virus. These immunological methods are not as specific as it might seem because some of the antibodies may "crossreact" within all the strains of a virus group, or among certain related strains but not others. The more virus specific sera you use in a test matrix, the better chance you have to narrow down which viruses are present.
Eventually, by repeating these steps and testing in experimental animals to see if the virus you have isolated recreates the original disease pathology, you will have identified it.
Today, the process is getting much quicker. The relative ease of isolating DNA and RNA from disease producing viruses, and sequencing the genes that are involved in important steps of viral antigen production or are involved in viral infectious process, has given us many highly specific tools for rapidly identifying pathogens. The use of a recently developed technique called the polymerase chain reaction (PCR) enables us to quickly generate replicates of viral genes and produce sequences that can be compared with known sequences as a way of identifying the virus. This is an oversimplified way of looking at it but the process now allows identification of viral unkowns in a period of hours or days that used to take days and weeks using the above classical methods. Another benefit of PCR - based viral diagnosis is that it is not necessary to succeed in the virus isolation and growth of virus in animals or cells to proceed. The viral genes are all replicated in the PCR device, no living cells are needed. The "primers" used to amplify genes in a PCR device are specific. The sequence of base pairs in the primers can be selected from published gene sequences for strains of virus you may want to test against your unknown. Only if some homology [match of the nucleotide base pair sequences of the DNA/RNA] occurs, will there be replication of DNA/RNA. Then if you get replication you can use more and more specific primers to narrow down the group, species and strain of the virus until the unknown is known. It is even possible to quickly identify viruses that have never before been seen using DNA/RNA PCR methods.
Try the links in the MadSci Library for more information on Virology.