Isolation and Identification of Viruses

virions. The infection can become systemic if it gets into the plant’s vascular system. The cycle starts over when the virus infects a new plant host.

There are certain features of the viral growth curve. It starts with an inoculation phase, prior to the penetration of virus particles. During the eclipse phase, viruses have penetrated the cells and there are no free virions. Then there is a burst phase, when virus particles are released. The burst size becomes the number of virions released after the burst phase. The viral titer is the concentration of viral particles. The culture declines if there are no bacteria to penetrate anymore.

ISOLATION AND IDENTIFICATION OF VIRUSES

Because viruses require a host, it can be difficult to culture them without a host cell. They are allowed to infect the host and are harvested after separating them from host cells after they’ve been infected and after the virus particles have been allowed to replicate. A filter is used to allow viruses but not bacteria to filter into the filtrate.

Viruses can be grown within a living organism, in which it is called in vivo. If they are grown in vitro, it is done inside a test tube or on an agar plate. Bacteriophages are cultured in a culture or plate of bacteria, called a bacterial lawn. There will be a clear zone or plaque where the bacteria have died off on the agar plate.

Animals will grow viruses in vivo. It can be done to identify certain pathogenic viruses, to help produce vaccines, and in research settings. An embryo from a chicken is often used for these types of settings. Remember that viruses have tissue tropism so they must be grown in the presence of specific tissues.

Tissue cultures are used to grow certain viruses. A commonly used immortal cell line tissue culture is the HeLa cell line, which was isolated in the middle of the twentieth century from a woman who had cervical cancer. This cell line provides consistency in growing human cells in a culture medium. These are used to grow certain animal viruses.

Samples of viruses can be prepared in an infected cell line, embryo, or whole host. There will be certain cytopathic effects from the infection, which involve changes in the

cells’ features or adherence to the culture medium surface. There can also be cell lysis as a result of the infection. The kind of cytopathic effect depends on what virus is being studied. Researchers look for specific cytopathic effects in the cell culture to identify the virus being studied.

The hemagglutinin assay is one way of determining if a virus is present in a patient’s blood or serum. The serum or the liquid component of blood is isolated and mixed with blood. If the virus is present in the serum, there will be agglutination or clumping of red blood cells after the serum and test blood cells are mixed. It can be seen with the naked

eye.

In most cases, indirect hemagglutination is used, which involves the ability not to detect the viruses themselves but to detect antibodies that are directed at the viruses. Antibodies are mixed with the serum, binding to virus particles. When this happens, no hemagglutination can occur. This is called a hemagglutinin inhibition assay.

In the nucleic acid amplification test or NAAT, specific nucleic acid sequences of the viral particles are detected. An example of this is the polymerase chain reaction. Many copies of the viral DNA segments are gotten and the nucleic acid segments are detected. Another similar test is the reverse transcriptase PCR test. Reverse transcriptase is an enzyme used to make a DNA sequence from the RNA present in the virus particle. This allows for amplification of the DNA segments and analysis of the presence of the virus.

In an enzyme immunoassay or EIA, antibodies will attach to antigens. We will talk more about this process later. There is an enzyme attached to the antibody that can cause a specific reaction to occur. The enzymatic reaction that will then happen after the antigen and antibody have attached leads to a colored end-product. The color will then be detected.