Star Team

Hi, I'm currently working in the tissue culture and viral isolation section of virology lab. The virology lab consists of tissue culture, viral isolation, immunofluorescence and HIV labs. Here, I'll be using the example of HSV to describe the principle of viral isolation.

Isolation of the Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)

The diagnosis of the HSV-1 & 2 can be made from the isolation of the virus from lesions found in patient, by growing in cell culture. Swabs from the penile, vaginal or oral lesions are commonly used. The swabs are sent in Hank's transport media to the laboratory, so that the viruses remain alive, in order for us to isolate them. Different viruses grow best in different cell lines. For example, the dengue virus is sensitive to C6/36 mosquito cell line, but not in mammalian cell lines.

For HSV-1 & 2, it is most sensitive to cell lines from Mink Lung (ML) and Human Embryonic Lung (HEL). Basically, the sample will be inoculated into healthy ML and HEL cell line and incubated in 36°C and observed for cytopathic effect (CPE) daily. Healthy cell line refers to the absence of cell degeneration and cell line with around 70% confluency, when viewed under light microscope is used. This can be vague at times, but with enough experience one should be able to gauge the confluency of the cell line. Uninoculated tube of the same batch of cell line for ML and HEL will be incubated, which acts as a control. This allows us to decide whether any morphological changes in the inoculated cell are due to viral infection or degeneration of cell line. Typically, CPE for HSV infected cells occurs within 7 days after inoculation, after which the virus is identified by fluorescent-antibody staining of the infected cells. However, some slow-growing viruses, such as enterovirus and measles would require a longer time to show CPE, 21 days and 14 days respectively.

CPE is a result of virus infection of cell, which leads to many changes to host cells. Some changes include, altered shape, cell lysis, membrane fusion, and apoptosis. In the case of HSV-1 & 2 infections, a 'ballooning' effect can be seen under the light microscope. The cells will round up and look like tiny balloons. This is indicative of viral infection. Thus, a confirmatory test using direct immunofluorescence test (IF) is carried out. Since there are two types of HSV, monoclonal antibody is used to identify the type of HSV. The principle of this test is similar to the CMV IF test I posted earlier on. The only difference is that the monoclonal antibody is conjugated with Fluorescein isothiocyanate (FITC), which exhibits green fluorescence when excited by ultraviolet light, indicating the presence of HSV infected cells.

Yea, I guess that's about it.


Yong Yang (TG02)
0503196H