A measure of successful virus entry

There are numerous techniques that can be used to examine the interactions between the virus and the cell. These techniques can generally fall into two categories: direct and indirect observation. Direct observation uses familiar techniques such as thin-section microscopy, cryo-electron microscopy, tomography, and other less familiar techniques like freeze-fracture immunolabeling to examine virus-cell interactions. When properly prepared and processed, the interactions between the virus and cell seen in the microscope are the actual interactions that occurred at the time of fixation. By examining different times during the infection process, the possible mechanism and pathway of virus entry can be elucidated. Unfortunately, thin-sections and other direct observation techniques cannot distinguish between a successful infection process, and that of an unproductive interaction. This makes microscopy very subjective and is the primary problem when observing virus-cell interactions, as even with a relatively low particle/pfu ratio of 1:10, most observations are of virus-cell interactions that do not lead to a successful infection. For SINV, the SVHR strain can be purified to a particle/pfu ratio of 1:1, (28) and this virus was used in EM studies of virus infection that led to the direct cell penetration hypothesis of virus entry. Many of the previous observations that have been made using EM are of viruses preparation made with strains or samples with high or undetermined particle/pfu ratio and therefore are difficult to interpret.

When direct observation is not possible, or cannot answer the question being asked, techniques that take advantage of indirect or secondary reporters have been used. Some indirect reporters that have been used as a measure of successful virus entry include virus RNA production, virus protein synthesis and virus production. While these tools are useful and have shed light on many virus-cell interactions, they are at a disadvantage insomuch that they cannot determine if a virus has not entered the cell as the events assayed are not entry. Many of these biochemical reporters are significantly downstream of the initial events of virus adsorption and entry. As a result, each step between entry and the reporter has the potential to be inhibited giving a false negative. To give a time-scale to RNA translation and protein synthesis, super infection inhibition is detectable 15 minutes post infection. This implies that entry is a very fast process, and that the genome is quickly unpacked and rapidly available for processing. The speed of infection, the problems of indirect reporters, and the limits of direct observation need to be carefully considered when building an assay to measure successful virus entry.