Enumeration of an Extremely High Particle-to-PFU Ratio for Varicella-Zoster Virus

Varicella-zoster virus (VZV) is renowned for its low titers. Yet investigations to explore the low infectivity are hampered by the fact that the VZV particle-to-PFU ratio has never been determined with precision. Herein, we accomplish that task by applying newer imaging technology. More than 300 images were taken of VZV-infected cells on 4 different samples at high magnification. We enumerated the total number of viral particles within 25 cm² of the infected monolayer at 415 million. Based on these numbers, the VZV particle:PFU ratio was approximately 40,000:1 for a cell-free inoculum.A precise ratio of particles to PFU of varicella-zoster virus (VZV) has never been determined, even though VZV was first isolated in cell culture by the Nobel laureate T. H. Weller in 1952 (21). His group determined that VZV replicated in a few embryonic tissues and in amnion cells. Subsequently, Taylor-Robinson and Caunt found that VZV replication was restricted to a small number of mainly embryonic cells by testing more than 20 primary and continuous cell lines (19). A decade later, VZV was propagated in melanoma cell lines, which are derived from the neural crest (8). In all of these cultured cells, the titer was found to be low, particularly when compared with that of the closely related herpes simplex type 1 virus (HSV-1). Again, in sharp contrast with HSV-1, the virus remained strongly cell associated.The term particle/PFU ratio refers to the number of viral particles required to form one plaque in a plaque assay. It is a measure of the efficiency by which a virus infects cultured cells. Early in the 1960s, investigators began using negative staining electron microscopy to count viral particles in inoculum material and compare those counts to the measured titer, thereby measuring ratios for a few animal viruses (6). For example, the ratio for HSV-1 is around 10:1 (10, 20). Due to the strong cell association of VZV infection of cultured cells, no precise VZV particle/PFU ratio has ever been determined. The lack of any widely accepted VZV ratio severely limits our ability to assess whether mutated or recombinant viruses produce more or fewer complete infectious particles in cultured cells (4, 5, 15, 17). In other words, if an attenuated virus has a lower titer, we do not know whether fewer viral particles are produced per square centimeter of cellular monolayer (without a change in the particle/PFU ratio) or alternatively fewer infectious viral particles are produced overall (with a higher particle/PFU ratio).In this report, we successfully define a VZV particle/PFU ratio by imaging viral particles with advanced scanning electron microscopic (SEM) technology not available during our earlier investigations of viral structure (12). We demonstrate that the VZV ratio is much higher than that for other common human viruses grown in cultured cells and remarkably higher than that for HSV. Finally, this report documents evidence of an ever-widening difference between HSV and VZV replication and assembly in cultured cells (7, 13, 18).