Enhancement of Infectivity of Murine Cytomegalovirus in Vitro by Centrifugal Inoculation

Abstract

Centrifugation of murine cytomegalovirus inocula from a variety of sources onto secondary mouse embryo cell monolayers at 1,900 × g for 30 min regularly revealed 10- to 100-fold more infectious virus than could be found in the same materials using standard inoculation methods. Virus demonstrable only by centrifugation was present throughout the entire growth cycle in a constant proportion to virus measured without centrifugation. Extracellular growth curves of both populations revealed an 18- to 21-hr latent period, followed by a long-linear increase over the next 12 hr; final yield was 30 plaque-forming units (PFU) per cell. Centrifugation of cells prior to inoculation or after standard adsorption and removal of inoculum failed to result in any significant change in measured virus titer. However, even after 4-hr adsorption, the supernatant inoculum could be transferred and centrifuged onto a fresh monolayer resulting in the same increment of measurable virus. Neutralizing antibody and interferon were equally efficacious against 100 PFU of virus as defined by either method. Thus, this newly identified population of cytomegalo-virus represents the vast majority of potentially infectious units and appears to differ solely in ease of adsorption onto cell monolayers.

Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic “honeycomb” surface coat

“…To image viral core structure outside of whole virions, cells were abruptly inoculated with massive amounts of virus (100-500 pfu/cell) by centrifugal inoculation or "spinoculation" (Gordon et al, 1960;Weiss and Dressler, 1960;Osborn and Walker, 1968;Hudson et al, 1976;Tenser, 1978;Smith, 1981;Thiele et al, 1987). This involves centrifuging a suspension of viruses down onto already plated cells, then warming them up to allow viral fusion.…”

Diagnosis of cytomegalovirus infection by detection of the early antigen of cytomegalovirus in cell cultures

“…Out of the 18 specimens positive only by early antigen detection, 11 were from patients, whose other urine specimens contained CMV or who had diagnostic rise of CMV antibodies in paired sera, which suggested that the early antigen positive findings were true positive results. In four of the 18 cases, virus isolation could not be completed due to bacterial or fungal contamination.…”

The cytotoxic response to murine cytomegalovirus: requirements for the generation of MCMV‐specific target cells

“…The total number of MEF susceptible to lysis by MCMV-immune T cells and the amount of ^'Cr released at a given effector:target ratio were increased if MHF were adsorbed with MCMV under centrifugation. This may be explained by an increase in the number of virions entering each MEF under centrifugation (14) but other unknown factors may be involved. Titrations of MCMV input to MEF using the centrifugation protocol showed increased MCMV-immune T cell-mediated lysis of infected MEF targets with increasing virus input, particularly if CSS-treated MEF were used.…”