Determination of Infectious Retrovirus Concentration from Colony-Forming Assay with Quantitative Analysis

Abstract

The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP؉E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ؎ 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

Viral Dynamics during Primary Simian Immunodeficiency Virus Infection: Effect of Time-Dependent Virus Infectivity

“…According to our model estimates, the initial infection rate, ␤ 0 , is higher in ramp-up plasma recipient animals than in set-point plasma recipients in agreement with the findings that ramp-up plasma is more infectious than set-point plasma (26). The fact that 20 vRNA copies from ramp-up plasma could initiate infection is surprising given that prior studies have found that fewer than 0.1% of virions in plasma or culture media are infectious (5,22,30,42). However, a recent study (38) found that rapid dissociation of HIV-1 from cultured cells severely limits infectivity assays and masks the inherent high infectivity of virions.…”

Efficiency of Human Immunodeficiency Virus Type 1 Postentry Infection Processes: Evidence against Disproportionate Numbers of Defective Virions

“…This difference may mean that most particles (i) are defective and cannot complete the necessary infection steps or (ii) simply never come into contact with a permissive cell. Based on experimental observations of retrovirus properties and theoretical models using murine leukemia virus vectors, the second alternative is probably more likely (2,4,5,17,30,37). Further support for an underestimate of infectious particles comes from observations that titers can be increased by procedures such as the addition of polycations (46), vesicular stomatitis virus G (VSV-G) pseudotype formation (10,38), or spinoculation (38).…”

Quantification system for the viral dynamics of a highly pathogenic simian/human immunodeficiency virus based on an in vitro experiment and a mathematical model

“…Here, we have estimated the production rates of both infectious and non-infectious virus, allowing for a novel quantitative specification of the fraction of newly generated virus that is infectious. This fundamental quantity is important in understanding the role and influence of defective virus particles [48][49][50]52]; and, to our knowledge, this has not been measured before for any virus strain. We determined the theoretical minimum value for the proportion of infectious virions among newly produced virus, p, to be 8.62 × 10 -6 , by calculating the ratio of the infectious to total viral production rates k 50 / k. The ratio of the production rates, however, is actually p multiplied by a, where a is the conversion factor from RNA count of infectious virions to TCID 50 (i.e., roughly the fraction of infectious virions that are actually measured in a TCID 50 titration assay).…”

“…It is widely believed that retroviruses are predominantly defective, with less than 0.1% of virions in plasma or culture media being infectious [47][48][49]. On the other hand, it has recently been suggested that HIV-1 virions, for example, are inherently highly infectious, but that slow viral diffusion in liquid media and rapid dissociation of virions from cells severely limit infections in cultures (i.e., in assays measuring infectivity) [50,51].…”