New mathematical approaches to quantify human infectious viruses from environmental media using integrated cell culture-qPCR.

Quantifying infectious viruses by cell culture depends on visualizing cytopathic effect, or for integrated cell culture-PCR, attaining confidence a PCR-positive signal is the result of virus growth and not inoculum carryover. This study developed mathematical methods to calculate infectious virus numbers based on viral growth kinetics in cell culture. Poliovirus was inoculated into BGM cell monolayers at 10 concentrations from 0.001 to 1000 PFU/ml. Copy numbers of negative-strand RNA, a marker of infectivity for single-stranded positive RNA viruses, were measured over time by qRT-PCR. Growth data were analyzed by two approaches. First, data were fit with a continuous function to estimate directly the initial virus number, expressed as genomic copies. Such estimates correlated with actual inoculum numbers across all concentrations (R(2)=0.62, n=17). Second, the length of lag phase appeared to vary inversely with inoculum titers; hence, standard curves to predict inoculum virus numbers were derived based on three definitions of lag time: (1) time of first detection of (-)RNA, (2) second derivative maximum of the fitted continuous function, and (3) time when the fitted curve crossed a threshold (-)RNA concentration. All three proxies yielded standard curves with R(2)=0.69-0.90 (n=17). The primary advantage of these growth kinetics approaches is being able to quantify virions that are unambiguously infectious, a particular advantage for viruses that do not produce CPE. 2009 Elsevier B.V. All rights reserved.