Absolute quantification of infecting viral particles by chip-based digital polymerase chain reaction

In silico and empirical quantification of viruses is paramount for obtaining information on viral populations that have a major impact on biogeochemical cycles. The uncultured Pelagibacter virus vSAG 37-F6 discovered via single-virus genomics is one of the most abundant and cosmopolitan marine viruses; however, little is understood about its temporal variation. Here, we estimated the absolute number of infecting 37-F6 viruses in coastal bacterioplankton from the Mediterranean Sea by using a novel, feasible SYBR Green I chip-based digital PCR (SYBR dPCR) technique, not implemented before for enumerating (uncultured) microbes. Quantitative SYBR dPCR estimated 450-3480 genome copies of virus 37-F6 in cells/mL (i.e. infecting viruses) and a total of ≈10-400 putative infected cells/mL with a potential C release of 0.12-4.9 pg/ml in the analysed samples. Considering that virus 37-F6 is ubiquitous and abundant in all Tara samples, an enormous amount of C could be transformed by this virus through the 'viral shunt'. Thus, this SYBR dPCR technique has enabled the absolute quantification of an ecologically relevant uncultured virus in nature and the estimation of its potential contribution on biogeochemical cycles. Overall, our study also shows that this approach has a broad applicability for quantifying any other target loci in Microbiology and Virology.