Based upon analysis of multiple influenza sequence alignments, pan-influenza virus RT-PCR primer sets were developed that were capable of amplifying all three influenza virus species (A, B, and C) and subtypes (HxNy) from different animal hosts (e.g., human, avian, and swine) and distinguishing essential molecular features using base composition signatures. Additional primer pairs were designed that broadly amplified all known members of a particular species, but that did not cross-amplify members of different species (e.g., pan-influenza A and pan-influenza B primers). A surveillance panel of eight primer pairs (Table S1 ) was selected comprising one pan-influenza primer pair targeting the PB1 segment, five pan-influenza A primer pairs targeting NP, M1, PA, and the NS segments, and two pan-influenza B primer pairs targeting NP and PB2 segments. All primers used in this study had a thymine nucleotide at the 59 end to minimize addition of non-templated adenosines during amplification using Taq polymerase [24] . The sensitivity of each RT-PCR primer pair was determined using known quantities of a synthetic calibrant RNA template as described previously [20] . Each of the primer pairs was sensitive to as few as twenty copies of the calibrant RNA and several primers were sensitive to five copies (Table S1 ).