Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts

The epidemiological and clinical impacts of influenza C virus infection may have been underestimated by conventional viral culture screening alone. To evaluate a newly developed real-time polymerase chain reaction (PCR) assay as a tool for diagnosing influenza C virus infection. The primers and probe for real-time PCR were designed to amplify the conserved region of the nucleoprotein gene based on the aligned sequences of nine isolates from 1967 to 2010. Respiratory specimens from children collected between January 2010 and August 2010 were examined for the presence of influenza C virus by cell culture and real-time PCR. Specimens that were positive for the virus using real-time PCR were further examined using an infectivity assay with embryonated hen's eggs. Of the 1203 specimens examined, 34 (2.8%) tested positive for the influenza C virus by cell culture and 51 (4.2%) tested positive by real-time PCR. The mean viral load and infectivity titer in specimens that tested positive using cell culture were 3.97 × 108 copies/ml and 5.43 × 105 EID50/ml, respectively, and those in specimens that were negative using cell culture were 2.18 × 106 copies/ml and 3.67 × 102 EID50/ml, respectively. In the clinical specimens with viral loads less than 105 copies/ml, it was not possible to isolate the virus using embryonated hen's eggs. The copy number-to-EID50 ratio of the clinical specimens was much higher, ranging from 32 to 278,000, than those of culture fluid, ranging from 2.3 to 13.5. The real-time PCR assay described here can be used as a sensitive method for diagnosing influenza C virus infection.