title: Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts.
abstract: BACKGROUND
  The epidemiological and clinical impacts of influenza C virus infection may have been underestimated by conventional viral culture screening alone.
  
  
  OBJECTIVE
  To evaluate a newly developed real-time polymerase chain reaction (PCR) assay as a tool for diagnosing influenza C virus infection.
  
  
  METHODS
  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.
  
  
  RESULTS
  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 3.97×10(8)copies/ml and 5.43×10(5)EID(50)/ml, respectively, and those in specimens that were negative using cell culture were 2.18×10(6)copies/ml and 3.67×10(2)EID(50)/ml, respectively. In the clinical specimens with viral loads less than 10(5)copies/ml, it was not possible to isolate the virus using embryonated hen's eggs. The copy number-to-EID(50) 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.
  
  
  CONCLUSIONS
  The real-time PCR assay described here can be used as a sensitive method for diagnosing influenza C virus infection.
authors.affiliation: Department of Infectious Diseases, Yamagata University Faculty of Medicine, Iida-Nishi 2-2-2, Yamagata 990-9585, Japan. matuzaki@med.id.yamagata-u.ac.jp
authors.forename: Yoko
authors.initials: Y
authors.lastname: Matsuzaki
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ikeda
authors.forename: Chieko
authors.initials: C
authors.lastname: Abiko
authors.forename: Yoko
authors.initials: Y
authors.lastname: Aoki
authors.forename: Katsumi
authors.initials: K
authors.lastname: Mizuta
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Shimotai
authors.forename: Kanetsu
authors.initials: K
authors.lastname: Sugawara
authors.forename: Seiji
authors.initials: S
authors.lastname: Hongo
mesh_terms: D000293:Adolescent; D000818:Animals; D002642:Chick Embryo; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D009982:Gammainfluenzavirus / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019562:Viral Load / Q000379:methods*; D014776:Virus Cultivation / Q000379:methods*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral