Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus

Abstract

Swine influenza viruses (SIV) have been shown to sporadically infect humans and are infrequently identified by the Influenza Division of the Centers for Disease Control and Prevention (CDC) after being received as unsubtypeable influenza A virus samples. Real-time reverse transcriptase PCR (rRT-PCR) procedures for detection and characterization of North American lineage (N. Am) SIV were developed and implemented at CDC for rapid identification of specimens from cases of suspected infections with SIV. These procedures were utilized in April 2009 for detection of human cases of 2009 A (H1N1) pandemic (pdm) influenza virus infection. Based on genetic sequence data derived from the first two viruses investigated, the previously developed rRT-PCR procedures were optimized to create the CDC rRT-PCR Swine Flu Panel for detection of the 2009 A (H1N1) pdm influenza virus. The analytical sensitivity of the CDC rRT-PCR Swine Flu Panel was shown to be 5 copies of RNA per reaction and 10(-1.3 - -0.7) 50% infectious doses (ID(50)) per reaction for cultured viruses. Cross-reactivity was not observed when testing human clinical specimens or cultured viruses that were positive for human seasonal A (H1N1, H3N2) and B influenza viruses. The CDC rRT-PCR Swine Flu Panel was distributed to public health laboratories in the United States and internationally from April 2009 until June 2010. The CDC rRT-PCR Swine Flu Panel served as an effective tool for timely and specific detection of 2009 A (H1N1) pdm influenza viruses and facilitated subsequent public health response implementation.

Fig. 1

Schematic diagram representing the genotypes of N. Am tr-SIV H1N1, H1N2, and H3N2 subtypes and 2009 A (H1N1) pdm influenza viruses. Gray, yellow, green, and pink gene segments represent genes originating from human seasonal, avian, classical swine, and Eurasian swine influenza viruses, respectively.

Fig. 2

CDC evaluation of inconclusive specimens (n = 281) submitted by U.S. public health laboratories. Results obtained by the CDC Influenza Division for 160 specimens with InfA C_T values > 30 and 121 specimens with C_T values ≤ 30 confirmed by the CDC rRT-PCR Swine Flu Panel and/or genetic sequence characterization are shown.

Fig. 3

Nucleotide sequence alignment of swH1 primer and probe regions from specimens and of viruses positive for 2009 A (H1N1) pdm influenza virus that tested negative by the swH1 assay. Only locations where nucleotide differences were observed are indicated. The numbers of specimens identified with each pattern of nucleic acid differences were as follows: for pattern 1, 19; for pattern 2, 2; for pattern 3, 1; for pattern 4, 1; for pattern 5, 1; for pattern 6, 1; and for pattern 7, 1. The primer and probe locations are indicated according to the HA gene coding domain sequence of 2009 A (H1N1) pdm influenza virus strain A/California/07/2009 (FJ966974).