PMC

MSSCP genotyping method differentiates pandemic and seasonal strains of influenza virus A/H1N1. Products of hemagglutinin gene amplification obtained for pandemic A/H1N1/v and reference seasonal strains of influenza virus A/H1N1 were denatured, and ssDNA was separated in a 9% polyacrylamide gel using the MSSCP method under optimal electrophoretic conditions. DNA bands were visualized with silver stain. Strains are indicated as follows: pandemic (P) strains were A/Mexico/4486/09 (lane P1), A/England/195/09 (lane P2), A/Gdansk/037/2009(H1N1) (lane P3), and A/Gdansk/036/2009 (H1N1) (lane P4). Reference seasonal (S) strains were A/Brisbane/59/2007 (lane S1) A/Hong Kong/2652/2006 (lane S20), A/New Caledonia/20/1999 (lane S3), and A/Solomon Islands/3/2006 (lane S4).

Sensitivity of MSSCP identification of double A/H1N1 infection. (A) To evaluate the minimum coinfection detection level, PCR products of seasonal (s) and pandemic (p) genotypes were mixed in proportions ranging from 50% seasonal to 50% pandemic to 99.9% seasonal to 0.1% pandemic. All samples were denatured and resolved under optimal electrophoretic conditions using the MSSCP method. DNA bands were visualized by silver stain. ssDNA bands indicated by arrowheads, representing seasonal (red) and pandemic (blue) strains, were cut out from the gel, and DNA was recovered and sequenced. (B) Fragments of DNA sequence reads representing seasonal (red) and pandemic (blue) A/H1N1 strains obtained from ssDNA bands. Strain-specific regions are indicated by letters above the histograms.

Identification of minor genetic variants of A/H1N1 strains by MSSCP method and sequencing. (A) DNA sequence reads of samples found to be coinfected after direct Sanger sequencing (samples 8, 14, 15, and 19) prior to MSSCP analysis. DNA sequences correspond to sample 8 (predominantly seasonal strain) and samples 14, 15, and 19 (predominantly pandemic strains). (B) Sanger sequencing of isolates from four coinfected samples (samples 8, 14, 15, and 19) after MSSCP-based minor variant enrichment. PCR products were denatured and separated in a 9% polyacrylamide gel using the MSSCP method under optimal electrophoretic conditions. DNA bands were visualized with silver stain. The ssDNA bands indicated by arrowheads, representing seasonal (s; red) and pandemic (p; blue) strains, were cut out from the gel, and DNA was recovered and sequenced. DNA sequencing identified the presence of both seasonal (red) and pandemic (blue) isolate sequences, proving the presence of coinfection not detected by direct sequencing in all analyzed samples. Strain-specific regions are indicated by letters above the histograms.

Simultaneous detection of seasonal and pandemic A/H1N1 strains in specimens collected in 2009. Products of hemagglutinin gene amplification obtained for pandemic A/H1N1/v and reference seasonal strains of influenza virus A/H1N1 were denatured, and ssDNA was separated in a 9% polyacrylamide gel using the MSSCP method under optimal electrophoretic conditions. DNA bands were visualized with silver stain. The presence of both pandemic and seasonal H1N1 virus strains in four samples indicated coinfection (marked with arrows). The following lanes contain the indicated clinical specimen (sample number): 1, United States visitor (110); 2, Canada visitor (193); 3, contact with Canada visitor (195) (); 4, Canada visitor (244); 5, United Kingdom visitor (253); 6, contact with Canada visitor (256) (); 7, Canada visitor and contact with Canada visitor (272) (); 8, Spain visitor (360); 9, without symptoms (383); 10, without symptoms (384); 11, Spain visitor (415); 12, Holland visitor (462); 13, India visitor (667); 14, Mexico and United States visitor (682); 15, northeast Poland (692); 16, postmortem sample, central Poland (702); 17, northwest Poland (779); 18, central Poland (751); 19, east Poland (723); 20, southwest Poland (911); 21, central Poland (837); 22, southeast Poland (888); and 23, north Poland (736). Seasonal (S) reference strains were A/Brisbane/59/2007 (lane S1), A/Fukushima/141/2006 (lane S2), A/Fukushima/97/2006 (lane S3), A/Hong Kong/2652/2006 (lane S4), A/New Caledonia/20/1999 (lane S5), A/St. Petersburg/10/2007 (lane S6), A/Solomon Islands/3/2006 (lane S7), and A/Taiwan/1/1986 (lane S8). Pandemic (P) viruses were A/Gdansk/037/2009 (H1N1) (lane P1) and A/Gdansk/036/2009 (H1N1) (lane P2).