Skip to Content
Merck
  • In situ molecular identification of the influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City.

In situ molecular identification of the influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City.

BMC infectious diseases (2013-01-19)
Rodolfo Ocadiz-Delgado, Martha Estela Albino-Sanchez, Enrique Garcia-Villa, Maria Guadalupe Aguilar-Gonzalez, Carlos Cabello, Dora Rosete, Fidencio Mejia, Maria Eugenia Manjarrez-Zavala, Carmen Ondarza-Aguilera, Rosa Ma Rivera-Rosales, Patricio Gariglio
ABSTRACT

In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City's hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1) triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. In order to detect A (H1N1) virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1) Neuraminidase (NA) RNA using in situ RT-PCR. We detected NA transcripts in 100% of the previously diagnosed A (H1N1)-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1)-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1)-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. We have successfully amplified specific influenza A (H1N1) NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1) outbreak in Mexico City.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Neuraminidase from Clostridium perfringens (C. welchii), Type X, lyophilized powder, ≥50 units/mg protein (using 4MU-NANA)
Sigma-Aldrich
Neuraminidase from Clostridium perfringens (C. welchii), Type VIII, lyophilized powder, 10-20 units/mg protein (using 4MU-NANA), 3.5-8.0 units/mg protein (mucin)
Sigma-Aldrich
α(2→3) Neuraminidase from Streptococcus pneumoniae, buffered aqueous solution
Sigma-Aldrich
α(2→3,6) Neuraminidase from Clostridium perfringens (C. welchii), recombinant, expressed in E. coli, buffered aqueous solution, ≥250 units/mg protein
Sigma-Aldrich
Neuraminidase from Clostridium perfringens (C. welchii), Suitable for manufacturing of diagnostic kits and reagents, Type V, lyophilized powder
Sigma-Aldrich
Neuraminidase from Clostridium perfringens (C. welchii), Type VI, lyophilized powder, 6-15 units/mg protein (using 4MU-NANA), 2-10 units/mg protein (mucin)
Sigma-Aldrich
Neuraminidase Agarose from Clostridium perfringens (C. welchii), Type VI-A, ammonium sulfate suspension
Sigma-Aldrich
α(2→3,6,8,9) Neuraminidase from Arthrobacter ureafaciens, Proteomics Grade, suitable for MALDI-TOF MS
Sigma-Aldrich
Neuraminidase from Vibrio cholerae, Type II, buffered aqueous solution, 8-24 units/mg protein (Lowry, using NAN-lactose)
Sigma-Aldrich
Neuraminidase from Vibrio cholerae, Type III, buffered aqueous solution, 0.2 μm filtered, 1-5 units/mg protein (Lowry, using NAN-lactose)
Sigma-Aldrich
α(2→3,6,8,9) Neuraminidase from Arthrobacter ureafaciens, recombinant, expressed in E. coli, buffered aqueous solution