Two complete 1918 influenza A/H1N1 pandemic virus genomes characterized by next-generation sequencing using RNA isolated from formalin-fixed, paraffin-embedded autopsy lung tissue samples along with evidence of secondary bacterial co-infection

ABSTRACT The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50–100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus -influenza virus co-infection. IMPORTANCE This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus -influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.