Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses

Abstract

Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms.

Circulation and characterization of seasonal influenza viruses in Cambodia, 2012‐2015

“…Viral RNA extracted from MDCK supernatant was used to sequence the HA gene of all influenza isolates at the NIC laboratory using Sanger sequencing. At the WHOCC (Melbourne), a single‐reaction, multiplex RT‐PCR method that amplifies the HA, NA, and M genomic segments of seasonal influenza A and B viruses for next‐generation sequencing was used, as previously described . Nucleotide sequences from the coding regions of the HA genes of A(H3N2), A(H1N1)pdm09, and influenza B viruses were aligned using the Mafft multiple aligner V1.3.7 in the Geneious V10.0.9 software package (http://www.geneious.com).…”

“…The complete matrix gene was sequenced from representative influenza A viruses using previously described methods, to ascertain the presence of mutations (eg, Ser31Asn) associated with resistance to the adamantine class of inhibitors.…”

“…Cambodian data accessed from the Global Initiative on Sharing All Influenza Data (GISAID-https ://www.gisaid.org/) multiplex RT-PCR method that amplifies the HA, NA, and M genomic segments of seasonal influenza A and B viruses for next-generation sequencing was used, as previously described. 15 Nucleotide sequences from the coding regions of the HA genes of A(H3N2), A(H1N1)pdm09, and influenza B viruses were aligned using the Mafft multiple aligner V1.3.7 in the Geneious V10.0.9 software package (www.genei ous.com). Sequences originating from Cambodia, surrounding countries, and representative reference sequences were downloaded from the EpiFlu™ Database (www.gisaid.org).…”

Detection of adamantane-sensitive influenza A(H3N2) viruses in Australia, 2017: a cause for hope?

“…Using Sanger sequencing [6], next generation sequencing [7] or pyrosequencing [8] we found that from 1 March to 30 June 2017, all 310 tested Australian A(H3N2) viruses encoded M2 N31 indicating adamantane-resistance (M2 N31 viruses). However, in July, August and September 2017, two of 201 (1.0%), 10 of 115 (8.7%) and three of 145 (2.1%) tested A(H3N2) virus isolates, respectively, encoded M2 S31 indicating adamantane-sensitivity (M2 S31 viruses).…”

A six‐plex droplet digital RT‐PCR assay for seasonal influenza virus typing, subtyping, and lineage determination

“…15,16 There are real-time multiplex qRT-PCR assays for typing and subtyping seasonal influenza viruses. Some can even detect influenza B virus and determine influenza B viral lineage, 17,18 but none of these assays allows simultaneous typing and subtyping/lineage differentiation of all 4 seasonal influenza viruses in a single qRT-PCR reaction. Further, ddRT-PCR does not require a standard curve for quantification and it is less susceptible to PCR inhibitors.…”