Proteotyping to Establish Gene Origin within Reassortant Influenza Viruses

Abstract

The application of a rapid and direct proteotyping approach with which to identify the gene origin of viral antigens in a reassortant influenza strain is demonstrated. The reassortant strain, constructed for a vaccine against type A 2009 H1N1 pandemic influenza, contains genes derived from a wild-type pandemic strain (A/California/7/2009) and an egg adapted high-growth strain (denoted NYMC X-157) derived from an earlier A/Puerto Rico/8/34 strain. The proteotyping approach employs modern proteomics methods and high resolution mass spectrometry to correctly establish that the genes of the surface antigens, hemagglutinin and neuraminidase, are derived from the A/California/7/2009 strain while those for nucleoprotein and matrix protein M1 antigens are derived from the NYMC X-157 strain. This is achieved for both gel-separated antigens and those from a whole vaccine digest. Furthermore, signature peptides detected in the mass spectra of the digested antigens enable the engineered reassortant strain to be identified as a type A virus of the H1N1 subtype in accord with earlier studies. The results demonstrate that proteotyping approach provides a more direct and rapid approach over RT-PCR with which to characterize reassortant strains of the influenza virus at the molecular protein level. Given that these strains pose the greatest risk to human and animal health and have been responsible for all human pandemics of the 20th and 21st centuries, there is a vital need for the origins and evolutionary history of these strains to be rapidly established.

Origins of the reassortant 2009 pandemic influenza virus through proteotyping with mass spectrometry

“…A few months into the 2009 pandemic, mutations in the matrix gene were found to further invalidate or limit the suitability of RT‐PCR assays developed specifically for the type A pandemic H1N1 virus . The mass spectrometric approach, by comparison, does not require primers as it analyses the viral proteins directly, even within whole virus digests . The detection of more than one signature or indicator peptide, and the associated conservation of sequence of these peptides within viral proteins across strains of a particular host, minimize limitations associated with mutations that would impair their detection.…”

“…Peptides obtained from tryptic digestion (1 µl) were diluted within a solution (4 µl) of a MALDI matrix (5 mg · mL −1 α‐cyano‐4‐hydroxycinnaminic acid in 50% ACN with 0.1% TFA) and 1 µl was spotted onto a MALDI sample plate (MTP AnchorChip™ 400/384 TF, Bruker Daltonics, Billerica, MA, USA) and air dried. MALDI FT‐ICR mass spectra were recorded on a 7 T Bruker APEX‐Qe instrument (Bruker Daltonics, Billerica, MA, USA) in the positive ion mode as previously described …”

“…MALDI FT-ICR mass spectra were recorded on a 7 T Bruker APEX-Qe instrument (Bruker Daltonics, Billerica, MA, USA) in the positive ion mode as previously described. [12][13][14][15][16][17][18][19] same period, were downloaded from the Influenza Virus Resource Database. [23] A three year period was used since novel antigenic variants have been shown to arise as a result of antigenic drift and circulate for approximately three years.…”

“…[29,30] The mass spectrometric approach, by comparison, does not require primers as it analyses the viral proteins directly, even within whole virus digests. [12][13][14][15][16][17][18] The detection of more than one signature or indicator peptide, and the associated conservation of sequence of these peptides within viral proteins across strains of a particular host, minimize limitations associated with mutations that would impair their detection.…”

“…Signature peptides represent proteolytic segments of a viral protein that are conserved in sequence, for a particular type, subtype and lineage [16] of the virus, and unique in mass when the mass of all proteolytic peptides from viral proteins of all other strains, both human and animal, including proteins associated with common contaminants, are considered. The approach has been shown to be able to differentiate seasonal from pandemic influenza strains, [17] identify the gene origin of reassorted human influenza viruses [18] and identify reassorted viruses from non-reassorted ones with the assistance of two computer algorithms written for that purpose. [19] Here, host-specific signatures are identified and detected that enable the swine origin of genes encoding the viral proteins within a reassorted 2009 pandemic-like strain to be determined in a primary clinical isolate.…”