Screening of influenza mutations using base-specific cleavage and MALDI mass spectrometry.
BACKGROUND
The hemagglutinin (HA) and neuraminidase (NA) genes encode surface glycoproteins of influenza virus. These two proteins are involved in pathogenicity and are the primary targets of the immune system. Mutations in the HA and NA genes can result in antigenic drift in an influenza viral strain. A comparative sequencing method using MALDI MS combined with base-specific cleavage has been applied for the surveillance of these viral mutations. This approach shows advantages in high throughput and efficiency than the traditional direct sequencing methods in targeted sequence analysis.


METHODS
Base-specific cleavage assay with RNAse A was combined with MALDI-MS for the analysis of the HA and NA genes of 2 influenza viral strains. The mass peak patterns from the spectra were compared with the in silico digest result of reference gene sequences from the database to achieve comparative sequencing and screening of novel mutations.


RESULTS
The HA and NA genes of two influenza lab strains were comparatively sequenced using the base-specific cleavage and MALDI-MS approach. Mutations could be exactly identified if more than one observation (mass peak changes) were detected in the spectrum. Mutations with only one observation could be located in a small area for further validation.


CONCLUSIONS
We showed a proof of a principle that base-specific combined with MALDI-MS comparative sequencing approach can be utilized for targeted sequence analysis and potentially rapid and cost effective screening of emerging viral mutations.
Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Xiang
X
Gao
Richard J
RJ
Sugrue
Boon-Huan
BH
Tan
Kai
K
Tang
D006388:Hemagglutinins / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007251:Influenza, Human / Q000821:virology; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics; D009975:Orthomyxoviridae / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006388:Hemagglutinins; D009439:Neuraminidase