Global surveillance of emerging Influenza virus genotypes by mass spectrometry.
BACKGROUND
Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.


RESULTS
Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.


CONCLUSIONS
Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.
Sampath
Rangarajan
R
Ibis Biosciences Inc., A Wholly Owned Subsidiary of Isis Pharmaceuticals, Carlsbad, California, United States of America. rsampath@ibisbio.com
Russell
Kevin L
KL
Massire
Christian
C
Eshoo
Mark W
MW
Harpin
Vanessa
V
Blyn
Lawrence B
LB
Melton
Rachael
R
Ivy
Cristina
C
Pennella
Thuy
T
Li
Feng
F
Levene
Harold
H
Hall
Thomas A
TA
Libby
Brian
B
Fan
Nancy
N
Walcott
Demetrius J
DJ
Ranken
Raymond
R
Pear
Michael
M
Schink
Amy
A
Gutierrez
Jose
J
Drader
Jared
J
Moore
David
D
Metzgar
David
D
Addington
Lynda
L
Rothman
Richard
R
Gaydos
Charlotte A
CA
Yang
Samuel
S
St George
Kirsten
K
Fuschino
Meghan E
ME
Dean
Amy B
AB
Stallknecht
David E
DE
Goekjian
Ginger
G
Yingst
Samuel
S
Monteville
Marshall
M
Saad
Magdi D
MD
Whitehouse
Chris A
CA
Baldwin
Carson
C
Rudnick
Karl H
KH
Hofstadler
Steven A
SA
Lemon
Stanley M
SM
Ecker
David J
DJ
D005838:Genotype; D009980:Influenza A virus / Q000145:classification / Q000235:genetics*; D011159:Population Surveillance; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*