Rapid identification viruses from nasal pharyngeal aspirates in acute viral respiratory infections by RT-PCR and electrospray ionization mass spectrometry

a b s t r a c t Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot evaluation compared performance characteristics of the RT-PCR and electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to conventional virologic methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N = 192) attending an emergency department during the 2007-2008 respiratory season consented, and "excess" frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8 h. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly

Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit

Rapid Identification of Emerging Pathogens: Coronavirus

New surveillance approach can analyze >900 polymerase chain reactions per day

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

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.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.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

PhD

dissertationThe regulation of expression of the pyelonephritis-associated pili (pap) pilin gene in Escherichia coli was investigated using a single copy lacZYA fusion system. The expression of pap-encoded pili is under the control of a heritable phase variation mechanism. Pap phase-variation is transcriptionally regulated and responsive to changes in the carbon source used for growth and the incubation temperature. Pilin phase variation occurs without DNA rearrangement of pap DNA sequences. Analysis of the DNA regulatory region upstream of the papBA promoter showed that there are two nearly perfect 27 base pair inverted repeats separated by 89 base pairs. Each inverted repeat contains a single Dam (deoxyadenosine methylase) methylation site (GATC1028 and GATC1130 sites). Using a pap-lac gene fusion, as well as primer extension analysis of papB mRNA, it was found that the dam gene product is required for pap transcription. The methylation states of the GATC028 and GATC1130 sites in DNAs isolated from transcriptionally phase on and phase off cells were different as evidenced by Southern blot experiments. The GATC1028 site was found to be unmethylated only in DNA isolated from phase on populations. Conversely, GATC1130 sites were unmethylated in DNA isolated from phase off populations. E. coli mutants were isolated that failed to protect both the GATC1028 and GATC 1130 sites from methylation by Dam. The defective gene(s) in these mutants was cloned and designated as mbf for methylation blocking factor. The mbf gene is located at 19.6 min on the E. coli chromosome. In mbf+ E. coli the GATC1130 site is umethylated (the phase off methylation state) in the absence of any pap-encoded proteins. Methylation protection of GATC1028 (the phase on methylation state) requires both mbf and papI, a regulatory gene within the pap operon, indicating that pap pilin phase variation requires the action of at least two genes, papI and mbf. A model is presented for pap pilin phase variation which predicts that the GATC methylation state regulates Mbf and PapI binding to pap DNA and that binding of Mbf and PapI inhibits the methylation of GATC sites by Dam methylase

MicC, a Second Small-RNA Regulator of Omp Protein Expression in Escherichia coli

In a previous bioinformatics-based search for novel small-RNA genes encoded by the Escherichia coli genome, we identified a region, IS063, located between the ompN and ydbK genes, that encodes an ∼100-nucleotide small-RNA transcript. Here we show that the expression of this small RNA is increased at a low temperature and in minimal medium. Twenty-two nucleotides at the 5′ end of this transcript have the potential to form base pairs with the leader sequence of the mRNA encoding the outer membrane protein OmpC. The deletion of IS063 increased the expression of an ompC-luc translational fusion 1.5- to 2-fold, and a 10-fold overexpression of the small RNA led to a 2- to 3-fold repression of the fusion. Deletion and overexpression of the IS063 RNA also resulted in increases and decreases, respectively, in OmpC protein levels. Taken together, these results suggest that IS063 is a regulator of OmpC expression; thus, the small RNA has been renamed MicC. The antisense regulation was further demonstrated by the finding that micC mutations were suppressed by compensatory mutations in the ompC mRNA. MicC was also shown to inhibit ribosome binding to the ompC mRNA leader in vitro and to require the Hfq RNA chaperone for its function. We suggest that the MicF and MicC RNAs act in conjunction with the EnvZ-OmpR two-component system to control the OmpF/OmpC protein ratio in response to a variety of environmental stimuli

Reverse transcription polymerase chain reaction and electrospray ionization mass spectrometry for identifying acute viral upper respiratory tract infections

Abstract Diagnosis of respiratory viruses traditionally relies on culture or antigen detection. We aimed to demonstrate capacity of the reverse transcription polymerase chain reaction/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) platform to identify clinical relevant respiratory viruses in nasopharyngeal aspirate (NPA) samples and compare the diagnostic performance characteristics relative to conventional culture-and antigen-based methods. An RT-PCR/ESI-MS respiratory virus surveillance kit designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, Adenoviridae types A-F, Coronaviridae, human bocavirus, and human metapneumovirus was evaluated using both mock-ups and frozen archived NPA (N = 280), 95 of which were positive by clinical virology methods. RT-PCR/ESI-MS detected 74/95 (77.9%) known positive samples and identified an additional 13/185 (7%) from culture-negative samples. Viruses that are nondetectable with conventional methods were also identified. Viral load was semiquantifiable and ranged from 2400 to N320 000 copies/mL. Time to results was 8 h. RT-PCR/ESI-MS showed promise in rapid detection of respiratory viruses and merits further evaluation for use in clinical settings