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

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