Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.

The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called ‘bisulfite treatment’, which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies.The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.

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.

The Walter Reed Army Medical Center has experienced an influx of traumatically injured patients either infected or colonized with Acinetobacter baumannii. Using multilocus polymerase chain reaction (PCR) and mass spectrometry to genotype isolates, we found an atypical and evolving strain distribution, distinct from those found at nonmilitary hospitals in the United States.

Abstract. Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus , causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque ( Macaca fascicularis ) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.