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Find video protocols related to scientific articles indexed in Pubmed.
High-Content Image-Based Screening of a Signal Transduction Pathway Inhibitor Small-Molecule Library against Highly Pathogenic RNA Viruses.
J Biomol Screen
PUBLISHED: 10-25-2014
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High-content image-based screening was developed as an approach to test a small-molecule library of compounds targeting signal transduction pathways for antiviral activity against multiple highly pathogenic RNA viruses. Of the 2843 compounds screened, 120 compounds exhibited ?60% antiviral activity. Four compounds (E225-0969, E528-0039, G118-0778, and G544-0735), which were most active against Rift Valley fever virus (RVFV) and showed broad-spectrum antiviral activity, were selected for further evaluation for their concentration-response profile and cytotoxicity. These compounds did not show any visible cytotoxicity at the highest concentration of compound tested (200 µM). All four of these compounds were more active than ribavirin against several viruses. One compound, E225-0969, had the lowest effective concentration (EC50 = 1.9-8.92 µM) for all the viruses tested. This compound was 13- and 43-fold more inhibitory against RVFV and Chikungunya virus (CHIKV), respectively, than ribavirin. The highest selectivity index (>106.2) was for E225-0969 against CHIKV. Time-of-addition assays suggested that all four lead compounds targeted early steps in the viral life cycle (entry and/or replication) but not virus egress. Overall, this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals against highly pathogenic viruses.
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High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.
PLoS Negl Trop Dis
PUBLISHED: 08-21-2014
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High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ? 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology.
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Molecular characterization of plasmid pMoma1of Moraxella macacae, a newly described bacterial pathogen of macaques.
Folia Microbiol. (Praha)
PUBLISHED: 08-06-2014
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We report the complete nucleotide sequence and characterization of a small cryptic plasmid of Moraxella macacae 0408225, a newly described bacterial species within the family Moraxellaceae and a causative agent of epistaxis in macaques. The complete nucleotide sequence of the plasmid pMoma1 was determined and found to be 5,375 bp in size with a GC content of 37.4 %. Computer analysis of the sequence data revealed five open reading frames encoding putative proteins of 54.4 kDa (ORF1), 17.6 kDa (ORF2), 13.3 kDa (ORF3), 51.6 kDa (ORF4), and 25.0 kDa (ORF5). ORF1, ORF2, and ORF3 encode putative proteins with high identity (72, 42, and 55 %, respectively) to mobilization proteins of plasmids found in other Moraxella species. ORF3 encodes a putative protein with similarity (about 40 %) to several plasmid replicase (RepA) proteins. The fifth open reading frames (ORF) was most similar to hypothetical proteins with unknown functions, although domain analysis of this sequence suggests it belongs to the Abi-like protein family. Upstream of the repA gene, a 470-bp intergenic region, was identified that contained an AT-rich section and two sets of tandem direct and indirect repeats, consistent with a putative origin of replication site. In contrast to other plasmids of Moraxella, the occurrence of pMoma1 in M. macacae isolates appears to be common as PCR testing of 14 clinical isolates from two different research institutions all contained the plasmid.
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Identification and characterization of Highlands J virus from a Mississippi sandhill crane using unbiased next-generation sequencing.
J. Virol. Methods
PUBLISHED: 02-21-2014
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Advances in massively parallel DNA sequencing platforms, commonly termed next-generation sequencing (NGS) technologies, have greatly reduced time, labor, and cost associated with DNA sequencing. Thus, NGS has become a routine tool for new viral pathogen discovery and will likely become the standard for routine laboratory diagnostics of infectious diseases in the near future. This study demonstrated the application of NGS for the rapid identification and characterization of a virus isolated from the brain of an endangered Mississippi sandhill crane. This bird was part of a population restoration effort and was found in an emaciated state several days after Hurricane Isaac passed over the refuge in Mississippi in 2012. Post-mortem examination had identified trichostrongyliasis as the possible cause of death, but because a virus with morphology consistent with a togavirus was isolated from the brain of the bird, an arboviral etiology was strongly suspected. Because individual molecular assays for several known arboviruses were negative, unbiased NGS by Illumina MiSeq was used to definitively identify and characterize the causative viral agent. Whole genome sequencing and phylogenetic analysis revealed the viral isolate to be the Highlands J virus, a known avian pathogen. This study demonstrates the use of unbiased NGS for the rapid detection and characterization of an unidentified viral pathogen and the application of this technology to wildlife disease diagnostics and conservation medicine.
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Genomic variability of monkeypox virus among humans, Democratic Republic of the Congo.
Emerging Infect. Dis.
PUBLISHED: 01-25-2014
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Monkeypox virus is a zoonotic virus endemic to Central Africa. Although active disease surveillance has assessed monkeypox disease prevalence and geographic range, information about virus diversity is lacking. We therefore assessed genome diversity of viruses in 60 samples obtained from humans with primary and secondary cases of infection from 2005 through 2007. We detected 4 distinct lineages and a deletion that resulted in gene loss in 10 (16.7%) samples and that seemed to correlate with human-to-human transmission (p = 0.0544). The data suggest a high frequency of spillover events from the pool of viruses in nonhuman animals, active selection through genomic destabilization and gene loss, and increased disease transmissibility and severity. The potential for accelerated adaptation to humans should be monitored through improved surveillance.
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Molecular diversity and predictability of Vibrio parahaemolyticus along the Georgian coastal zone of the Black Sea.
Front Microbiol
PUBLISHED: 01-01-2014
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Vibrio parahaemolyticus is a leading cause of seafood-related gastroenteritis and is also an autochthonous member of marine and estuarine environments worldwide. One-hundred seventy strains of V. parahaemolyticus were isolated from water and plankton samples collected along the Georgian coast of the Black Sea during 28 months of sample collection. All isolated strains were tested for presence of tlh, trh, and tdh. A subset of strains were serotyped and tested for additional factors and markers of pandemicity. Twenty-six serotypes, five of which are clinically relevant, were identified. Although all 170 isolates were negative for tdh, trh, and the Kanagawa Phenomenon, 7 possessed the GS-PCR sequence and 27 the 850 bp sequence of V. parahaemolyticus pandemic strains. The V. parahaemolyticus population in the Black Sea was estimated to be genomically heterogeneous by rep-PCR and the serodiversity observed did not correlate with rep-PCR genomic diversity. Statistical modeling was used to predict presence of V. parahaemolyticus as a function of water temperature, with strongest concordance observed for Green Cape site samples (Percent of total variance = 70, P < 0.001). Results demonstrate a diverse population of V. parahaemolyticus in the Black Sea, some of which carry pandemic markers, with increased water temperature correlated to an increase in abundance of V. parahaemolyticus.
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Microbial water quality of recreational lakes near Tbilisi, Georgia.
J Water Health
PUBLISHED: 05-28-2013
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Microbial safety of recreational water is one of the major human public health issues in developing countries. Three water bodies, the Tbilisi Sea, Kumisi and Lisi lakes, in the South Caucasus region near Tbilisi, Georgia, were monitored in 2006-2009 to determine microbiological quality using standard methods. Microbial pollution indicators were determined in parallel with phytoplankton abundance and measurement of a number of physical-chemical parameters. Kumisi Lake, a brackish water body in an active agricultural area, appeared to be the most polluted, whereas the Tbilisi Sea, a freshwater reservoir was the least polluted. High values for fecal indicators in all three lakes in summer and early autumn were revealed. In our study, total enterococci counts (TEC) appeared to be a better indicator than either fecal or total coliform counts for the evaluation of fresh and brackish microbial water quality. We found significant correlation between total Vibrio counts and TEC for all three water bodies. Prevalence of somatic coliphages and V. cholerae-specific phages as additional water pollution indicator significantly correlated with abundance of the host bacteria. Particular phytoplankton groups in the lakes responded to the changes of fecal indicators; however, no correlation was observed between dominant zooplankton taxonomic groups and microbial parameters.
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Crimean-Congo hemorrhagic fever: History, epidemiology, pathogenesis, clinical syndrome and genetic diversity.
Antiviral Res.
PUBLISHED: 05-15-2013
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Crimean-Congo hemorrhagic fever (CCHF) is the most important tick-borne viral disease of humans, causing sporadic cases or outbreaks of severe illness across a huge geographic area, from western China to the Middle East and southeastern Europe and throughout most of Africa. CCHFV is maintained in vertical and horizontal transmission cycles involving ixodid ticks and a variety of wild and domestic vertebrates, which do not show signs of illness. The virus circulates in a number of tick genera, but Hyalomma ticks are the principal source of human infection, probably because both immature and adult forms actively seek hosts for the blood meals required at each stage of maturation. CCHF occurs most frequently among agricultural workers following the bite of an infected tick, and to a lesser extent among slaughterhouse workers exposed to the blood and tissues of infected livestock and medical personnel through contact with the body fluids of infected patients. CCHFV is the most genetically diverse of the arboviruses, with nucleotide sequence differences among isolates ranging from 20% for the viral S segment to 31% for the M segment. Viruses with diverse sequences can be found within the same geographic area, while closely related viruses have been isolated in far distant regions, suggesting that widespread dispersion of CCHFV has occurred at times in the past, possibly by ticks carried on migratory birds or through the international livestock trade. Reassortment among genome segments during co-infection of ticks or vertebrates appears to have played an important role in generating diversity, and represents a potential future source of novel viruses. In this article, we first review current knowledge of CCHFV, summarizing its molecular biology, maintenance and transmission, epidemiology and geographic range. We also include an extensive discussion of CCHFV genetic diversity, including maps of the range of the virus with superimposed phylogenetic trees. We then review the features of CCHF, including the clinical syndrome, diagnosis, treatment, pathogenesis, vaccine development and laboratory animal models of CCHF. The paper ends with a discussion of the possible future geographic range of the virus. For the benefit of researchers, we include a Supplementary Table listing all published reports of CCHF cases and outbreaks in the English-language literature, plus some principal articles in other languages, with total case numbers, case fatality rates and all CCHFV strains on GenBank.
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Genome Sequence of Weissella ceti NC36, an Emerging Pathogen of Farmed Rainbow Trout in the United States.
Genome Announc
PUBLISHED: 02-14-2013
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Novel Weissella sp. bacteria have recently been reported to be associated with disease outbreaks in cultured rainbow trout (Oncorhynchus mykiss) at commercial farms in China, Brazil, and the United States. Here we present the first genome sequence of this novel Weissella species, isolated from the southeastern United States.
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Genome Sequence of Moraxella macacae 0408225, a Novel Bacterial Species Isolated from a Cynomolgus Macaque with Epistaxis.
Genome Announc
PUBLISHED: 02-14-2013
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Moraxella macacae is a recently described bacterial species that has been associated with at least two outbreaks of epistaxis in macaques. Here we present the first genome sequence of this novel species, isolated from a symptomatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases.
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Entry of Yersinia pestis into the viable but nonculturable state in a low-temperature tap water microcosm.
PLoS ONE
PUBLISHED: 01-24-2011
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Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.
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Current status of human arboviral diseases in Turkey.
Vector Borne Zoonotic Dis.
PUBLISHED: 12-06-2010
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Infections caused by viruses transmitted via blood-feeding arthropods (arthropod-borne or arboviruses) have gained considerable attention and importance during the last decades due to their resurgence, impact on public health, and changing epidemiologic features. The complex transmission cycles affected by environmental, technological, and ecological changes place arboviral infections in the realm of emerging and reemerging infections that intermittantly reappear in epidemic form or display tendency to extend beyond endemic zones. A number of previously undetected arboviral diseases have emerged in Turkey during the last decade, although, in some cases, serologic evidence has been provided earlier. Since Crimean-Congo hemorrhagic fever first emerged in Turkey in 2002, there are now more than 4400 laboratory-confirmed cases of the disease. In addition, convincing evidence has accumulated to suggest that pathogenic flaviviruses, including West Nile virus, are in circulation. Recent studies have also revealed human exposure, central nervous system infections, and outbreaks of febrile diseases by sandfly fever viruses. In this study, reports published in local and international journals on surveillance and epidemiology of medically important arboviruses and associated diseases from Turkey have been reviewed, and current data on tick, mosquito, and sandfly vectors are incorporated.
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Prevalence of hypermucoid Klebsiella pneumoniae among wild-caught and captive vervet monkeys (Chlorocebus aethiops sabaeus) on the island of St. Kitts.
J. Wildl. Dis.
PUBLISHED: 08-07-2010
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Invasive, hypermucoid Klebsiella pneumoniae causes severe abscess formation in humans and in certain species of nonhuman primates. We conducted a survey of captive and wild-caught African green monkeys, or vervets (Chlorocebus aethiops sabaeus), on the Caribbean island of St. Kitts to assess their carriage rate of Klebsiella spp. Forty percent of rectal swabs from captive monkeys were positive for K. pneumoniae, and 20% of wild-caught animals were positive. Two wild-caught monkeys (4%) were positive for K. oxytoca, and one monkey (2%) was found to be infected with a hypermucoid rmpA-positive K. pneumoniae strain. Genotyping of this strain showed that it had an indistinguishable random amplified polymorphic DNA fingerprint to a strain that caused fatal abscessation in several African green monkeys in a research colony in the USA in 2005. This is the first report of hypermucoid K. pneumoniae isolation from a wild population of nonhuman primates and represents a potential health risk to these animals, as well as to the humans who come in contact with them.
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Arbovirus detection in insect vectors by rapid, high-throughput pyrosequencing.
PLoS Negl Trop Dis
PUBLISHED: 05-20-2010
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Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown.
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Application of the Ibis-T5000 pan-Orthopoxvirus assay to quantitatively detect monkeypox viral loads in clinical specimens from macaques experimentally infected with aerosolized monkeypox virus.
Am. J. Trop. Med. Hyg.
PUBLISHED: 02-06-2010
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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.
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Rapid identification of vector-borne flaviviruses by mass spectrometry.
Mol. Cell. Probes
PUBLISHED: 02-05-2010
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Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.
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Identification of pathogenic Vibrio species by multilocus PCR-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of Georgia.
Appl. Environ. Microbiol.
PUBLISHED: 01-29-2010
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The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected.
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Molecular epidemiology of Crimean-Congo hemorrhagic fever virus in Turkey: occurrence of local topotype.
Virus Res.
PUBLISHED: 01-15-2010
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The goal of this study was to investigate the molecular epidemiology of Crimean-Congo hemorrhagic fever virus (CCHFV) in Turkey. The study was performed on a total of 48 confirmed human CCHF cases from 2006 to 2008. The majority of the CCHF viral strains in Turkey were found to belong to the European lineage. Local CCHF viral strains are grouped into two main clusters, which can be further divided into two sub-groups. We also identified an AP92-like virus causing clinical disease in Corum (a mid-Anatolian province). Phylogenetic analysis revealed that the most recent CCHFV infections were caused by intrinsic (or native) CCHF viral strains, which we identified as the local topotype. Comparison of deduced amino acid sequences of S-segment RNAs indicated that the local topotype was derived from viruses of previous years, most likely by a low rate recombination. No genetic differences, based on S- and M-segment RNA sequences, were found between human and tick viral isolates. This data suggest that replication of CCHFV in the tick vector, whether Rhiphicephalus spp. or Hyalomma spp., has no effect on the viral genomic structure.
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Characterization of a Moraxella species that causes epistaxis in macaques.
Vet. Microbiol.
PUBLISHED: 01-14-2010
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Bacteria of the genus Moraxella have been isolated from a variety of mammalian hosts. In a prior survey of bacteria that colonize the rhesus macaque nasopharynx, performed at the Tulane National Primate Research Center, organisms of the Moraxella genus were isolated from animals with epistaxis, or "bloody nose syndrome." They were biochemically identified as Moraxella catarrhalis, and cryopreserved. Another isolate was obtained from an epistatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases. Based on differences in colony and cell morphologies between rhesus and human M. catarrhalis isolates, we hypothesized that the nonhuman primate Moraxella might instead be a different species. Despite morphological differences, the rhesus isolates, by several biochemical tests, were indistinguishable from M. catarrhalis. Analysis of the cynomolgus isolate by Vitek 2 Compact indicated that it belonged to a Moraxella group, but could not differentiate among species. However, sequencing of the 16S ribosomal RNA gene from four representative rhesus isolates and the cynomolgus isolate showed closest homology to Moraxella lincolnii, a human respiratory tract inhabitant, with 90.16% identity. To examine rhesus macaques as potential hosts for M. catarrhalis, eight animals were inoculated with human M. catarrhalis isolates. Only one of the animals was colonized and showed disease, whereas four of four macaques became epistatic after inoculation with the rhesus Moraxella isolate. The nasopharyngeal isolates in this study appear uniquely adapted to a macaque host and, though they share many of the phenotypic characteristics of M. catarrhalis, appear to form a genotypically distinct species.
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Epidemiology of invasive Klebsiella pneumoniae with hypermucoviscosity phenotype in a research colony of nonhuman primates.
Comp. Med.
PUBLISHED: 12-26-2009
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Invasive Klebsiella pneumoniae with hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that, over the past 20 y, has resulted in a distinct clinical syndrome characterized by pyogenic liver abscesses sometimes complicated by bacteremia, meningitis, and endophthalmitis. Infections occur predominantly in Taiwan and other Asian countries, but HMV K. pneumoniae is considered an emerging infectious disease in the United States and other Western countries. In 2005, fatal multisystemic disease was attributed to HMV K. pneumoniae in African green monkeys (AGM) at our institution. After identification of a cluster of subclinically infected macaques in March and April 2008, screening of all colony nonhuman primates by oropharyngeal and rectal culture revealed 19 subclinically infected rhesus and cynomolgus macaques. PCR testing for 2 genes associated with HMV K. pneumoniae, rmpA and magA, suggested genetic variability in the samples. Random amplified polymorphic DNA analysis on a subset of clinical isolates confirmed a high degree of genetic diversity between the samples. Environmental testing did not reveal evidence of aerosol or droplet transmission of the organism in housing areas. Further research is needed to characterize HMV K. pneumoniae, particularly with regard to genetic differences among bacterial strains and their relationship to human disease and to the apparent susceptibility of AGM to this organism.
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Detection of toxigenic Vibrio cholerae O1 in freshwater lakes of the former Soviet Republic of Georgia.
Environ Microbiol Rep
PUBLISHED: 09-03-2009
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Three freshwater lakes, Lisi Lake, Kumisi Lake and Tbilisi Sea, near Tbilisi, Georgia, were studied from January 2006 to December 2007 to determine the presence of Vibrio cholerae employing both bacteriological culture method and direct detection methods, namely PCR and direct fluorescent antibody (DFA). For PCR, DNA extracted from water samples was tested for presence of V. cholerae and genes coding for selected virulence factors. Vibrio cholerae non-O1/non-O139 was routinely isolated by culture from all three lakes; whereas V. cholerae O1 and O139 were not. Water samples collected during the summer months from Lisi Lake and Kumisi Lake were positive for both V. cholerae and V. cholerae ctxA, tcpA, zot, ompU and toxR by PCR. Water samples collected during the same period from both Lisi and Kumisi Lake were also positive for V. cholerae serogroup O1 by DFA. All of the samples were negative for V. cholerae serotype O139. The results of this study provide evidence for an environmental presence of toxigenic V. cholerae O1, which may represent a potential source of illness as these lakes serve as recreational water in Tbilisi, Georgia.
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Rapid real-time PCR assays for detection of Klebsiella pneumoniae with the rmpA or magA genes associated with the hypermucoviscosity phenotype: screening of nonhuman primates.
J Mol Diagn
PUBLISHED: 07-30-2009
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The relationship of mucoviscosity-associated (magA) and/or regulator of mucoid phenotype (rmpA) genes to the Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been reported. We previously demonstrated that rmpA+ K. pneumoniae can cause serious disease in African green monkeys and isolated rmpA+ and magA+ HMV K. pneumoniae from other species of non-human primates. To rapidly screen African green monkeys/non-human primates for these infections, we developed three real-time PCR assays. The first was K. pneumoniae-specific, targeting the khe gene, while the others targeted rmpA and magA. Primer Express 2 was used with the three K. pneumoniae genes to generate sequence-specific TaqMan/TaqMan-Minor Groove Binder assays. Oral/rectal swabs and necropsy samples were collected; swabs were used for routine culture and DNA extraction. K. pneumoniae colonies were identified on the Vitek 2 with DNA tested using the K. pneumoniae-specific assays. Testing of 45 African green monkeys resulted in 19 khe+ samples from 14 animals with none positive for either rmpA or magA. Of these 19 khe+ samples, five were culture-positive, but none were HMV "string test"-positive. Subsequent testing of 307 non-human primates resulted in 64 HMV K. pneumoniae isolates of which 42 were rmpA+ and 15 were magA+. Non-human primate testing at the U.S. Army Medical Research Institute of Infectious Diseases demonstrated the ability to screen both live and necropsied animals for K. pneumoniae by culture and real-time PCR to determine HMV genotype.
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Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 02-18-2009
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Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria.
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Rapid and high-throughput pan-Orthopoxvirus detection and identification using PCR and mass spectrometry.
PLoS ONE
PUBLISHED: 02-11-2009
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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.
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Ebola virus genome plasticity as a marker of its passaging history: a comparison of in vitro passaging to non-human primate infection.
PLoS ONE
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To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.
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Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry.
PLoS ONE
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Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.
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