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1.  Emerging and Resistant Infections 
Annals of the American Thoracic Society  2014;11(Suppl 4):S193-S200.
The lungs are a major target for infection and a key battleground in the fight against the development of antimicrobial drug–resistant pathogens. Ventilator-associated pneumonia (VAP) is associated with mortality rates of 24–50%. The optimal duration of antibiotic therapy against VAP is unknown, but prolonged courses are associated with the emergence of bacterial resistance. De-escalation strategies in which treatment is discontinued based on signs of clinical resolution, fixed durations of therapy (generally 7–8 d), or serum procalcitonin responses have been shown to decrease antibiotic consumption. Outcomes are comparable to longer treatment courses, with the possible exception of VAP due to nonfermenting, gram-negative bacilli such as Pseudomonas aeruginosa. Staphylococcus aureus is a leading cause of VAP and other infections. Outcomes after S. aureus infection are shaped by the interplay between environmental, bacterial, and host genetic factors. It is increasingly clear that mechanisms of pathogenesis vary in different types of S. aureus infections. Genome-scale studies of S. aureus strains, host responses, and host genetics are redefining our understanding of the pathogenic mechanisms underlying VAP. Genome-sequencing technologies are also revolutionizing our understanding of the molecular epidemiology, evolution, and transmission of influenza. Deep sequencing using next-generation technology platforms is defining the remarkable genetic diversity of influenza strains within infected hosts. Investigators have demonstrated that antiviral drug-resistant influenza may be present prior to the initiation of treatment. Moreover, drug-resistant minor variant influenza strains can be transmitted from person to person in the absence of selection pressure. Studies of lung infections and the causative pathogens will remain at the cutting edge of clinical and basic medical research.
doi:10.1513/AnnalsATS.201402-069PL
PMCID: PMC4200571  PMID: 25148425
pneumonia; ventilator-associated pneumonia; Staphylococcus aureus; influenza
2.  Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea 
The Journal of General Virology  2013;94(Pt 12):2609-2615.
Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae.
doi:10.1099/vir.0.055939-0
PMCID: PMC3836499  PMID: 24062532
3.  A large-scale immuno-epidemiological simulation of influenza A epidemics 
BMC Public Health  2014;14(1):1019.
Background
Agent based models (ABM) are useful to explore population-level scenarios of disease spread and containment, but typically characterize infected individuals using simplified models of infection and symptoms dynamics. Adding more realistic models of individual infections and symptoms may help to create more realistic population level epidemic dynamics.
Methods
Using an equation-based, host-level mathematical model of influenza A virus infection, we develop a function that expresses the dependence of infectivity and symptoms of an infected individual on initial viral load, age, and viral strain phenotype. We incorporate this response function in a population-scale agent-based model of influenza A epidemic to create a hybrid multiscale modeling framework that reflects both population dynamics and individualized host response to infection.
Results
At the host level, we estimate parameter ranges using experimental data of H1N1 viral titers and symptoms measured in humans. By linearization of symptoms responses of the host-level model we obtain a map of the parameters of the model that characterizes clinical phenotypes of influenza infection and immune response variability over the population. At the population-level model, we analyze the effect of individualizing viral response in agent-based model by simulating epidemics across Allegheny County, Pennsylvania under both age-specific and age-independent severity assumptions.
Conclusions
We present a framework for multi-scale simulations of influenza epidemics that enables the study of population-level effects of individual differences in infections and symptoms, with minimal additional computational cost compared to the existing population-level simulations.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1019) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2458-14-1019
PMCID: PMC4194421  PMID: 25266818
4.  Niakha virus: A novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal 
Virology  2013;444(0):80-89.
Members of the family Rhabdoviridae have been assigned to eight genera but many remain unassigned. Rhabdoviruses have a remarkably diverse host range that includes terrestrial and marine animals, invertebrates and plants. Transmission of some rhabdoviruses often requires an arthropod vector, such as mosquitoes, midges, sandflies, ticks, aphids and leafhoppers, in which they replicate. Herein we characterize Niakha virus (NIAV), a previously uncharacterized rhabdovirus isolated from phebotomine sandflies in Senegal. Analysis of the 11,124 nt genome sequence indicates that it encodes the five common rhabdovirus proteins with alternative ORFs in the M, G and L genes. Phylogenetic analysis of the L protein indicate that NIAV’s closest relative is Oak Vale rhabdovirus, although in this analysis NIAV is still so phylogenetically distinct that it might be classified as distinct from the eight currently recognized Rhabdoviridae genera. This observation highlights the vast, and yet not fully recognized diversity, of this family.
doi:10.1016/j.virol.2013.05.035
PMCID: PMC3755043  PMID: 23773405
Niakha virus (NIAV); Rhabdoviridae; phlebotomine sandflies; complement fixation; West Africa
5.  Potential involvement of Brugia malayi cysteine proteases in the maintenance of the endosymbiotic relationship with Wolbachia 
Graphical abstract
Highlights
•Bm-cpl-3 and Bm-cpl-6 are involved during development and endosymbiosis.•In tetracycline-treated worms Bm-cpl-3 and -6 are regulated in a bimodal pattern.•Reduction in Bm-cpl-3 and -6 levels resulted in hindered microfilarial development.•Reduction in Bm-cpl-3 and -6 levels resulted in reduced Wolbachia DNA levels.•These enzymes might be strong drug target candidates.
Brugia malayi, a parasitic nematode that causes lymphatic filariasis, harbors endosymbiotic intracellular bacteria, Wolbachia, that are required for the development and reproduction of the worm. The essential nature of this endosymbiosis led to the development of anti-Wolbachia chemotherapeutic approaches for the treatment of human filarial infections. Our study is aimed at identifying specific proteins that play a critical role in this endosymbiotic relationship leading to the identification of potential targets in the adult worms. Filarial cysteine proteases are known to be involved in molting and embryogenesis, processes shown to also be Wolbachia dependent. Based on the observation that cysteine protease transcripts are differentially regulated in response to tetracycline treatment, we focused on defining their role in symbiosis. We observe a bimodal regulation pattern of transcripts encoding cysteine proteases when in vitro tetracycline treated worms were examined. Using tetracycline-treated infertile female worms and purified embryos we established that the first peak of the bimodal pattern corresponds to embryonic transcripts while the second takes place within the hypodermis of the adult worms. Localization studies of the native proteins corresponding to Bm-cpl-3 and Bm-cpl-6 indicate that they are present in the area surrounding Wolbachia, and, in some cases, the proteins appear localized within the bacteria. Both proteins were also found in the inner bodies of microfilariae. The possible role of these cysteine proteases during development and endosymbiosis was further characterized using RNAi. Reduction in Bm-cpl-3 and Bm-cpl-6 transcript levels was accompanied by hindered microfilarial development and release, and reduced Wolbachia DNA levels, making these enzymes strong drug target candidates.
doi:10.1016/j.ijpddr.2014.08.001
PMCID: PMC4266806  PMID: 25516837
Filaria; Wolbachia; Symbiosis; Cysteine proteases
6.  A multi-reservoir model of influenza evolution 
Journal of critical care  2009;24(3):e33-e34.
doi:10.1016/j.jcrc.2009.06.040
PMCID: PMC4123430  PMID: 25110389
7.  The human mycobiome in health and disease 
Genome Medicine  2013;5(7):63.
The mycobiome, referring primarily to the fungal biota in an environment, is an important component of the human microbiome. Despite its importance, it has remained understudied. New culture-independent approaches to determine microbial diversity, such as next-generation sequencing methods, are greatly broadening our view of fungal importance. An integrative analysis of current studies shows that different body sites harbor specific fungal populations, and that diverse mycobiome patterns are associated with various diseases. By interfacing with other biomes, as well as with the host, the mycobiome probably contributes to the progression of fungus-associated diseases and plays an important role in health and disease.
doi:10.1186/gm467
PMCID: PMC3978422  PMID: 23899327
8.  Genome Scale Evolution of Myxoma Virus Reveals Host-Pathogen Adaptation and Rapid Geographic Spread 
Journal of Virology  2013;87(23):12900-12915.
The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.
doi:10.1128/JVI.02060-13
PMCID: PMC3838154  PMID: 24067966
9.  Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range 
Virology Journal  2014;11:97.
Background
The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes.
Findings
Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d’Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 – 588 nt) of unknown function in the 5’ region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication.
Conclusions
In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.
doi:10.1186/1743-422X-11-97
PMCID: PMC4038087  PMID: 24884700
Nidoviruses; Mesonivirus; Insect-specific; Phylogeny; Next generation sequencing
10.  Widespread Colonization of the Lung by Tropheryma whipplei in HIV Infection 
Rationale: Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV.
Objectives: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota.
Methods: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects.
Measurements and Main Results: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple’s disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple’s disease.
Conclusions: Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.
doi:10.1164/rccm.201211-2145OC
PMCID: PMC3734615  PMID: 23392441
human; microbiome; metagenome; 16S ribosomal RNA; bronchoalveolar lavage
11.  Comparison of the Respiratory Microbiome in Healthy Nonsmokers and Smokers 
Rationale: Results from 16S rDNA-encoding gene sequence–based, culture-independent techniques have led to conflicting conclusions about the composition of the lower respiratory tract microbiome.
Objectives: To compare the microbiome of the upper and lower respiratory tract in healthy HIV-uninfected nonsmokers and smokers in a multicenter cohort.
Methods: Participants were nonsmokers and smokers without significant comorbidities. Oral washes and bronchoscopic alveolar lavages were collected in a standardized manner. Sequence analysis of bacterial 16S rRNA-encoding genes was performed, and the neutral model in community ecology was used to identify bacteria that were the most plausible members of a lung microbiome.
Measurements and Main Results: Sixty-four participants were enrolled. Most bacteria identified in the lung were also in the mouth, but specific bacteria such as Enterobacteriaceae, Haemophilus, Methylobacterium, and Ralstonia species were disproportionally represented in the lungs compared with values predicted by the neutral model. Tropheryma was also in the lung, but not the mouth. Mouth communities differed between nonsmokers and smokers in species such as Porphyromonas, Neisseria, and Gemella, but lung bacterial populations did not.
Conclusions: This study is the largest to examine composition of the lower respiratory tract microbiome in healthy individuals and the first to use the neutral model to compare the lung to the mouth. Specific bacteria appear in significantly higher abundance in the lungs than would be expected if they originated from the mouth, demonstrating that the lung microbiome does not derive entirely from the mouth. The mouth microbiome differs in nonsmokers and smokers, but lung communities were not significantly altered by smoking.
doi:10.1164/rccm.201210-1913OC
PMCID: PMC3734620  PMID: 23491408
lung; microbiome; smoking; bronchoscopy; metagenome
12.  Comparative Analysis of the Complete Genome Sequence of the California MSW Strain of Myxoma Virus Reveals Potential Host Adaptations 
Journal of Virology  2013;87(22):12080-12089.
Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.
doi:10.1128/JVI.01923-13
PMCID: PMC3807925  PMID: 23986601
13.  Mammalian Adaptation in the PB2 Gene of Avian H5N1 Influenza Virus 
Journal of Virology  2013;87(19):10884-10888.
The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host range determinant for infection of mammals. Here, we report that the E-to-K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tracts of mice and was complete by day 6 postinoculation. This mutation correlated with efficient replication of the virus in mice.
doi:10.1128/JVI.01016-13
PMCID: PMC3807384  PMID: 23864613
14.  Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design 
Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.
Author Summary
With the adaptation of chikungunya virus (CHIKV) to transmission by the Aedes albopictus mosquito, a pandemic has occurred resulting in four to six million human infections, and the virus continues to become endemic in new regions, most recently in the Caribbean. CHIKV can cause debilitating polyarthralgia, lasting for weeks to years, and there are currently no licensed vaccines or antiviral therapies available. While an investigational live-attenuated vaccine (LAV) exists, problems with reactogenicity have precluded its licensure. The purpose of the current study was to: i) devise an in vitro passage procedure that reliably generates a panel of CHIKV envelope glycoprotein mutations for screening as vaccine candidates; ii) determine the position of the mutations in the three-dimensional structure of the alphavirus spike complex and their effect on electrostatic potential; iii) determine the attenuation characteristics of each mutation in a murine model of CHIKV musculoskeletal disease; and iv) to identify in vitro assays examining the dependency of infection upon HS that correlate with attenuation and localization in the glycoprotein spike. This approach provides a paradigm for the rational design of future LAVs for CHIKV and other mosquito-borne viruses, by deliberately selecting and combining attenuating processes.
doi:10.1371/journal.pntd.0002719
PMCID: PMC3930508  PMID: 24587470
15.  Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus 
Journal of Virology  2013;87(14):8064-8074.
Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.
doi:10.1128/JVI.00240-13
PMCID: PMC3700204  PMID: 23678180
16.  Presence of Oseltamivir-Resistant Pandemic A/H1N1 Minor Variants Before Drug Therapy With Subsequent Selection and Transmission 
The Journal of Infectious Diseases  2012;206(10):1504-1511.
A small proportion (1%–1.5%) of 2009 pandemic influenza A/H1N1 virus strains (A[H1N1]pdm09) are oseltamivir resistant, almost exclusively because of a H275Y mutation in the neuraminidase protein. However, many individuals infected with resistant strains had not received antivirals. Whether drug-resistant viruses are initially present as minor variants in untreated individuals before they emerge as the dominant strain in a virus population is of great importance for predicting the speed at which resistance will arise. To address this issue, we used ultra-deep sequencing of viral populations from serial nasopharyngeal specimens from an immunocompromised child and from 2 individuals in a household outbreak. We observed that the Y275 mutation was present as a minor variant in infected hosts before the onset of therapy. We also found evidence for the transmission of this drug-resistant variant with drug-susceptible viruses. These observations provide important information on the relative fitness of the Y275 mutation in the absence of oseltamivir treatment.
doi:10.1093/infdis/jis571
PMCID: PMC3475640  PMID: 22966122
17.  Migratory Flyway and Geographical Distance are Barriers to the Gene Flow of Influenza Virus among North American Birds 
Ecology letters  2011;15(1):24-33.
Despite the importance of migratory birds in the ecology and evolution of avian influenza virus (AIV), there is a lack of information on the patterns of AIV spread at the intra-continental scale. We applied a variety of statistical phylogeographic techniques to a plethora of viral genome sequence data to determine the strength, pattern, and determinants of gene flow in AIV sampled from wild birds in North America. These analyses revealed a clear isolation-by-distance of AIV among sampling localities. In addition, we show that phylogeographic models incorporating information on the avian flyway of sampling proved a better fit to the observed sequence data than those specifying homogeneous or random rates of gene flow among localities. In sum, these data strongly suggest that the intra-continental spread of AIV by migratory birds is subject to major ecological barriers, including spatial distance and avian flyway.
doi:10.1111/j.1461-0248.2011.01703.x
PMCID: PMC3228906  PMID: 22008513
avian influenza; phylogeography; evolution; gene flow; ecological barriers; flyways; spatial distance
18.  Large-scale sequencing and the natural history of model human RNA viruses 
Future virology  2012;7(6):563-573.
RNA virus exploration within the field of medical virology has greatly benefited from technological developments in genomics, deepening our understanding of viral dynamics and emergence. Large-scale first-generation technology sequencing projects have expedited molecular epidemiology studies at an unprecedented scale for two pathogenic RNA viruses chosen as models: influenza A virus and dengue. Next-generation sequencing approaches are now leading to a more in-depth analysis of virus genetic diversity, which is greater for RNA than DNA viruses because of high replication rates and the absence of proofreading activity of the RNA-dependent RNA polymerase. In the field of virus discovery, technological advancements and metagenomic approaches are expanding the catalogs of novel viruses by facilitating our probing into the RNA virus world.
doi:10.2217/fvl.12.45
PMCID: PMC3653332  PMID: 23682295
dengue; influenza; intrahost diversity; metagenomics; molecular epidemiology; next-generation sequencing; viral genomics
19.  A Potential Role for the Interaction of Wolbachia Surface Proteins with the Brugia malayi Glycolytic Enzymes and Cytoskeleton in Maintenance of Endosymbiosis 
The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium of the genus Wolbachia. The Wolbachia represent an attractive target for the control of filarial induced disease as elimination of the bacteria affects molting, reproduction and survival of the worms. The molecular basis for the symbiotic relationship between Wolbachia and their filarial hosts has yet to be elucidated. To identify proteins involved in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other bacterial systems. Two WSP-like proteins (wBm0152 and wBm0432) were localized to various host tissues of the B. malayi female adult worms and are present in the excretory/secretory products of the worms. We provide evidence that both of these proteins bind specifically to B. malayi crude protein extracts and to individual filarial proteins to create functional complexes. The wBm0432 interacts with several key enzymes involved in the host glycolytic pathway, including aldolase and enolase. The wBm0152 interacts with the host cytoskeletal proteins actin and tubulin. We also show these interactions in vitro and have verified that wBm0432 and B. malayi aldolase, as well as wBm0152 and B. malayi actin, co-localize to the vacuole surrounding Wolbachia. We propose that both WSP protein complexes interact with each other via the aldolase-actin link and/or via the possible interaction between the host's enolase and the cytoskeleton, and play a role in Wolbachia distribution during worm growth and embryogenesis.
Author Summary
The human filarial parasite Brugia malayi harbors a Wolbachia endosymbiotic bacterium that is required for normal reproduction and development. However, the molecular basis of how this essential endosymbiotic relationship is maintained is not understood. As a first step in trying to understand the molecular interactions that might be essential in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other systems. Our aim was to determine whether there are any functional interactions between some of these WSPs and the proteins produced by the host parasite cells. We found that two of the WSP family members specifically interact with proteins produced by the host. Wolbachia wBm0432 interacted with several key enzymes involved in the host glycolytic pathway, the primary energy-producing pathway in the cell. Wolbachia wBm0152 interacted with the host cytoskeleton. These findings suggest that WSP family proteins might play important roles in both optimization of the energy production pathway in B. malayi as well as in anchoring the endosymbiont to the host's cytoskeleton.
doi:10.1371/journal.pntd.0002151
PMCID: PMC3617236  PMID: 23593519
20.  Analysis of Transcriptional Regulation of Tetracycline Responsive Genes in Brugia malayi 
The Wolbachia endosymbiont of the human filarial parasites is necessary for parasite reproduction, making it an attractive chemotherapeutic target. Previous studies have demonstrated that mRNA levels of several nuclearly encoded genes are altered as a result of exposure to antibiotics that eliminate the endosymbiont, suggesting that they may be involved in maintaining the parasite-endosymbiont relationship. Here, we tested the hypothesis that the increase in mRNA levels of certain nuclearly encoded genes of Brugia malayi in response to tetracycline treatment involved specific regulatory elements present in the promoters of these genes. The promoters of three such genes (BmRPL13, BmRPS4 and BmHSP70) were tested for tetracycline responsiveness utilizing a homologous transient transcription system. Reporter gene expression driven by all three promoters was up-regulated in transfected embryos exposed to tetracycline. Substitution mutagenesis was employed to map the cis-acting elements responsible for this response in the BmHSP70 promoter. Tetracycline responsiveness was found to be distinct from the cis-acting elements involved in regulating the stress response from the BmHSP70 promoter; rather, tetracycline responsiveness was mediated by a TATAA-box like element. This study represents the first demonstration of small molecule-mediated gene regulation of a native B. malayi promoter.
doi:10.1016/j.molbiopara.2011.09.004
PMCID: PMC3205945  PMID: 21944995
filariasis; transfection; promoter; Wolbachia
21.  Evolutionary History and Attenuation of Myxoma Virus on Two Continents 
PLoS Pathogens  2012;8(10):e1002950.
The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.
Author Summary
The text-book example of the evolution of virulence is the attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe in the 1950s. However, the key work on this topic, most notably by Frank Fenner and his colleagues, occurred before the availability of genome sequence data. The evolutionary genetic basis to the major changes in virulence in both the Australian and European epidemics is therefore largely unknown. We provide, for the first time, key details on the genome-wide changes that underpin this landmark example of pathogen emergence and virulence evolution. By sequencing and comparing MYXV genomes, including the original strains released in the 1950s, we show that (i) MYXV evolved rapidly in both Australia and Europe, producing one of the highest rates of evolutionary change ever recorded for a DNA virus, (ii) that changes in virulence were caused by mutations in multiple genes, often involving losses of gene function due to insertions and deletions, and that (iii) strains of the same virulence were defined by different mutations, such that both attenuated and virulent MYXV strains are produced by a variety genetic pathways, and generating convergent evolution for phenotype but not genotype.
doi:10.1371/journal.ppat.1002950
PMCID: PMC3464225  PMID: 23055928
22.  Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi 
International journal for parasitology  2011;41(10):1053-1061.
The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesize that they might be involved in the Wolbachia-Brugia symbiotic relationship. Notably, immunolocalization studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localization by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.
doi:10.1016/j.ijpara.2011.05.008
PMCID: PMC3160744  PMID: 21782817
Filariasis; Brugia malayi; Wolbachia; Symbiotic relationship; Wolbachia surface protein; Phage display panning
23.  Molecular characterization of a new species in the genus Alphacoronavirus associated with mink epizootic catarrhal gastroenteritis 
The Journal of General Virology  2011;92(Pt 6):1369-1379.
A coronavirus (CoV) previously shown to be associated with catarrhal gastroenteritis in mink (Mustela vison) was identified by electron microscopy in mink faeces from two fur farms in Wisconsin and Minnesota in 1998. A pan-coronavirus and a genus-specific RT-PCR assay were used initially to demonstrate that the newly discovered mink CoVs (MCoVs) were members of the genus Alphacoronavirus. Subsequently, using a random RT-PCR approach, full-genomic sequences were generated that further confirmed that, phylogenetically, the MCoVs belonged to the genus Alphacoronavirus, with closest relatedness to the recently identified but only partially sequenced (fragments of the polymerase, and full-length spike, 3c, envelope, nucleoprotein, membrane, 3x and 7b genes) ferret enteric coronavirus (FRECV) and ferret systemic coronavirus (FRSCV). The molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with epizootic catarrhal gastroenteritis outbreaks in mink and demonstrate that MCoVs possess high genomic variability and relatively low overall nucleotide sequence identities (91.7 %) between contemporary strains. Additionally, the new MCoVs appeared to be phylogenetically distant from human (229E and NL63) and other alphacoronaviruses and did not belong to the species Alphacoronavirus 1. It is proposed that, together with the partially sequenced FRECV and FRSCV, they comprise a new species within the genus Alphacoronavirus.
doi:10.1099/vir.0.025353-0
PMCID: PMC3168282  PMID: 21346029
24.  In vivo transfection of developmentally competent Brugia malayi infective larvae✯ 
Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.
doi:10.1016/j.ijpara.2010.10.005
PMCID: PMC3046311  PMID: 21118694
Filariasis; Transfection; HSP70; Transgenesis; Gaussia; Luciferase
25.  Deep Sequencing Reveals Mixed Infection with 2009 Pandemic Influenza A (H1N1) Virus Strains and the Emergence of Oseltamivir Resistance 
The Journal of Infectious Diseases  2011;203(2):168-174.
Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.
doi:10.1093/infdis/jiq040
PMCID: PMC3071067  PMID: 21288815

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