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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  Extensive Geographical Mixing of 2009 Human H1N1 Influenza A Virus in a Single University Community▿† 
Journal of Virology  2011;85(14):6923-6929.
Despite growing interest in the molecular epidemiology of influenza virus, the pattern of viral spread within individual communities remains poorly understood. To determine the phylogeography of influenza virus in a single population, we examined the spatial diffusion of H1N1/09 influenza A virus within the student body of the University of California, San Diego (UCSD), sampling for a 1-month period between October and November 2009. Despite the highly focused nature of our study, an analysis of complete viral genome sequences revealed between 24 and 33 independent introductions of H1N1/09 into the UCSD community, comprising much of the global genetic diversity in this virus. These data were also characterized by a relatively low level of on-campus transmission as well as extensive spatial mixing, such that there was little geographical clustering by either student residence or city ZIP code. Most notably, students experiencing illness on the same day and residing in the same dorm possessed phylogenetically distinct lineages. H1N1/09 influenza A virus is therefore characterized by a remarkable spatial fluidity, which is likely to impede community-based methods for its control, including class cancellations, quarantine, and chemoprophylaxis.
doi:10.1128/JVI.00438-11
PMCID: PMC3126550  PMID: 21593168
18.  A Deep Sequencing Approach to Comparatively Analyze the Transcriptome of Lifecycle Stages of the Filarial Worm, Brugia malayi 
Background
Developing intervention strategies for the control of parasitic nematodes continues to be a significant challenge. Genomic and post-genomic approaches play an increasingly important role for providing fundamental molecular information about these parasites, thus enhancing basic as well as translational research. Here we report a comprehensive genome-wide survey of the developmental transcriptome of the human filarial parasite Brugia malayi.
Methodology/Principal Findings
Using deep sequencing, we profiled the transcriptome of eggs and embryos, immature (≤3 days of age) and mature microfilariae (MF), third- and fourth-stage larvae (L3 and L4), and adult male and female worms. Comparative analysis across these stages provided a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite. Genome-wide assessment of the overall transcriptional variability indicated that the cuticle collagen family and those implicated in molting exhibit noticeably dynamic stage-dependent patterns. Of particular interest was the identification of genes displaying sex-biased or germline-enriched profiles due to their potential involvement in reproductive processes. The study also revealed discrete transcriptional changes during larval development, namely those accompanying the maturation of MF and the L3 to L4 transition that are vital in establishing successful infection in mosquito vectors and vertebrate hosts, respectively.
Conclusions/Significance
Characterization of the transcriptional program of the parasite's lifecycle is an important step toward understanding the developmental processes required for the infectious cycle. We find that the transcriptional program has a number of stage-specific pathways activated during worm development. In addition to advancing our understanding of transcriptome dynamics, these data will aid in the study of genome structure and organization by facilitating the identification of novel transcribed elements and splice variants.
Author Summary
Lymphatic filariasis, also known as elephantiasis, is a tropical disease affecting over 120 million people worldwide. More than 40 million people live with painful, disfiguring symptoms that can cause severe debilitation and social stigma. The disease is caused by infection with thread-like filarial nematodes (roundworms) that have a complex parasitic lifecycle involving both human and mosquito hosts. In the study, the authors profiled the transcriptome (the set of genes transcribed into messenger RNA rather than all of those in the genome) of the human filarial worm Brugia malayi in different lifecyle stages using deep sequencing technology. The analysis revealed major transitions in RNA expression from eggs through larval stages to adults. Using statistical approaches, the authors identified groups of genes with distinct life stage dependent transcriptional patterns, with particular emphasis on genes displaying sex-biased or germline-enriched patterns and those displaying significant changes during larval development. This study presents a first comprehensive analysis of the lifecycle transcriptome of B. malayi, providing fundamental molecular information that should help researchers better understand parasite biology and could provide clues for the development of more effective interventions.
doi:10.1371/journal.pntd.0001409
PMCID: PMC3236722  PMID: 22180794
19.  The early diversification of influenza A/H1N1pdm 
PLoS Currents  2009;1:RRN1126.
Background Since its initial detection in April 2009, the A/H1N1pdm influenza virus has spread rapidly in humans, with over 5,700 human deaths. However, little is known about the evolutionary dynamics of H1N1pdm and its geographic and temporal diversification.
Methods Phylogenetic analysis was conducted upon the concatenated coding regions of whole-genome sequences from 290 H1N1pdm isolates sampled globally between April 1 – July 9, 2009, including relatively large samples from the US states of Wisconsin and New York.
Results At least 7 phylogenetically distinct viral clades have disseminated globally and co-circulated in localities that experienced multiple introductions of H1N1pdm. The epidemics in New York and Wisconsin were dominated by two different clades, both phylogenetically distinct from the viruses first identified in California and Mexico, suggesting an important role for founder effects in determining local viral population structures.
Conclusions Determining the global diversity of H1N1pdm is central to understanding the evolution and spatial spread of the current pandemic, and to predict its future impact on human populations. Our results indicate that H1N1pdm has already diversified into distinct viral lineages with defined spatial patterns.
doi:10.1371/currents.RRN1126
PMCID: PMC2773564  PMID: 20029664
20.  Phylogeography of the Spring and Fall Waves of the H1N1/09 Pandemic Influenza Virus in the United States▿ §  
Journal of Virology  2010;85(2):828-834.
Spatial variation in the epidemiological patterns of successive waves of pandemic influenza virus in humans has been documented throughout the 20th century but never understood at a molecular level. However, the unprecedented intensity of sampling and whole-genome sequencing of the H1N1/09 pandemic virus now makes such an approach possible. To determine whether the spring and fall waves of the H1N1/09 influenza pandemic were associated with different epidemiological patterns, we undertook a large-scale phylogeographic analysis of viruses sampled from three localities in the United States. Analysis of genomic and epidemiological data reveals distinct spatial heterogeneities associated with the first pandemic wave, March to July 2009, in Houston, TX, Milwaukee, WI, and New York State. In Houston, no specific H1N1/09 viral lineage dominated during the spring of 2009, a period when little epidemiological activity was observed in Texas. In contrast, major pandemic outbreaks occurred at this time in Milwaukee and New York State, each dominated by a different viral lineage and resulting from strong founder effects. During the second pandemic wave, beginning in August 2009, all three U.S. localities were dominated by a single viral lineage, that which had been dominant in New York during wave 1. Hence, during this second phase of the pandemic, extensive viral migration and mixing diffused the spatially defined population structure that had characterized wave 1, amplifying the one viral lineage that had dominated early on in one of the world's largest international travel centers.
doi:10.1128/JVI.01762-10
PMCID: PMC3020026  PMID: 21068250
21.  Genomic and Protein Structural Maps of Adaptive Evolution of Human Influenza A Virus to Increased Virulence in the Mouse 
PLoS ONE  2011;6(6):e21740.
Adaptive evolution is characterized by positive and parallel, or repeated selection of mutations. Mouse adaptation of influenza A virus (IAV) produces virulent mutants that demonstrate positive and parallel evolution of mutations in the hemagglutinin (HA) receptor and non-structural protein 1 (NS1) interferon antagonist genes. We now present a genomic analysis of all 11 genes of 39 mouse adapted IAV variants from 10 replicate adaptation experiments. Mutations were mapped on the primary and structural maps of each protein and specific mutations were validated with respect to virulence, replication, and RNA polymerase activity. Mouse adapted (MA) variants obtained after 12 or 20–21 serial infections acquired on average 5.8 and 7.9 nonsynonymous mutations per genome of 11 genes, respectively. Among a total of 115 nonsynonymous mutations, 51 demonstrated properties of natural selection including 27 parallel mutations. The greatest degree of parallel evolution occurred in the HA receptor and ribonucleocapsid components, polymerase subunits (PB1, PB2, PA) and NP. Mutations occurred in host nuclear trafficking factor binding sites as well as sites of virus-virus protein subunit interaction for NP, NS1, HA and NA proteins. Adaptive regions included cap binding and endonuclease domains in the PB2 and PA polymerase subunits. Four mutations in NS1 resulted in loss of binding to the host cleavage and polyadenylation specificity factor (CPSF30) suggesting that a reduction in inhibition of host gene expression was being selected. The most prevalent mutations in PB2 and NP were shown to increase virulence but differed in their ability to enhance replication and demonstrated epistatic effects. Several positively selected RNA polymerase mutations demonstrated increased virulence associated with >300% enhanced polymerase activity. Adaptive mutations that control host range and virulence were identified by their repeated selection to comprise a defined model for studying IAV evolution to increased virulence in the mouse.
doi:10.1371/journal.pone.0021740
PMCID: PMC3128085  PMID: 21738783
23.  Functional analysis of putative operons in Brugia malayi 
Operons are a common mode of gene organization in Caenorhabditis elegans. Similar gene arrangements suggest that functional operons may exist in Brugia malayi. To definitively test this hypothesis, a bicistronic reporter vector consisting of an upstream firefly luciferase gene and a downstream renilla luciferase gene was constructed. The genome was then surveyed to identify 16 gene pairs that were likely to represent operons. Two of four domains upstream of the 5’ gene from these clusters exhibited promoter activity. When constructs replicating the promoter and intergenic arrangement found in the native putative operon were transfected into embryos, both firefly and renilla activities were detected, while constructs with the promoter alone or intergenic region alone produced no activity from the downstream reporter. These data confirm that functional operons exist in B. malayi. Mutation of three U-rich element homologues present in one of the operons resulted in a decrease in downstream renilla reporter activity, suggesting that these were important in mRNA maturation. Hemi-nested reverse transcriptase-PCR assays demonstrated that while the mRNA encoding the native downstream open reading frame of one operon contained an SL1 spliced leader at its 5’ end, the renilla gene mRNA produced from the corresponding transgenic construct did not.
doi:10.1016/j.ijpara.2009.07.001
PMCID: PMC2813416  PMID: 19631652
Filariasis; Trans-splicing; Transfection; Biolositics; Operon
24.  Unseasonal Transmission of H3N2 Influenza A Virus During the Swine-Origin H1N1 Pandemic▿ †  
Journal of Virology  2010;84(11):5715-5718.
The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.
doi:10.1128/JVI.00018-10
PMCID: PMC2876599  PMID: 20237080
25.  Mixed Infection and the Genesis of Influenza Virus Diversity▿  
Journal of Virology  2009;83(17):8832-8841.
The emergence of viral infections with potentially devastating consequences for human health is highly dependent on their underlying evolutionary dynamics. One likely scenario for an avian influenza virus, such as A/H5N1, to evolve to one capable of human-to-human transmission is through the acquisition of genetic material from the A/H1N1 or A/H3N2 subtypes already circulating in human populations. This would require that viruses of both subtypes coinfect the same cells, generating a mixed infection, and then reassort. Determining the nature and frequency of mixed infection with influenza virus is therefore central to understanding the emergence of pandemic, antigenic, and drug-resistant strains. To better understand the potential for such events, we explored patterns of intrahost genetic diversity in recently circulating strains of human influenza virus. By analyzing multiple viral genome sequences sampled from individual influenza patients we reveal a high level of mixed infection, including diverse lineages of the same influenza virus subtype, drug-resistant and -sensitive strains, those that are likely to differ in antigenicity, and even viruses of different influenza virus types (A and B). These results reveal that individuals can harbor influenza viruses that differ in major phenotypic properties, including those that are antigenically distinct and those that differ in their sensitivity to antiviral agents.
doi:10.1128/JVI.00773-09
PMCID: PMC2738154  PMID: 19553313

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