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1.  A prospective study of freezing of gait with early Parkinson disease in Chinese patients 
Medicine  2016;95(26):e4056.
Supplemental Digital Content is available in the text
This study investigated the risk factors for freezing of gait (FOG) in the early stage of Parkinson disease in China, using a sample of 248 patients who were followed for 3 years. Part III of the Unified Parkinson Disease Rating Scale and the modified Hoehn-Yahr grading scale were used to evaluate the severity of motor symptoms. Nonmotor symptoms were assessed using the Hamilton Anxiety Rating Scale, Hamilton Depression Rating Scale (HAMD), and Non-Motor Symptoms Scale (NMSS). The end-point was the presence of FOG at the end of follow-up; patients with FOG were classified as freezers. The risk factors for FOG were analyzed at the end of the first, second, and third years after baseline. There were 40 freezers (16.13%) 1 year later, 98 (39.52%) 2 years later, and 128 (51.61%) 3 years later. FOG 3 years later was associated with the following variables: depression (P = 0.003), older age, living in the countryside, lower education, akinetic-rigid style, lower limbs as site of onset, early use of levodopa, higher daily dose of levodopa, and not using amantadine or selegiline and dopamine receptor agonists (P < 0.001). Early use of amantadine, selegiline, and dopamine receptor agonists was negatively related to FOG (P < 0.001). Binary logistic regression found that FOG was associated with lower education (odds ratio [OR] = 0.012, P < 0.001), akinetic-rigid style (OR = 4.881, P = 0.024), not using dopamine receptor agonists (OR = 4.324, P = 0.035), cognitive disturbances (OR = 0.331, P = 0.007), and sleep disorders (OR = 2.418, P = 0.036). However, the cardiovascular domain of the NMSS (OR = 2.729, P = 0.001) was the only risk factor for FOG 1 year later. Two years later, FOG was associated with mixed style (OR = 0.189, P = 0.005), lower limbs as site of onset (OR = 4.772, P = 0.008), not using dopamine receptor agonists (OR = 0.031, P < 0.001), and the anxiety/somatic domain of the HAMD (OR = 0.596, P = 0.033). Scores at baseline, patients with Parkinson disease were more likely to experience FOG if: they were older, or from the countryside; had an akinetic-rigid style, anxiety, or higher NMSS scores; they used levodopa early or did not use amantadine or selegiline; their lower limbs were the site of onset; or they had more severe motor disability or higher HAMD scores at baseline.
PMCID: PMC4937955  PMID: 27368041
freezing of gait; Parkinson disease; risk factors
2.  Extreme Precipitation and Emergency Room Visits for Gastrointestinal Illness in Areas with and without Combined Sewer Systems: An Analysis of Massachusetts Data, 2003–2007 
Environmental Health Perspectives  2015;123(9):873-879.
Combined sewer overflows (CSOs) occur in combined sewer systems when sewage and stormwater runoff are released into water bodies, potentially contaminating water sources. CSOs are often caused by heavy precipitation and are expected to increase with increasing extreme precipitation associated with climate change.
The aim of this study was to assess whether the association between heavy rainfall and rate of emergency room (ER) visits for gastrointestinal (GI) illness differed in the presence of CSOs.
For the study period 2003–2007, time series of daily rate of ER visits for GI illness and meteorological data were organized for three exposure regions: a) CSOs impacting drinking water sources, b) CSOs impacting recreational waters, c) no CSOs. A distributed lag Poisson regression assessed cumulative effects for an 8-day lag period following heavy (≥ 90th and ≥ 95th percentile) and extreme (≥ 99th percentile) precipitation events, controlling for temperature and long-term time trends.
The association between extreme rainfall and rate of ER visits for GI illness differed among regions. Only the region with drinking water exposed to CSOs demonstrated a significant increased cumulative risk for rate (CRR) of ER visits for GI for all ages in the 8-day period following extreme rainfall: CRR: 1.13 (95% CI: 1.00, 1.28) compared with no rainfall.
The rate of ER visits for GI illness was associated with extreme precipitation in the area with CSO discharges to a drinking water source. Our findings suggest an increased risk for GI illness among consumers whose drinking water source may be impacted by CSOs after extreme precipitation.
Jagai JS, Li Q, Wang S, Messier KP, Wade TJ, Hilborn ED. 2015. Extreme precipitation and emergency room visits for gastrointestinal illness in areas with and without combined sewer systems: an analysis of Massachusetts data, 2003–2007. Environ Health Perspect 123:873–879;
PMCID: PMC4559956  PMID: 25855939
3.  Comparison of the effectiveness of duloxetine in depressed patients with and without a family history of affective disorders in first-degree relatives 
Shanghai Archives of Psychiatry  2015;27(4):237-245.
It remains unclear whether or not a positive family history of affective disorders predicts the effectiveness of antidepressant treatment of depression.
Assess the relationship of a family history of affective disorders to the efficacy of duloxetine in the treatment of depressive disorder.
Seventy-seven patients with depressive disorder (as defined by the 10th edition of the International Classification of Diseases, ICD-10) were enrolled in the study and treated with standard doses of duloxetine for 12 weeks. Among these patients 37 had a family history of affective disorder in first-degree relatives and 40 did not. The Hamilton Depression rating scale (HAMD-17), Hamilton Anxiety rating scale (HAMA), Side Effects Rating Scale (SERS), Snaith-Hamilton Pleasure Scale (SHAPS), and Beck Depression Inventory (BDI) were assessed at baseline and at the end of the 2nd, 4th, 6th, 8th, and 12th week after enrollment. Repeated measures analysis of variance and logistic regression were used to analyze the association between a family history of affective disorders and the efficacy of duloxetine.
Patients with a positive family history of affective disorders had an earlier age of onset, a longer duration of illness, a higher level of psychic anxiety, and more prominent anhedonia. Repeated measures analysis of variance showed a significant improvement in the severity of depression over the 12 weeks but no differences in the magnitude or speed of improvement between the two groups. Treatment was considered effective (i.e., drop in baseline HAMD-17 total score of ≥50%) in 75.7% of those with a family history of affective disorders and in 77.5% of those without a family history (X2=0.04, p=0.850).
Family history of affective disorders is not associated with the effectiveness of duloxetine in the acute treatment of depressive disorder.
PMCID: PMC4621289  PMID: 26549960
family history;  depressive disorders;  duloxetine; China
4.  Social Media as a Sensor of Air Quality and Public Response in China 
Recent studies have demonstrated the utility of social media data sources for a wide range of public health goals, including disease surveillance, mental health trends, and health perceptions and sentiment. Most such research has focused on English-language social media for the task of disease surveillance.
We investigated the value of Chinese social media for monitoring air quality trends and related public perceptions and response. The goal was to determine if this data is suitable for learning actionable information about pollution levels and public response.
We mined a collection of 93 million messages from Sina Weibo, China’s largest microblogging service. We experimented with different filters to identify messages relevant to air quality, based on keyword matching and topic modeling. We evaluated the reliability of the data filters by comparing message volume per city to air particle pollution rates obtained from the Chinese government for 74 cities. Additionally, we performed a qualitative study of the content of pollution-related messages by coding a sample of 170 messages for relevance to air quality, and whether the message included details such as a reactive behavior or a health concern.
The volume of pollution-related messages is highly correlated with particle pollution levels, with Pearson correlation values up to .718 (n=74, P<.001). Our qualitative results found that 67.1% (114/170) of messages were relevant to air quality and of those, 78.9% (90/114) were a firsthand report. Of firsthand reports, 28% (32/90) indicated a reactive behavior and 19% (17/90) expressed a health concern. Additionally, 3 messages of 170 requested that action be taken to improve quality.
We have found quantitatively that message volume in Sina Weibo is indicative of true particle pollution levels, and we have found qualitatively that messages contain rich details including perceptions, behaviors, and self-reported health effects. Social media data can augment existing air pollution surveillance data, especially perception and health-related data that traditionally requires expensive surveys or interviews.
PMCID: PMC4400579  PMID: 25831020
air pollution; public health surveillance; social media; data mining; text mining; natural language processing
5.  Sequencing and Analyses of All Known Human Rhinovirus Genomes Reveal Structure and Evolution 
Science (New York, N.Y.)  2009;324(5923):55-59.
Infection by human rhinovirus (HRV) is a major cause of upper and lower respiratory tract disease worldwide and displays considerable phenotypic variation. We examined diversity by completing the genome sequences for all known serotypes (n = 99). Superimposition of capsid crystal structure and optimal-energy RNA configurations established alignments and phylogeny. These revealed conserved motifs; clade-specific diversity, including a potential newly identified species (HRV-D); mutations in field isolates; and recombination. In analogy with poliovirus, a hypervariable 5′ untranslated region tract may affect virulence. A configuration consistent with nonscanning internal ribosome entry was found in all HRVs and may account for rapid translation. The data density from complete sequences of the reference HRVs provided high resolution for this degree of modeling and serves as a platform for full genome-based epidemiologic studies and antiviral or vaccine development.
PMCID: PMC3923423  PMID: 19213880
6.  VIGOR extended to annotate genomes for additional 12 different viruses 
Nucleic Acids Research  2012;40(Web Server issue):W186-W192.
A gene prediction program, VIGOR (Viral Genome ORF Reader), was developed at J. Craig Venter Institute in 2010 and has been successfully performing gene calling in coronavirus, influenza, rhinovirus and rotavirus for projects at the Genome Sequencing Center for Infectious Diseases. VIGOR uses sequence similarity search against custom protein databases to identify protein coding regions, start and stop codons and other gene features. Ribonucleicacid editing and other features are accurately identified based on sequence similarity and signature residues. VIGOR produces four output files: a gene prediction file, a complementary DNA file, an alignment file, and a gene feature table file. The gene feature table can be used to create GenBank submission. VIGOR takes a single input: viral genomic sequences in FASTA format. VIGOR has been extended to predict genes for 12 viruses: measles virus, mumps virus, rubella virus, respiratory syncytial virus, alphavirus and Venezuelan equine encephalitis virus, norovirus, metapneumovirus, yellow fever virus, Japanese encephalitis virus, parainfluenza virus and Sendai virus. VIGOR accurately detects the complex gene features like ribonucleicacid editing, stop codon leakage and ribosomal shunting. Precisely identifying the mat_peptide cleavage for some viruses is a built-in feature of VIGOR. The gene predictions for these viruses have been evaluated by testing from 27 to 240 genomes from GenBank.
PMCID: PMC3394299  PMID: 22669909
7.  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.
PMCID: PMC3168282  PMID: 21346029
8.  Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury 
PLoS ONE  2012;7(4):e34946.
Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.
Methodology/Principal Findings
Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.
The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.
PMCID: PMC3329538  PMID: 22529961
9.  Genetic Dissection of the Canq1 Locus Governing Variation in Extent of the Collateral Circulation 
PLoS ONE  2012;7(3):e31910.
Native (pre-existing) collaterals are arteriole-to-arteriole anastomoses that interconnect adjacent arterial trees and serve as endogenous bypass vessels that limit tissue injury in ischemic stroke, myocardial infarction, coronary and peripheral artery disease. Their extent (number and diameter) varies widely among mouse strains and healthy humans. We previously identified a major quantitative trait locus on chromosome 7 (Canq1, LOD = 29) responsible for 37% of the heritable variation in collateral extent between C57BL/6 and BALB/c mice. We sought to identify candidate genes in Canq1 responsible for collateral variation in the cerebral pial circulation, a tissue whose strain-dependent variation is shared by similar variation in other tissues.
Methods and Findings
Collateral extent was intermediate in a recombinant inbred line that splits Canq1 between the C57BL/6 and BALB/c strains. Phenotyping and SNP-mapping of an expanded panel of twenty-one informative inbred strains narrowed the Canq1 locus, and genome-wide linkage analysis of a SWRxSJL-F2 cross confirmed its haplotype structure. Collateral extent, infarct volume after cerebral artery occlusion, bleeding time, and re-bleeding time did not differ in knockout mice for two vascular-related genes located in Canq1, IL4ra and Itgal. Transcript abundance of 6 out of 116 genes within the 95% confidence interval of Canq1 were differentially expressed >2-fold (p-value<0.05÷150) in the cortical pia mater from C57BL/6 and BALB/c embryos at E14.5, E16.5 and E18.5 time-points that span the period of collateral formation.
These findings refine the Canq1 locus and identify several genes as high-priority candidates important in specifying native collateral formation and its wide variation.
PMCID: PMC3295810  PMID: 22412848
10.  Genetic Architecture Underlying Variation in Extent and Remodeling of the Collateral Circulation 
Circulation research  2010;107(4):558-568.
Collaterals are arteriole-to-arteriole anastomoses that connect adjacent arterial trees. They lessen ischemic tissue injury by serving as endogenous bypass vessels when the trunk of one tree becomes narrowed by vascular disease. The number and diameter (“extent”) of native (pre-existing) collaterals, plus their amount of lumen enlargement (growth/remodeling) in occlusive disease, show remarkably wide variation among inbred mouse strains (eg, C57BL/6 and BALB/c), resulting in large differences in tissue injury in models of occlusive disease. Evidence suggests similar large differences exist among healthy humans.
To identify candidate loci responsible for genetic-dependent collateral variation.
Methods and Results
Cerebral collateral number and diameter were determined in 221 C57BL/6-x-BALB/c F2 progeny, followed by linkage analysis to identify quantitative trait loci (QTL) for collateral number and diameter. Four QTL were obtained for collateral number, including epistasis between two loci. A QTL that was identical to the strongest QTL for collateral number on chromosome 7 (LOD=29, effect size=37%) was also mapped for collateral diameter (LOD=17, effect size=30%). Chromosome substitution strain analysis confirmed this locus. We also obtained a unique QTL on chromosome 11 for collateral remodeling after middle cerebral artery occlusion. Association mapping within the chromosome 7 QTL interval using collateral traits measured for 15 inbred strains, delineated 172k (p=0.00002) and 290k (p=0.0004) base-pair regions on chromosome 7 containing 2 and 9 candidate genes, respectively.
We conclude that collateral extent and remodeling are unique, highly heritable complex traits, with one QTL predominantly affecting native collateral number and diameter.
PMCID: PMC2924933  PMID: 20576932
Collateral vessels; genetics; quantitative trait loci; cerebral circulation; arteriogenesis
11.  QTL Underlying Voluntary Exercise in Mice: Interactions with the “Mini Muscle” Locus and Sex 
Journal of Heredity  2009;101(1):42-53.
Exercise improves many aspects of human health, yet many people remain inactive even when exercise is prescribed. We previously created a backcross (BC) between mice selectively bred for high levels of voluntary wheel running (VWR) and fixed for “mini muscle” (MM), a recessive mutation causing ∼50% reduction in triceps surae mass. We previously showed that BC mice having the MM trait ran faster and further than mice without MM and that MM maps to chromosome 11. Here, we genotyped the BC with genome-wide single nucleotide polymorphisms to identify quantitative trait loci (QTL) controlling voluntary exercise and tissue and body mass traits and to determine whether these QTL interact with the MM locus or with sex. We detected 3 VWR QTL, representing the first voluntary exercise QTL mapped using this high running selection line, and 5 tissue mass QTL. Several interactions between trait QTL and the MM locus as well as sex were also identified. These results begin to explain the genetic architecture of VWR and further support MM as a locus having major effects, including its main effects on the muscle phenotype, its pleiotropic effects on wheel running and tissue mass traits, and through its interactions with other QTL and with sex.
PMCID: PMC2800800  PMID: 19666993
artificial selection; complex trait; genetic architecture; physical activity; running
12.  Genomic mapping of social behavior traits in a F2 cross derived from mice selectively bred for high aggression 
BMC Genetics  2010;11:113.
Rapid response to selection was previously observed in mice selected for high levels of inter-male aggression based on number of attacks displayed in a novel social interaction test after isolation housing. Attack levels in this high aggression line (NC900) increased significantly within just four generations of selective breeding, suggesting the presence of a locus with large effect. We conducted an experiment using a small (n ≈ 100) F2 cross between the ICR-derived, non-inbred NC900 strain and the low aggression inbred strain C57BL/6J, genotyped for 154 fully informative SNPs, to determine if a locus with large effect controls the high-aggression selection trait. A second goal was to use high density SNP genotyping (n = 549,000) in the parental strains to characterize residual patterns of heterozygosity within NC900, and evaluate regions that are identical by descent (IBD) between NC900 and C57BL/6J, to determine what impacts these may have on accuracy and resolution of quantitative trait locus (QTL) mapping in the F2 cross.
No evidence for a locus with major effect on aggressive behavior in mice was identified. However, several QTL with genomewide significance were mapped for aggression on chromosomes 7 and 19 and other social behavior traits on chromosomes 4, 7, 14, and 19. High density genotyping revealed that 28% of the genome is still segregating among the six NC900 females used to originate the F2 cross, and that segregating regions are present on every chromosome but are of widely different sizes. Regions of IBD between NC900 and C57BL/6J are found on every chromosome but are most prominent on chromosomes 10, 16 and X. No significant differences were found for amounts of heterozygosity or prevalence of IBD in QTL regions relative to global analysis.
While no major gene was identified to explain the rapid selection response in the NC900 line, transgressive variation (i.e. where the allele from the C57BL/6J increased attack levels) and a significant role for dominant gene action were hallmarks of the genetic architecture for aggressive behavior uncovered in this study. The high levels of heterozygosity and the distribution of minor allele frequency observed in the NC900 population suggest that maintenance of heterozygosity may have been under selection in this line.
PMCID: PMC3022667  PMID: 21194443
13.  VIGOR, an annotation program for small viral genomes 
BMC Bioinformatics  2010;11:451.
The decrease in cost for sequencing and improvement in technologies has made it easier and more common for the re-sequencing of large genomes as well as parallel sequencing of small genomes. It is possible to completely sequence a small genome within days and this increases the number of publicly available genomes. Among the types of genomes being rapidly sequenced are those of microbial and viral genomes responsible for infectious diseases. However, accurate gene prediction is a challenge that persists for decoding a newly sequenced genome. Therefore, accurate and efficient gene prediction programs are highly desired for rapid and cost effective surveillance of RNA viruses through full genome sequencing.
We have developed VIGOR (Viral Genome ORF Reader), a web application tool for gene prediction in influenza virus, rotavirus, rhinovirus and coronavirus subtypes. VIGOR detects protein coding regions based on sequence similarity searches and can accurately detect genome specific features such as frame shifts, overlapping genes, embedded genes, and can predict mature peptides within the context of a single polypeptide open reading frame. Genotyping capability for influenza and rotavirus is built into the program. We compared VIGOR to previously described gene prediction programs, ZCURVE_V, GeneMarkS and FLAN. The specificity and sensitivity of VIGOR are greater than 99% for the RNA viral genomes tested.
VIGOR is a user friendly web-based genome annotation program for five different viral agents, influenza, rotavirus, rhinovirus, coronavirus and SARS coronavirus. This is the first gene prediction program for rotavirus and rhinovirus for public access. VIGOR is able to accurately predict protein coding genes for the above five viral types and has the capability to assign function to the predicted open reading frames and genotype influenza virus. The prediction software was designed for performing high throughput annotation and closure validation in a post-sequencing production pipeline.
PMCID: PMC2942859  PMID: 20822531
14.  Evolutionary Dynamics of Human Rotaviruses: Balancing Reassortment with Preferred Genome Constellations 
PLoS Pathogens  2009;5(10):e1000634.
Group A human rotaviruses (RVs) are a major cause of severe gastroenteritis in infants and young children. Yet, aside from the genes encoding serotype antigens (VP7; G-type and VP4; P-type), little is known about the genetic make-up of emerging and endemic human RV strains. To gain insight into the diversity and evolution of RVs circulating at a single location over a period of time, we sequenced the eleven-segmented, double-stranded RNA genomes of fifty-one G3P[8] strains collected from 1974 to 1991 at Children's Hospital National Medical Center, Washington, D. C. During this period, G1P[8] strains typically dominated, comprising on average 56% of RV infections each year in hospitalized children. A notable exception was in the 1976 and 1991 winter seasons when the incidence of G1P[8] infections decreased dramatically, a trend that correlated with a significant increase in G3P[8] infections. Our sequence analysis indicates that the 1976 season was characterized by the presence of several genetically distinct, co-circulating clades of G3P[8] viruses, which contained minor but significant differences in their encoded proteins. These 1976 lineages did not readily exchange gene segments with each other, but instead remained stable over the course of the season. In contrast, the 1991 season contained a single major clade, whose genome constellation was similar to one of the 1976 clades. The 1991 clade may have gained a fitness advantage after reassorting with as of yet unidentified RV strain(s). This study reveals for the first time that genetically distinct RV clades of the same G/P-type can co-circulate and cause disease. The findings from this study also suggest that, although gene segment exchange occurs, most reassortant strains are replaced over time by lineages with preferred genome constellations. Elucidation of the selective pressures that favor maintenance of RVs with certain sets of genes may be necessary to anticipate future vaccine needs.
Author Summary
Rotaviruses are the most important cause of severe diarrhea in infants and young children. Due to the segmented nature of their genomes, rotaviruses can exchange (reassort) genes during co-infections, a feature that is predicted to generate new, possibly more dangerous virus strains. However, the amount of gene reassortment occurring in nature is not known, as very few rotavirus genomes have been sequenced. To better understand the genetic make-up of rotaviruses circulating at a single location over a period of time, we sequenced the genomes of fifty-one isolates recovered from sick children from 1974 to 1991 at Children's Hospital National Medical Center, Washington, D. C. By analyzing these sequences, we found that several distinct groups (clades) of rotaviruses co-circulated and caused disease in a single epidemic season. In contrast to what was previously thought, very few rotaviruses exchanged gene segments with each other; instead, the genome constellations of the viruses remained relatively stable. We also discovered that these distinct rotavirus clades encode different viral proteins, which may be important in the development of effective vaccines. Together, the findings from this first large-scale rotavirus genomics project provide unparalleled insight into how these pathogens evolve during their spread through the human population.
PMCID: PMC2760143  PMID: 19851457
15.  Bovine-Like Coronaviruses Isolated from Four Species of Captive Wild Ruminants Are Homologous to Bovine Coronaviruses, Based on Complete Genomic Sequences▿  
Journal of Virology  2006;82(24):12422-12431.
We sequenced and analyzed the full-length genomes of four coronaviruses (CoVs), each from a distinct wild-ruminant species in Ohio: sambar deer (Cervus unicolor), a waterbuck (Kobus ellipsiprymnus), a sable antelope (Hippotragus niger), and a white-tailed deer (Odocoileus virginianus). The fecal samples from the sambar deer, the waterbuck, and the white-tailed deer were collected during winter dysentery outbreaks and sporadic diarrhea cases in 1993 and 1994 (H. Tsunemitsu, Z. R. el-Kanawati, D. R. Smith, H. H. Reed, and L. J. Saif, J. Clin. Microbiol. 33:3264-3269, 1995). A fecal sample from a sable antelope was collected in 2003 from an Ohio wild-animal habitat during the same outbreak when a bovine-like CoV from a giraffe (GiCoV) was isolated (M. Hasoksuz, K. Alekseev, A. Vlasova, X. Zhang, D. Spiro, R. Halpin, S. Wang, E. Ghedin, and L. J. Saif, J. Virol. 81:4981-4990, 2007). For two of the CoVs (sambar deer and waterbuck), complete genomes from both the cell culture-adapted and gnotobiotic-calf-passaged strains were also sequenced and analyzed. Phylogenetically, wild-ruminant CoVs belong to group 2a CoVs, with the closest relatedness to recent bovine CoV (BCoV) strains. High nucleotide identities (99.4 to 99.6%) among the wild-ruminant strains and recent BCoV strains (BCoV-LUN and BCoV-ENT, isolated in 1998) further confirm the close relatedness. Comparative genetic analysis of CoVs of captive wild ruminants with BCoV strains suggests that no specific genomic markers are present that allow discrimination between the bovine strains and bovine-like CoVs from captive wild ruminants; furthermore, no specific genetic markers were identified that defined cell cultured or calf-passaged strains or the host origin of strains. The results of this study confirm prior reports of biologic and antigenic similarities between bovine and wild-ruminant CoVs and suggest that cattle may be reservoirs for CoVs that infect captive wild ruminants or vice versa and that these CoVs may represent host range variants of an ancestral CoV.
PMCID: PMC2593316  PMID: 18842722
16.  Draft Genome of the Filarial Nematode Parasite Brugia malayi 
Science (New York, N.Y.)  2007;317(5845):1756-1760.
Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ~90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ~11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ~350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.
PMCID: PMC2613796  PMID: 17885136
17.  Complete genomic sequences, a key residue in the spike protein and deletions in non-structural protein 3b of US strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus 
Virology  2006;358(2):424-435.
Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of porcine respiratory coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506-706) that elicites neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522-744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.
PMCID: PMC1850758  PMID: 17023013
transmissible gastroenteritis virus; porcine respiratory coronavirus; complete genomic sequence; virulence; attenuation; tropism shift
18.  Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets 
PLoS ONE  2007;2(11):e1189.
We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.
PMCID: PMC2063515  PMID: 18000556
19.  Biologic, Antigenic, and Full-Length Genomic Characterization of a Bovine-Like Coronavirus Isolated from a Giraffe▿  
Journal of Virology  2007;81(10):4981-4990.
Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.
PMCID: PMC1900194  PMID: 17344285
20.  The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode 
PLoS Biology  2005;3(4):e121.
Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.
Analysis of this Wolbachia genome, which resides within filarial parasites, offers insight into endosymbiont evolution and the promise of new strategies for the elimination of human filarial disease
PMCID: PMC1069646  PMID: 15780005

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