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1.  Population Structure in Nontypeable Haemophilus influenzae 
Nontypeable Haemophilus influenzae (NTHi) frequently colonize the human pharynx asymptomatically, and are an important cause of otitis media in children. Past studies have identified typeable H. influenzae as being clonal, but the population structure of NTHi has not been extensively characterized. The research presented here investigated the diversity and population structure in a well-characterized collection of NTHi isolated from the middle ears of children with otitis media or the pharynges of healthy children in three disparate geographic regions. Multilocus sequence typing identified 109 unique sequence types among 170 commensal and otitis media-associated NTHi isolates from Finland, Israel, and the US. The largest clonal complex contained only five sequence types, indicating a high level of genetic diversity. The eBURST v3, ClonalFrame 1.1, and structure 2.3.3 programs were used to further characterize diversity and population structure from the sequence typing data. Little clustering was apparent by either disease state (otitis media or commensalism) or geography in the ClonalFrame phylogeny. Population structure was clearly evident, with support for eight populations when all 170 isolates were analyzed. Interestingly, one population contained only commensal isolates, while two others consisted solely of otitis media isolates, suggesting associations between population structure and disease.
doi:10.1016/j.meegid.2012.11.023
PMCID: PMC4053184  PMID: 23266487
Nontypeable Haemophilus influenzae; NTHi; Population structure; Otitis media; Multilocus sequence typing; MLST
2.  Polymorphisms in the SUFU gene are Associated with Organ Injury Protection and Sepsis Severity in Patients with Enterobacteriacea Bacteremia 
Background
Organ injury including acute kidney injury (AKI) and acute lung Injury (ALI) are major contributors to mortality and morbidity in the setting of sepsis. Hedgehog pathway has been recognized as an important mediator in repair of organ injury. There are some clinical predictors associated with the development of organ injury in sepsis; however few host genetic risk factors have been identified and candidate genes for organ injury susceptibility and severity are largely unknown.
Methods
A prospective cohort study in a tertiary care hospital included 250 adult hospitalized patients with Enterobacteriacea bacteremia. We selected a panel of 69 tagging SNPs for genes in the Hedgehog signaling pathway using the TagSNP functionality of the SNPInfo web server and designed a panel on the GoldenGate Veracode genotyping assay (Illumina). We confirmed Illumina data using Taqman allelic discrimination assays. We assessed SNPs in combination with clinical variables for associations with outcomes and organ injury.
Results
Significant associations were identified using logistic regression models, controlling for age, race and gender. From the 69 tagging SNPs, 5 SNPs were associated with renal function and 2 with APACHEII score after false discovery rate correction. After multivariate analysis SNPs rs10786691 (p=0.03), rs12414407 (p=0.026), rs10748825 (p=0.01), and rs7078511 (p=0.006), all in the suppressor of fused homolog (SUFU) gene, correlated with renal function. Likewise, SUFU SNPs rs7907760 (p=0.009) and rs10748825 (p=0.029) were associated with APACHEII score. SNPs rs12414407 and rs1078825 are in linkage disequilibrium (LD) with rs2296590, a SNP in the 5′-UTR region that is within a predicted transcription factor bind site for CCAAT-enhancer-binding proteins. In multivariate analyses functional SNP rs2296590 was correlated with renal function (p=0.004) and APACHEII score (p=0.049).
Conclusions
Host susceptibility factors play an important role in sepsis development and sepsis related organ injury. Polymorphisms in the SUFU gene (encoding for a negative regulator of the hedgehog signaling pathway) are associated with protection from Enterobacteriacea bacteremia related organ injury and sepsis severity.
doi:10.1016/j.meegid.2013.03.025
PMCID: PMC3669235  PMID: 23538333
Gram negative sepsis; organ injury; polymorphism; SUFU
3.  The Myxovirus Resistance A (MxA) Gene −88G>T Single Nucleotide Polymorphism Is Associated with Prostate Cancer 
Background
Myxovirus (influenza virus) resistance A (MxA) is an interferon stimulated antiviral protein that is required for a complete antiviral response. MxA polymorphism (rs2071430) is located within an Interferon Stimulated Response Element (ISRE) at position −88 in the gene’s promoter region, and it has been associated with increased susceptibility to infections and various diseases. In general, the low promoter activity genotype (GG) promotes susceptibility, whereas the high promoter activity genotype (TT) confers protection to Hepatitis C viral infection. MxA’s role in prostate cancer is not fully understood. Previous literature has shown that MxA may be a mediator of the effect of IFN on normal and tumor cell motility. MxA may act as a tumor suppressor and the level of expression may be a predictor of metastatic potential. Based on this information, in this study we investigated the association of this functional polymorphism (rs2071430) in MxA with prostate cancer.
Methods
Sample size and power was calculated using the PGA software. Genomic DNA from a controls (n=140) and prostate cancer patients (n=164) were used for genotyping SNP rs2071430 on all samples. Statistical analysis was performed using logistic regression model.
Results
A significant association was observed between rs2071430 genotype GG and prostate cancer. Individuals harboring the GG genotype are at an increased risk of prostate cancer. Data stratification reveals that the mutant GT genotype offers either offers some protection against prostate cancer in Caucasians.
Conclusions
MxA SNP rs2071430 GG genotype is significantly associated with prostate cancer irrespective of race. However, data stratification also suggests that the GT genotype is under-represented in Caucasian subjects suggesting its role in protection against prostate cancer in Caucasians. Although MxA is primarily implicated in viral infection, but it may be also be associated with prostate cancer. Recent studies have implicated viral and bacterial infections with increased prostate cancer risk. Expression of the high promoter activity genotype may offer resistance to prostate cancer infection and possibly influence clinical outcomes.
doi:10.1016/j.meegid.2013.02.010
PMCID: PMC3669225  PMID: 23438650
MxA; Prostate; Cancer; polymorphism; rs2071430
4.  Cytogenetic analysis of Anopheles ovengensis revealed high structural divergence of chromosomes in the Anopheles nili group 
Cytogenetic analysis is an informative classical approach to understanding the relationships among members in a group of closely related species of mosquitoes. Anopheles ovengensis is a recently discovered species of the An. nili group and is one of the important malaria vectors in the African equatorial forest. This study characterized polytene chromosomes of An. ovengensis and compared them with polytene chromosomes of An. nili. Using fluorescent in situ hybridization and chromosome banding pattern comparison we have established correspondence between chromosomal arms of An. ovengensis and An. nili. Analysis of chromosome morphology in the two species revealed a limited similarity in the banding patterns. The most extensive reorganization occurs in pericentromeric and intercalary heterochromatin. Chromosomes of An. ovengensis are joined together by a diffuse chromocenter and they have two large regions of intercalary heterochromatin in arms 2L and 3R. In contrast, the chromocenter and intercalary heterochromatin are not seen in An. nili chromosomes. Comparative analysis of the arm association suggests the occurrence of a whole-arm translocation between the two members of the group. The observed, substantial reorganizations of chromosome structure implies either a rapid rate of chromosome evolution in the An. nili group, or that the two species belong to different taxonomic groups within subgenus Cellia.
doi:10.1016/j.meegid.2013.03.010
PMCID: PMC3669242  PMID: 23523820
malaria mosquito; Anopheles ovengensis; Anopheles nili; polytene chromosomes; fluorescent in situ hybridization
5.  No evidence for positive selection at two potential targets for malaria transmission-blocking vaccines in Anopheles gambiae s.s 
Human malaria causes nearly a million deaths in sub-Saharan Africa each year. The evolution of drug resistance in the parasite and insecticide resistance in the mosquito vector has complicated control measures and made the need for new control strategies more urgent. Anopheles gambiae s.s. is one of the primary vectors of human malaria in Africa, and parasite-transmission-blocking vaccines targeting Anopheles proteins have been proposed as a possible strategy to control the spread of the disease. However, the success of these hypothetical technologies would depend on the successful ability to broadly target mosquito populations that may be genetically heterogeneous. Understanding the evolutionary pressures shaping genetic variation among candidate target molecules offers a first step towards evaluating the prospects of successfully deploying such technologies. We studied the population genetics of genes encoding two candidate target proteins, the salivary gland protein saglin and the basal lamina structural protein laminin, in wild populations of the M and S molecular forms of A. gambiae in Mali. Through analysis of intraspecific genetic variation and interspecific comparisons, we found no evidence of positive natural selection at the genes encoding these proteins. On the contrary, we found evidence for particularly strong purifying selection at the laminin gene. These results provide insight into the patterns of genetic diversity of saglin and laminin, and we discuss these findings in relation to the potential development of these molecules as vaccine targets.
doi:10.1016/j.meegid.2013.01.006
PMCID: PMC3763813  PMID: 23357581
Malaria; Anopheles gambiae; Transmission-blocking vaccine; Plasmodium falciparum; Natural selection; Population genetics
6.  Natural selection maintains a stable polymorphism at the circumsporozoite protein locus of Plasmodium falciparum in a low endemic area 
Circumsporozoite protein gene sequences of Plasmodium falciparum were collected in 1996–1997 and in 2006–2007 from a single endemic area in Thailand. Repeat units were more similar within the same haplotype than between haplotypes, supporting the hypothesis that repeat arrays evolve by a process of concerted evolution. There was evidence that natural selection has favored amino acid changes in the Th2R and Th3R T-cell epitope regions. One haplotype in these epitopes, designated *5/*1, occurred in approximately 70% of sequences in both collection periods. The most common other haplotypes differed from *5/*1 by at least two amino acid replacements; and divergence in the epitopes was correlated with divergence in the repeats. These patterns are most consistent with balancing selection driven by interactions with the immune system of the vertebrate host, probably involving both T-cell recognition of the Th2R and Th3R epitopes and antibody responses to the repeats.
doi:10.1016/j.meegid.2009.02.006
PMCID: PMC4009382  PMID: 19460323
Balancing selection; Circumsporozoite protein; Concerted evolution; Genetic polymorphism; Malaria; Plasmodium falciparum
7.  Non-ribosomal phylogenetic exploration of Mollicute species: New insights into haemoplasma taxonomy 
Infection, Genetics and Evolution  2014;23(100):99-105.
Highlights
•The first gapA and dnaK phylogenetic analysis of Mollicute species.•Analysis includes a wide range of haemoplasmas species.•Analysis supports that haemoplasmas reside within a single clade.•Evidence of phylogenetic distance between the haemoplasmas and Mycoplasma species.
Nine species of uncultivable haemoplasmas and several Mycoplasma species were examined by partial sequencing of two protein-encoding housekeeping genes. Partial glyceraldehyde-3-phosphate dehydrogenase (gapA) and heat shock protein 70 (dnaK) gene sequences were determined for these Mollicute species; in total nine gapA sequences and ten dnaK sequences were obtained. Phylogenetic analyses of these sequences, along with those of a broad selection of Mollicute species downloaded from GenBank, for the individual genes, and for the gapA and dnaK concatenated data set, revealed a clear separation of the haemoplasmas from other species within the Mycoplasma genus; indeed the haemoplasmas resided within a single clade which was phylogenetically detached from the pneumoniae group of Mycoplasmas. This is the first report to examine the use of gapA and dnaK, as well as a concatenated data set, for phylogenetic analysis of the haemoplasmas and other Mollicute species. These results demonstrate a distinct phylogenetic separation between the haemoplasmas and Mycoplasmas that corresponds with the biological differences observed in these species, indicating that further evaluation of the haemoplasmas’ relationship with the Mycoplasma genus is required to determine whether reclassification of the haemoplasmas is necessary.
doi:10.1016/j.meegid.2014.02.001
PMCID: PMC3988868  PMID: 24518692
Mycoplasma; Phylogeny; gapA; dnaK; Hemotropic Mycoplasmas
8.  Comparative characterization of the virulence gene clusters (lipooligosacharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species 
Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosacharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.
doi:10.1016/j.meegid.2012.12.010
PMCID: PMC3622452  PMID: 23279811
Campylobacter; virulence gene clusters; lipooligosacharide; capsular polysaccharide; lateral gene transfer; genomics
9.  Mycobacterium leprae in Colombia described by SNP7614 in gyrA, two minisatellites and geography 
New cases of leprosy are still being detected in Colombia after the country declared achievement of the WHO defined ‘elimination’ status. To study the ecology of leprosy in endemic regions, a combination of geographic and molecular tools were applied for a group of 201 multibacillary patients including six multi-case families from eleven departments. The location (latitude and longitude) of patient residences were mapped. Slit skin smears and/or skin biopsies were collected and DNA was extracted. Standard agarose gel electrophoresis following a multiplex PCR-was developed for rapid and inexpensive strain typing of M. leprae based on copy numbers of two VNTR minisatellite loci 27-5 and 12-5. A SNP (C/T) in gyrA (SNP7614) was mapped by introducing a novel PCR-RFLP into an ongoing drug resistance surveillance effort. Multiple genotypes were detected combining the three molecular markers. The two frequent genotypes in Colombia were SNP7614(C)/27-5(5)/12-5(4) [C54] predominantly distributed in the Atlantic departments and SNP7614 (T)/27-5(4)/12-5(5) [T45] associated with the Andean departments. A novel genotype SNP7614 (C)/27-5(6)/12-5(4) [C64] was detected in cities along the Magdalena river which separates the Andean from Atlantic departments; a subset was further characterized showing association with a rare allele of minisatellite 23-3 and the SNP type 1 of M. leprae. The genotypes within intra-family cases were conserved. Overall, this is the first large scale study that utilized simple and rapid assay formats for identification of major strain types and their distribution in Colombia. It provides the framework for further strain type discrimination and geographic information systems as tools for tracing transmission of leprosy.
doi:10.1016/j.meegid.2012.12.015
PMCID: PMC3668693  PMID: 23291420
Leprosy; Mycobacterium leprae; SNP; gyrA; SNP7614; VNTR
10.  Molecular, ethno-spatial epidemiology of leprosy in China: Novel insights for tracing leprosy in endemic and non endemic provinces 
Leprosy continues to be detected at near stable rates in China even with established control programs, necessitating new knowledge and alternative methods to interrupt transmission. A molecular epidemiology investigation of 190 patients was undertaken to define M. leprae strain types and discern genetic relationships and clusters in endemic and non-endemic regions spanning seventeen provinces and two autonomous regions. The findings support multiple locus variable number of tandem repeat (VNTR) analysis as a useful tool in uncovering characteristic patterns across the multiethnic and divergent geographic landscape of China. Several scenarios of clustering of leprosy from township to provincial to regional levels were recognized, while recent occupational or remote migration showed geographical separation of certain strains. First, prior studies indicated that of the four major M. leprae subtypes defined by single nucleotide polymorphisms (SNPs), only type 3 was present in China, purportedly entering from Europe/West/Central Asia via the Silk Road. However, this study revealed VNTR linked strains that are of type 1 in Guangdong, Fujian and Guangxi in southern China. Second, a subset of VNTR distinguishable strains of type 3, co-exist in these provinces. Third, type 3 strains with rpoT VNTR allele of 4, detected in Japan and Korea were discovered in Jiangsu and Anhui in the east and in western Sichuan bordering Tibet. Fourth, considering the overall genetic diversity, strains of endemic counties of Qiubei, Yunnan; Xing Yi, Guizhou; and across Sichuan in southwest were related. However, closer inspection showed distinct local strains and clusters. Altogether, these insights, primarily derived from VNTR typing, reveal multiple and overlooked paths for spread of leprosy into, within and out of China and invoke attention to historic maritime routes in the South and East China Sea. More importantly, new concepts and approaches for prospective case finding and tracking of leprosy from county to national level have been introduced.
doi:10.1016/j.meegid.2012.12.009
PMCID: PMC3668695  PMID: 23291419
VNTR; Strain typing; Mycobacterium leprae; Leprosy; Transmission; Phylogeography; SNP typing; Maritime silk routes
11.  Inter and intra-specific diversity of parasites that cause lymphatic filariasis 
Lymphatic filariasis is caused by three closely related nematode parasites: Wuchereria bancrofti, Brugia malayi and Brugia timori. These species have many ecological variants that differ in several aspects of their biology such as mosquito vector species, host range, periodicity, and morphology. Although the genome of B. malayi (the first genome sequenced from a parasitic nematode) has been available for more than five years, very little is known about genetic variability among the lymphatic dwelling filariae. The genetic diversity among these worms is not only interesting from a biological perspective, but it may have important practical implications for the Global Program to Eliminate Lymphatic Filariasis, as the parasites may respond differently to diagnostic tests and/or medical interventions. Therefore, better information on their genetic variability is urgently needed. With improved methods for nucleic acid extraction and recent advances in sequencing chemistry and instrumentation, this gap can be filled relatively inexpensively. Improved information on filarial genetic diversity may increase the chances of success for lymphatic filariasis elimination programs.
doi:10.1016/j.meegid.2012.11.002
PMCID: PMC3801268  PMID: 23201850
12.  A nuclear single-nucleotide polymorphism (SNP) potentially useful for the separation of Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae) 
The design and application of rational strategies that rely on accurate species identification are pivotal for effective vector control. When morphological identification of the target vector species is impractical, the use of molecular markers is required. Here we describe a non-coding, single-copy nuclear DNA fragment that contains a single-nucleotide polymorphism (SNP) with the potential to distinguish the important domestic Chagas disease vector, Rhodnius prolixus, from members of the four sylvatic Rhodnius robustus cryptic species complex. A total of 96 primer pairs obtained from whole genome shotgun sequencing of the R. prolixus genome (12,626 random reads) were tested on 43 R. prolixus and R. robustus s.l. samples. One of the seven amplicons selected (AmpG) presented a SNP, potentially diagnostic for R. prolixus, on the 280th site. The diagnostic nature of this SNP was then performed on 154 R. prolixus and R. robustus s.l. samples aimed at achieving the widest possible geographic coverage. The results of a 60% majority rule Bayesian consensus tree and a median-joining network constructed based on the genetic variability observed reveal the paraphyletic nature of the R. robustus species complex, with respect to R. prolixus. AmpG region is located in the fourth intron of the Transmembrane protein 165 gene, which seems to be in the R. prolixus X chromosome. Other possible chromosomal locations of the AmpG region in the R. prolixus genome are also presented and discussed.
doi:10.1016/j.meegid.2012.10.018
PMCID: PMC3883588  PMID: 23219914
Rhodnius; cryptic species; SNP; nuclear DNA
13.  Global distribution and diversity of ovine-associated Staphylococcus aureus☆ 
Infection, Genetics and Evolution  2014;22(100):208-215.
Highlights
•97 global ovine S. aureus isolates characterised using MLST and spa typing.•Majority of 261 global ovine isolates belong to one of three clonal complexes (CC).•One CC has spread across the New World; two are restricted to Europe and Africa.•Clonal complex spread matches the route and timing of sheep domestication.•Patterns of clonal diversification of sheep isolates differ from human isolates.
Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n = 153), Africa (n = 28), South America (n = 14) and Australia (n = 1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump.
doi:10.1016/j.meegid.2013.09.008
PMCID: PMC3969713  PMID: 24035790
S. aureus; Ovine; Caprine; MLST; spa typing; Global diversity
14.  Multiple locus VNTR analysis highlights that geographical clustering and distribution of Dichelobacter nodosus, the causal agent of footrot in sheep, correlates with inter-country movements☆ 
Infection, Genetics and Evolution  2014;22(100):273-279.
Highlights
•Development of a portable MLVA technique to characterise Dichelobacter nodosus.•Seventy-seven isolates from three continents typed.•Isolates from countries with a long and short history of footrot.•Allele distribution between countries matches historical accounts of sheep movement.•D. nodosus has evolved via recombinational exchanges and clonal diversification.
Dichelobacter nodosus is a Gram-negative, anaerobic bacterium and the causal agent of footrot in sheep. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is a portable technique that involves the identification and enumeration of polymorphic tandem repeats across the genome. The aims of this study were to develop an MLVA scheme for D. nodosus suitable for use as a molecular typing tool, and to apply it to a global collection of isolates. Seventy-seven isolates selected from regions with a long history of footrot (GB, Australia) and regions where footrot has recently been reported (India, Scandinavia), were characterised. From an initial 61 potential VNTR regions, four loci were identified as usable and in combination had the attributes required of a typing method for use in bacterial epidemiology: high discriminatory power (D > 0.95), typeability and reproducibility. Results from the analysis indicate that D. nodosus appears to have evolved via recombinational exchanges and clonal diversification. This has resulted in some clonal complexes that contain isolates from multiple countries and continents; and others that contain isolates from a single geographic location (country or region). The distribution of alleles between countries matches historical accounts of sheep movements, suggesting that the MLVA technique is sufficiently specific and sensitive for an epidemiological investigation of the global distribution of D. nodosus.
doi:10.1016/j.meegid.2013.05.026
PMCID: PMC3969714  PMID: 23748018
Dichelobacter nodosus; Global distribution; MLVA; Ovine footrot
15.  Association between Specific HIV-1 Env Traits and Virologic Control in vivo 
HIV RNA levels are influenced by genetic characteristics of both the host and the virus. Here we applied machine learning techniques to determine if plasma-derived HIV-1 amino acid sequences can be used to predict spontaneous virologic control. We studied the relationship between HIV-1 env genotype and viral load in 20 chronically-infected patients undergoing treatment interruptions (SSITT, Swiss-Spanish Intermittent Treatment Trial) and in 104 primary HIV infected (PHI) patients before antiretroviral therapy (cART) and where applicable also after treatment stop. Extensive longitudinal sampling during the interruptions was performed in nine SSITT patients. Sequences obtained from these nine patients during the first virus rebound were used as a training data set and revealed a strong genetic signature (accuracy 98.6% in cross-validation) associated with control of viremia at levels below 5000 copies/mL of viral RNA maintained for at least two months after the final cART stop. The simple sequence pattern at gp120 positions 268E/358T was confirmed to be predictive of control in the clonal sequences originating from these patients during all subsequent rebounds. Sequences from the remaining 11 SSITT patients with less frequent sampling and from the PHI patients were used for external validation. High sensitivities (71-100%) and negative predictive values (80-100%) but low positive predictive values (12-40%) were achieved in the patient-wise analysis which was based on presence of the genetic pattern in all clones. These results suggest that presence of virus lacking the amino acid pattern 268E/358T is associated with VL >5000 at baseline of PHI and with low probability of spontaneous virologic control after treatment stop. Conversely, however, presence of 268E/358T does not predict control of viremia. These residues in HIV gp120 might affect in vivo HIV-1 fitness either at the level of Env function or influence susceptibility to adaptive or innate immune response.
doi:10.1016/j.meegid.2009.06.002
PMCID: PMC3918463  PMID: 19524069
HIV-1; envelope gp120; evolution; machine learning
16.  Phylogeography of Swine Influenza H3N2 in the United States: Translational Public Health for Zoonotic Disease Surveillance 
The field of phylogeography has received a lot of attention for its application to molecular evolution and geographic migration of species. More recent work has included infectious diseases especially zoonotic RNA viruses like influenza and rabies. Phylogeography of viruses has the potential to advance surveillance at agencies such as public health departments, agriculture departments, and wildlife agencies. However, little is known about how these agencies could use phylogeography for applied surveillance and the integration of animal and human sequence data. Here, we highlight its potential to support ‘translational public health’ that could bring sequence data to the forefront of surveillance. We focus on swine influenza H3N2 because of the recent link to a variant form in humans. We discuss the implications to applied surveillance and the need for an integrated biomedical informatics approach for adoption at agencies of animal and public health.
doi:10.1016/j.meegid.2012.09.015
PMCID: PMC3537880  PMID: 23137647
Molecular Sequence Data; Phylogeography; Epidemiology; Zoonoses; Influenza A Virus; H3N2 Subtype
17.  Analysis of salivary gland transcripts of the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis 
The saliva of blood sucking insects contains potent pharmacologically active components that assist them in counteracting the host hemostatic and inflammatory systems during blood feeding. In addition, sand fly salivary proteins affect host immunity and have the potential to be a vaccine against Leishmania infection. In the present study, the salivary gland transcripts of Lutzomyia ayacuchensis, a vector of cutaneous leishmaniasis in Ecuadorian and Peruvian Andes, were analyzed by sequencing randomly selected clones of the salivary gland cDNA library of this sand fly. This resulted in the identification of the most abundant transcripts coding for secreted proteins. These proteins were homologous to the salivary molecules present in other sand flies including the RGD-containing peptide, PpSP15/SL1 family protein, yellow-related protein, putative apyrase, antigen 5-related protein, D7 family protein, and 27 kDa salivary protein. Of note, homologues of maxadilan, an active vasodilator abundantly present in saliva of Lutzomyia longipalpis, were not identified. This analysis is the first description of salivary proteins from a sand fly of the subgenus Helcocyrtomyia and from vector of cutaneous leishmaniasis in the New World. The present analysis will provide further insights into the evolution of salivary components in blood sucking arthropods.
doi:10.1016/j.meegid.2012.08.024
PMCID: PMC3873855  PMID: 23000112
Lutzomyia ayacuchensis; Salivary gland; Transcript; Bioinformatics; cDNA library
18.  Population genetics of the malaria vector Anopheles aconitus in China and Southeast Asia 
Anopheles aconitus is a well-known vector of malaria and is broadly distributed in the Oriental Region, yet there is no information on its population genetic characteristics. In this study, the genetic differentiation among populations was examined using 140 mtDNA COII sequences from 21 sites throughout southern China, Myanmar, Vietnam, Thailand, Laos and Sri Lanka. The population in Sri Lanka has characteristic rDNA D3 and ITS2, mtDNA COII and ND5 haplotypes, and may be considered a distinct subspecies. Clear genetic structure was observed with highly significant genetic variation present among population groups in Southeast Asia. The greatest genetic diversity exists in Yunnan and Myanmar population groups. All population groups are significantly different from one another in pairwise Fst values, except northern Thailand with central Thailand. Mismatch distributions and extremely significant Fs values suggest that the populations passed through a recent demographic expansion. These patterns are discussed in relation to the likely biogeographic history of the region and compared to other Anopheles species.
doi:10.1016/j.meegid.2012.08.007
PMCID: PMC3488592  PMID: 22982161
Anopheles aconitus; subspecies; population structure; demographic expansion; mtDNA; rDNA; China; Southeast Asia
19.  Factors associated with genotype clustering of Mycobacterium tuberculosis isolates in an ethnically diverse region of southern California, United States 
Mycobacterium tuberculosis (Mtb) isolates with identical genotypes, found in different patients, are most likely the result of recent transmission. Mtb strains with closely related genotypes, called clonal complexes, are most likely derived from one another. We examined Mtb genotypes from southern California TB patients from 2005 through 2008 to complete the first comprehensive molecular epidemiology analysis of this complicated and ethnically diverse region. Mtb genotypes were characterized with spoligo-type and MIRU-12 typing. MIRU-VNTRplus was utilized to assign genotypes to global lineages and complete cluster analyses. Associations between patient characteristics and genotype clustering and clonal complexes were evaluated using logistic regression and frequency analysis. Of 832 Mtb isolates analyzed, 480 (58%) fell into 94 strain clusters. The majority of isolates were identified as being in the EA1 (31%), LAM (17%) and Haarlem (15%) lineages, but 13 different lineages were found in this region. TB patients with clustered isolates were more likely to be homeless (AOR 3.44, 95% CI 1.65, 7.18) and male (AOR 1.57, 95% CI 1.17, 2.10). Of the 480 clustered strains, 388 aggregated into six clonal complexes.
Over 45% of reported TB cases were clustered and likely resulted from recent transmission events. Patients with clustered Mtb isolates that were grouped into clonal complexes had unique socio-demographic characteristics. These data suggest that TB is being transmitted in relatively insular community networks defined by race/ethnicity and country of origin. The addition of clonal complex analysis to simple cluster analysis provides important public health insights into the local transmission of TB in ethnically diverse regions with diverse Mtb genotypes.
doi:10.1016/j.meegid.2012.08.022
PMCID: PMC3723456  PMID: 22982156
Tuberculosis; Molecular epidemiology; Social networks; Genotypes; Spoligotype; MIRU
20.  Quantifying variation in the potential for antibody-mediated apparent competition among nine genotypes of the rodent malaria parasite Plasmodium chabaudi☆ 
Infection, Genetics and Evolution  2013;20(100):270-275.
Highlights
•We measure antibody responses induced by 9 genotypes of Plasmodium chabaudi in mice.•In vitro antigens include an exoantigen and 2 recombinant malaria antigens.•Parasite genotypes vary significantly in the magnitude of antibody responses induced.•Cross-reactivity of anti-MSP119 responses is predicted by amino acid homology.•Differential antibody induction may predict the outcome of within-host competition.
Within-host competition among parasite genotypes affects epidemiology as well as the evolution of virulence. In the rodent malaria Plasmodium chabaudi, competition among genotypes, as well as clone-specific and clone-transcending immunity are well documented. However, variation among genotypes in the induction of antibodies is not well understood, despite the important role of antibodies in the clearance of malaria infection. Here, we quantify the potential for antibodies induced by one clone to bind another (i.e., to cause antibody-mediated apparent competition) for nine genetically distinct P. chabaudi clones. We hypothesised that clones would vary in the strength of antibody induction, and that the propensity for clone-transcending immunity between a pair of clones would increase with increasing genetic relatedness at key antigenic loci. Using serum collected from mice 35 days post-infection, we measured titres of antibody to an unrelated antigen, Keyhole Limpet Haemocyanin (KLH), and two malaria antigens: recombinant Apical Membrane Antigen-1 (AMA-1) and Merozoite Surface Protein-119 (MSP-119). Amino acid sequence homology within each antigenic locus was used as a measure of relatedness. We found significant parasite genetic variation for the strength of antibody induction. We also found that relatedness at MSP-119 but not AMA-1 predicted clone-transcending binding. Our results help explain the outcome of chronic-phase mixed infections and generate testable predictions about the pairwise competitive ability of P. chabaudi clones.
doi:10.1016/j.meegid.2013.09.013
PMCID: PMC3898986  PMID: 24056014
Plasmodium chabaudi; Cross-reactivity; Within-host competition; Antibody; Mixed infection; Virulence evolution
21.  Reconstructing the origin and transmission dynamics of the 1967–68 foot-and-mouth disease epidemic in the United Kingdom☆ 
Infection, Genetics and Evolution  2013;20(100):230-238.
Highlights
•Study characterised genomic sequences from archived FMDV samples collected during the 1960s.•The epidemic in 1967–68 occurred as a result of an independent introduction of FMDV into the UK.•Molecular clock used to identify viral sequences that accrued fewer than expected substitutions.•Study supports the use of sequences to trace RNA viruses during outbreaks and epidemics.
A large epidemic of foot-and-mouth disease (FMD) occurred in the United Kingdom (UK) over a seven month period in Northwest England from late 1967 to the summer of 1968. This was preceded by a number of smaller FMD outbreaks in the country, two in 1967, in Hampshire and Warwickshire and one in Northumberland during 1966. The causative agent of all four events was identified as FMD virus (FMDV) serotype O and the source of the large epidemic was attributed to infected bone marrow in lamb products imported from Argentina. However, the diagnostic tools available at the time were unable to entirely rule out connections with the earlier UK FMD outbreaks, as well as other potential sources from Europe. The aim of this study was to apply molecular sequencing to investigate the likely source of this epidemic using VP1 region and full genome (FG) sequences determined directly from clinical epithelium samples (n = 13) or cell culture isolates (n = 6), from this and contemporary outbreaks in the UK, Europe and South America. Analysis of the VP1 sequences provided evidence for at least three separate incursions of FMDV into the UK including one independent introduction that was responsible for the main 1967/68 epidemic. Analysis of FG sequences from the main 1967/68 outbreak (n = 10) revealed nucleotide substitutions at 94 genomic sites providing evidence for the linear accumulation of nucleotide substitutions (rate = 2.42 × 10−5 nt substitutions/site/day). However, there were five samples where this linear relationship was absent, indicating evolutional dormancy of the virus, presumably outside a host. These results help define the evolutionary dynamics of FMDV during an epidemic and contribute to the knowledge and understanding from which to base future outbreak control strategies.
doi:10.1016/j.meegid.2013.09.009
PMCID: PMC3898989  PMID: 24035793
Foot-and-mouth disease; Epidemic; Full-genome sequencing; Phylogenetics; United Kingdom
22.  Intrinsic features of Aedes aegypti genes affect transcriptional responsiveness of mosquito genes to dengue virus infection 
Dengue virus infection causes significant morbidity and mortality in humans world-wide. The Aedes aegypti mosquito is the major vector that spread dengue virus to humans. Interaction between dengue viruses and A. aegypti is a multi-factorial phenomena that is determined by both virus and mosquito genotypes. Although, studies have suggested significant association of mosquito vectorial capacity with population variation of dengue virus, specifications of the vector factors that may influence vector-virus compatibility have are very limited in the literature. Recently, we have shown that a large number of genes are differentially expressed between MOYO-S (susceptible) and MOYO-R (refractory) A. aegypti strains upon infection with dengue virus (JAM-1409 genotype). In the current study, we show that specific intrinsic features of A. aegypti genes are significantly associated with `responsiveness' of mosquito genes to dengue infection. Binomial logistic regression analysis further reveals differential marginal effects of these features on gene responsiveness of mosquito to the viral infection. Thus, our result shows that intrinsic features of genes significantly affect differential expression of A. aegypti genes to dengue infection. The information will benefit further investigation on evolution of genes among natural populations A. aegypti conferring differential susceptibility to dengue virus.
doi:10.1016/j.meegid.2012.04.027
PMCID: PMC3424315  PMID: 22579482
Aedes aegypti; vector-virus interaction; logit; gene context; codon bias; intron; co-evolution
23.  Translational Selection of Genes Coding for Perfectly Conserved Proteins among Three Mosquito Vectors 
The biased usage of synonymous codons affects translational efficiency of genes. We studied codon usage patterns of genes that are perfectly conserved at the amino acid level among three important mosquito vector species: Aedes aegypti (vector of dengue virus), Anopheles gambiae (vector of malaria) and Culex quinquefasciatus (vector of lymphatic filariasis and West Nile Virus). Although these proteins have same amino acid sequences, non-random usage of synonymous codons is evident among the orthologous genes. The coding sequences of these genes were simulated to generate random mutation sites to be further investigated for patterns of codon bias. It was found that codon usage bias is significantly higher in genes that represented perfectly conserved proteins than genes where variation was apparent at the amino acid sequence. Our results suggest that genes coding for perfectly conserved proteins are highly biased with optimized codons and may be under stringent translational selection in these vector species.
doi:10.1016/j.meegid.2012.06.005
PMCID: PMC3428495  PMID: 22705463
Vector mosquitoes; Selection; Codon bias; Translational efficiency; Codon context
24.  Genetic diversity in Treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws 
Pathogenic uncultivable treponemes, similar to syphilis-causing Treponema pallidum subspecies pallidum, include T. pallidum ssp. pertenue, T. pallidum ssp. endemicum and Treponema carateum, which cause yaws, bejel and pinta, respectively. Genetic analyses of these pathogens revealed striking similarity among these bacteria and also a high degree of similarity to the rabbit pathogen, T. paraluiscuniculi, a treponeme not infectious to humans. Genome comparisons between pallidum and non-pallidum treponemes revealed genes with potential involvement in human infectivity, whereas comparisons between pallidum and pertenue treponemes identified genes possibly involved in the high invasivity of syphilis treponemes. Genetic variability within syphilis strains is considered as the basis of syphilis molecular epidemiology with potential to detect more virulent strains, whereas genetic variability within a single strain is related to its ability to elude the immune system of the host. Genome analyses also shed light on treponemal evolution and on chromosomal targets for molecular diagnostics of treponemal infections.
doi:10.1016/j.meegid.2011.12.001
PMCID: PMC3786143  PMID: 22198325
Treponema pallidum; T pallidum ssp. Pertenue; T. pallidum ssp. endemicum; T. paraluiscuniculi; whole genome sequencing; molecular evolution; molecular diagnostics
25.  Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil 
São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n = 612 isolates clustered into groups of 2–84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates.
doi:10.1016/j.meegid.2012.10.015
PMCID: PMC3744825  PMID: 23201043
Mycobacterium tuberculosis; Population-structure; Spoligotyping; Brazil; Database

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