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1.  Retrotransposon OV-RTE-1 from the carcinogenic liver fluke Opisthorchis viverrini; potential target for DNA-based diagnosis 
Infections by the fish-borne liver flukes Opisthorchis viverrini and Clonorchis sinensis can lead to bile duct cancer. These neglected tropical disease pathogens occur in East Asia, with O. viverrini primarily in Thailand and Laos and C. sinensis in Cambodia, Vietnam, and China. Genomic information about these pathogens holds the potential to improve disease treatment and control. Transcriptome analysis indicates that mobile genetic elements are active in O. viverrini, including a novel non-Long Terminal Repeat (LTR) retrotransposon. A consensus sequence of this element, termed OV-RTE-1, was assembled from expressed sequence tags and PCR amplified genomic DNA. OV-RTE-1 was 3,330 bp in length, encoded 1,101 amino acid residues and exhibited hallmark structures and sequences of non-LTR retrotransposons including a single open reading frame encoding apurinic-apyrimidinic endonuclease (EN) and reverse transcriptase (RT). Phylogenetic analyses confirmed that OV-RTE-1 was member of the RTE clade of non-LTR retrotransposons. OV-RTE-1 is the first non-LTR retrotransposon characterized from the trematode family Opisthorchiidae. Sequences of OV-RTE-1 were targeted to develop a diagnostic tool for detection of infection by O. viverrini. PCR specific primers for detection of O. viverrini DNA showed 100% specificity and sensitivity for detection of as little as five femtograms of O. viverrini DNA whereas the PCR based approach showed 62% sensitivity and 100% specificity with clinical stool samples. The OV-RTE-1 specific PCR could be developed as a molecular diagnostic for Opisthorchis infection targeting parasite eggs in stool samples, especially in regions of mixed infection of O. viverrini and/or C. sinensis and minute intestinal flukes.
doi:10.1016/j.meegid.2013.12.015
PMCID: PMC3955739  PMID: 24394447
Opisthorchis viverrini; liver fluke; retrotransposon; diagnosis; feces
2.  Evolution of community- and healthcare-associated methicillin-resistant Staphylococcus aureus☆ 
Staphylococcus aureus is a prominent cause of human infections globally. The high prevalence of infections is compounded by antibiotic resistance—a significant problem for treatment. Methicillin-resistant S. aureus (MRSA) is endemic in hospitals and healthcare facilities worldwide, and is an increasingly common cause of community-associated bacterial infections in industrialized countries. Although much focus is placed on the role of S. aureus as a human pathogen, it is in fact a human commensal organism that has had a relatively long coexistence with the human host. Many S. aureus infections can be explained by host susceptibility or other predisposing risk factors. On the other hand, the emergence/re-emergence of successful S. aureus clones (referred to as epidemic waves) suggests a rapid bacterial adaption and evolution, which includes the emergence of antibiotic resistance and increased virulence and/or transmissibility. It is within this context that we review our understanding of selected S. aureus epidemic waves, and highlight the use of genome sequencing as a means to better understand the evolution of each lineage.
doi:10.1016/j.meegid.2013.04.030
PMCID: PMC3884050  PMID: 23648426
Staphylococcus aureus; MRSA; Epidemic; Genome sequencing; Antimicrobial resistance
3.  Comparative Genomics of Oral Isolates of Streptococcus mutans by in silico Genome Subtraction Does Not Reveal Accessory DNA Associated with Severe Early Childhood Caries 
Comparative genomics is a popular method for the identification of microbial virulence determinants, especially since the sequencing of a large number of whole bacterial genomes from pathogenic and non-pathogenic strains has become relatively inexpensive. The bioinformatics pipelines for comparative genomics usually include gene prediction and annotation and can require significant computer power. To circumvent this, we developed a rapid method for genome-scale in silico subtractive hybridization, based on blastn and independent of feature identification and annotation. Whole genome comparisons by in silico genome subtraction were performed to identify genetic loci specific to Streptococcus mutans strains associated with severe early childhood caries (S-ECC), compared to strains isolated from caries-free (CF) children.
The genome similarity of the 20 S. mutans strains included in this study, calculated by Simrank k-mer sharing, ranged from 79.5 to 90.9%, confirming this is a genetically heterogeneous group of strains. We identified strain-specific genetic elements in 19 strains, with sizes ranging from 200 bp to 39 kb. These elements contained protein-coding regions with functions mostly associated with mobile DNA. We did not, however, identify any genetic loci consistently associated with dental caries, i.e., shared by all the S-ECC strains and absent in the CF strains. Conversely, we did not identify any genetic loci specific with the healthy group. Comparison of previously published genomes from pathogenic and carriage strains of Neisseria meningitidis with our in silico genome subtraction yielded the same set of genes specific to the pathogenic strains, thus validating our method.
Our results suggest that S. mutans strains derived from caries active or caries free dentitions cannot be differentiated based on the presence or absence of specific genetic elements. Our in silico genome subtraction method is available as the Microbial Genome Comparison (MGC) tool, with a user-friendly JAVA graphical interface.
doi:10.1016/j.meegid.2013.11.003
PMCID: PMC3940162  PMID: 24291226
Comparative genomics; Software; Streptococcus mutans; Dental caries; Virulence; Pathogenesis
4.  Poxviruses and the Evolution of Host Range and Virulence 
Poxviruses as a group can infect a large number of animals. However, at the level of individual viruses, even closely related poxviruses display highly diverse host ranges and virulence. For example, variola virus, the causative agent of smallpox, is human-specific and highly virulent only to humans, whereas related cowpox viruses naturally infect a broad spectrum of animals and only cause relatively mild disease in humans. The successful replication of poxviruses depends on their effective manipulation of the host antiviral responses, at the cellular-, tissue- and species-specific levels, which constitutes a molecular basis for differences in poxvirus host range and virulence. A number of poxvirus genes have been identified that possess host range function in experimental settings, and many of these host range genes target specific antiviral host pathways. Herein, we review the biology of poxviruses with a focus on host range, zoonotic infections, virulence, genomics and host range genes as well as the current knowledge about the function of poxvirus host range factors and how their interaction with the host innate immune system contributes to poxvirus host range and virulence. We further discuss the evolution of host range and virulence in poxviruses as well as host switches and potential poxvirus threats for human and animal health.
doi:10.1016/j.meegid.2013.10.014
PMCID: PMC3945082  PMID: 24161410
5.  Complete genome sequence and molecular phylogeny of a newfound hantavirus harbored by the Doucet’s musk shrew (Crocidura douceti) in Guinea 
Elucidation of the molecular phylogeny of shrew-borne hantaviruses in sub-Saharan Africa has been hampered by the lack of full-length viral genomes. In this report, we present the complete genome analysis of a newfound hantavirus, designated Bowé virus, detected in ethanol-fixed intercostal muscle of a Doucet’s musk shrew (Crocidura douceti), captured in southwestern Guinea in February 2012. Full-length amino acid sequence comparison of the S-, M- and L-segment gene products revealed that Bowé virus differed by 24.1–53.4%, 17.0–59.9% and 14.6–39.7%, respectively, from all other representative rodent-, shrew- and mole-borne hantaviruses. Phylogenetic analysis, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that Bowé virus shared a common ancestry with Tanganya virus, a hantavirus detected in the Therese’s shrew (Crocidura theresae) in Guinea. Whole genome analysis of many more hantaviruses from sub-Saharan Africa are needed to better clarify how the radiation of African shrews might have contributed to the phylogeography of hantaviruses.
doi:10.1016/j.meegid.2013.08.016
PMCID: PMC3844077  PMID: 23994121
Hantavirus; Crocidura; Shrew; Phylogeny; Guinea
6.  Phylogenomics of Brazilian epidemic isolates of Mycobacterium abscessus subsp. bolletii reveals relationships of global outbreak strains 
Rapidly growing, non-tuberculous mycobacteria (NTM) in the Mycobacterium abscessus (MAB) species are emerging pathogens that cause various diseases including skin and respiratory infections. The species has undergone recent taxonomic nomenclature refinement, and is currently recognized as two subspecies, M. abscessus subsp. abscessus (MAB-A) and M. abscessus subsp. bolletii (MAB-B). The recently reported outbreaks of MAB-B in surgical patients in Brazil from 2004 to 2009 and in cystic fibrosis patients in the United Kingdom (UK) in 2006 to 2012 underscore the need to investigate the genetic diversity of clinical MAB strains. To this end, we sequenced the genomes of two Brazilian MAB-B epidemic isolates (CRM-0019 and CRM-0020) derived from an outbreak of skin infections in Rio de Janeiro, two unrelated MAB strains from patients with pulmonary infections in the United States (US) (NJH8 and NJH11) and one type MAB-B strain (CCUG 48898) and compared them to 25 publically available genomes of globally diverse MAB strains. Genome-wide analyses of 27,598 core genome single nucleotide polymorphisms (SNPs) revealed that the two Brazilian derived CRM strains are nearly indistinguishable from one another and are more closely related to UK outbreak isolates infecting CF patients than to strains from the US, Malaysia or France. Comparative genomic analyses of six closely related outbreak strains revealed geographic-specific large-scale insertion/deletion variation that corresponds to bacteriophage insertions and recombination hotspots. Our study integrates new genome sequence data with existing genomic information to explore the global diversity of infectious M. abscessus isolates and to compare clinically relevant outbreak strains from different continents.
doi:10.1016/j.meegid.2013.09.012
PMCID: PMC3853122  PMID: 24055961
Mycobacterium abscessus; comparative genomics; nontuberculous mycobacteria; NTM; bioinformatics
7.  Emergence of Acinetobacter baumannii international clone II in Brazil: reflection of a global expansion 
The aim of this study was to investigate the occurrence of carbapenem-resistant Acinetobacter baumannii international clones (IC) in Curitiba, Brazil, using multilocus sequence typing and trilocus PCR-based typing schemes. IC2 was the first emerging clone. This IC was detected in an isolate from 2003 of a PFGE type spread in at least two hospitals since 1999. Subsequently, IC2 waned while IC1 and clonal complex 15/104 prevailed. This is the first description of IC2 in Brazil and Latin America.
doi:10.1016/j.meegid.2013.09.028
PMCID: PMC3856948  PMID: 24121023
Acinetobacter baumannii; multilocus sequence typing; trilocus PCR-based typing; international clone; molecular epidemiology
8.  Purifying selection in Porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation 
Porcine Reproductive and Respiratory Syndrome Virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3,466 Type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS.
doi:10.1016/j.meegid.2013.09.022
PMCID: PMC3875317  PMID: 24084290
natural selection; evolution; PRRSV
9.  Association between TLR3 rs3775291 and resistance to HIV among highly exposed Caucasian intravenous drug users 
Background
TLR3 recognizes dsRNA and triggers immune responses against RNA and DNA viruses. A polymorphism in TLR3, rs3775291 (Leu412Phe), has been associated with the increased susceptibility to enteroviral myocarditis, protection against tick-borne encephalitis virus and HIV-1 infection. We investigated Caucasian intravenous drug users (IDUs) and blood donors in order to evaluate the associations between TLR3 genotypes and susceptibility to HIV infection.
Materials and methods
A total of 345 Caucasian IDUs were recruited, 50% of them were HIV positive, 89% HCV and 77% HBV positive. Based on their history of needle sharing, 20 of the HIV negative IDUs were classified as highly exposed HIV seronegatives (HESNs), 68 as non-HESNs and 85 as unexposed. The control group consisting of 497 blood donors tested negative for all three viruses. TLR3 rs3775291 were determined by using TaqMan Allelic Discrimination Assay.
Results
The TLR3 rs3775291 T allele frequency was similar among the HIV negative and HIV positive IDUs and blood donors – 36%, 31% and 34%, respectively. The frequency of persons possessing at least one TLR3 rs3775291 T allele was significantly higher in HESNs compared with blood donors and HIV positive IDUs (80% vs. 55%; p = 0.037 and 80% vs. 53%; p = 0.031, respectively). In the univariate analysis, persons who possessed at least one T allele had reduced odds of being HIV seropositive (OR = 0.29, 95% CI = 0.09–0.90). This association remained significant (OR = 0.25, 95% CI = 0.07–0.87) after the adjustment for other co-variates (HCV, HBV serostatus and duration of intravenous drug use).
Conclusions
The TLR3 rs3775291 T allele has a protective effect against HIV infection among HESNs IDUs.
doi:10.1016/j.meegid.2013.08.008
PMCID: PMC4001117  PMID: 23962581
TLR3; Leu412Phe; Intravenous drug users; Highly exposed HIV seronegatives
10.  The evolution and diversity of a low complexity vaccine candidate, merozoite surface protein 9 (MSP-9), in Plasmodium vivax and closely related species 
The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of msp-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, P. reichenowi, P. chabaudi, P. yoelii, P. berghei, P. coatneyi, P. gonderi, P. knowlesi, P. inui, P. simiovale, P. fieldi, P. cynomolgi and P. vivax and evaluated the signature of natural selection in all msp-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in Plasmodium vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other.
doi:10.1016/j.meegid.2013.09.011
PMCID: PMC4066979  PMID: 24044894
ABRA; Binding sites; Genetic diversity; Merozoite Surface Proteins; MSP-9; Plasmodium
11.  A panel of 16 full-length HCV genomes was characterized in China belonging to genotypes 1 to 6 including subtype 2f and two novel genotype 6 variants 
In this study, we characterized the full-length genomes of 16 HCV isolates obtained from patients in a single hospital in China using overlapping PCR followed by DNA sequencing. The obtained genomes are 9,414-9,628 nucleotides in length, and each genome contains a single ORF of 9,021-9,102 nucleotides. Nine genomes represent the common subtypes 1a, 1b, 2a, 2b, 3a, 3b, and 6a, while seven represent the infrequent lineages 1c, 2f, 4d, and 5a, and two novel genotype 6 variants. GZ51969 and GZ52540 are subtype 1b isolates belonging to two unique clusters designated A and B, which account for 29.5% and 59.5% of the 1b infections in China, respectively. ZS542 and GZ98799 represent the first two complete genomes of the provisionally assigned subtype 2f. ZS96 and ZS202 are novel genotype 6 variants that may qualify for two new subtypes. ZS17, ZS537, and ZS631 represent three alien subtypes, namely, 1c, 4d, and 5a, which were detected in China for the first time in this study and may have been recently introduced as a result of globalization. Taken together, these results confirmed a large variety of HCV taxonomic lineages in China through the sequencing of their full-length genomes. These lineages represent six genotypes, 11 subtypes, and two novel variants. They were characterized for achieving a better understanding of the HCV genetic variation patterns and for possible future research applications.
doi:10.1016/j.meegid.2013.08.014
PMCID: PMC4186718  PMID: 24012950
HCV; full-length genome; genotype; subtype; China
12.  Sex and rhythms in sandflies and mosquitoes: An appreciation of the work of Alexandre Afranio Peixoto (1963–2013) 
Highlights
•Lutzomyia longipalpis in Brazil has recently undergone complex speciation events.•Anopheles cruzii in southern Brazil has also undergone recent speciation.•The circadian clock mechanisms of both sandflies and mosquitoes have been described.
I will briefly discuss the work of Alexandre A. Peixoto on sandflies and mosquitoes, focusing initially on his contributions to the population biology and phylogenetics of Brazilian populations of these important hematophagous insects. I shall also review some of his work on the underlying molecular clocks that mediate rhythmic behaviour and physiology in these species.
doi:10.1016/j.meegid.2014.06.016
PMCID: PMC4265727  PMID: 25046172
Sandflies; Mosquitoes; Behaviour; Copulation songs; Population genetics; Phylogeny
13.  Fever versus Fever: the role of host and vector susceptibility and interspecific competition in shaping the current and future distributions of the sylvatic cycles of dengue virus and yellow fever virus 
Two different species of flaviviruses, dengue virus (DENV) and yellow fever virus (YFV), that originated in sylvatic cycles maintained in non-human primates and forest-dwelling mosquitoes have emerged repeatedly into sustained human-to-human transmission by Aedes aegypti mosquitoes. Sylvatic cycles of both viruses remain active, and where the two viruses overlap in West Africa they utilize similar suites of monkeys and Aedes mosquitoes. These extensive similarities render the differences in the biogeography and epidemiology of the two viruses all the more striking. First, the sylvatic cycle of YFV originated in Africa and was introduced into the New World, probably as a result of the slave trade, but is absent in Asia; in contrast, sylvatic DENV likely originated in Asia and has spread to Africa but not to the New World. Second, while sylvatic YFV can emerge into extensive urban outbreaks in humans, these invariably die out, whereas four different types of DENV have established human transmission cycles that are ecologically and evolutionarily distinct from their sylvatic ancestors. Finally, transmission of YFV among humans has been documented only in Africa and the Americas, whereas DENV is transmitted among humans across most of the range of competent Aedes vectors, which in the last decade has included every continent save Antarctica. This review summarizes current understanding of sylvatic transmission cycles of YFV and DENV, considers possible explanations for their disjunct distributions, and speculates on the potential consequences of future establishment of a sylvatic cycle of DENV in the Americas.
doi:10.1016/j.meegid.2013.03.008
PMCID: PMC3749261  PMID: 23523817
dengue virus; yellow fever virus; Aedes aegypti; sylvatic; arbovirus; emerging infectious disease
14.  Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum 
Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST = 0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r = 0.49, P=0.003, N = 83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India.
doi:10.1016/j.meegid.2013.07.009
PMCID: PMC3799960  PMID: 23871774
Plasmodium falciparum; India; Chloroquine resistance; Hitchhiking; Transmission area; Gene-flow
15.  Intra-Host Evolutionary Rates in HIV-1C env and gag during Primary Infection 
Background
HIV-1 nucleotide substitution rates are central for understanding the evolution of HIV-1. Their accurate estimation is critical for analysis of viral dynamics, identification of divergence time of HIV variants, inference of HIV transmission clusters, and modeling of viral evolution.
Methods
Intra-patient nucleotide substitution rates in HIV-1C gag and env gp120 V1C5 were analyzed in a longitudinal cohort of 32 individuals infected with a single viral variant. Viral quasispecies were derived by single genome amplification/sequencing from serially sampled blood specimens collected at median (IQR) of 5 (4–6) times per subject from enrollment (during Fiebig stages II to V) over a median (IQR) of 417 (351–471) days post-seroconversion (p/s). HIV-1C evolutionary rates were estimated by BEAST v.1.6.1 using a relaxed lognormal molecular clock model. The effect of antiretroviral therapy (ART) on substitution rates in gag and env was assessed in a subset of six individuals who started ARV therapy during the follow-up period.
Results
During primary HIV-1C infection, the intra-patient substitution rates were estimated at a median (IQR) of 5.22E-03 (3.28E-03–7.55E-03) substitutions per site per year of infection within gag, and 1.58E-02 (9.99E-03–2.04E-02) substitutions per site per year within env gp120 V1C5. The substitution rates in env gp120 V1C5 were higher than in gag (p<0.001, Wilcoxon signed rank test). The median (IQR) relative rates of evolution at codon positions 1, 2, and 3 were 0.73 (0.48–0.84), 0.67 (0.52–0.86), and 1.54 (1.21–1.71) in gag, and 1.01 (0.86–1.15), 1.05 (0.99–1.21), and 0.86 (0.67–0.94) in env gp120 V1C5, respectively. A first to the third position codon rate ratio > 1.0 within env was found in 25 (78.1%) cases, but only in 4 (12.5%) cases in gag, while a second to the third position codon rate ratio > 1.0 in env was observed in 26 (81.3%) cases, but in gag only in 2 (6.3%) cases (p<0.001 for both comparisons, Fisher’s exact test). No ART effect on substitution rates in gag and env was found, at least within the first 3–4 months after ART initiation. Individuals with early viral set point ≥ 4.0 log10 copies/ml had higher substitution rates in env gp120 V1C5 (median (IQR) 1.88E-02 (1.54E-02–2.46E-02) vs. 1.04E (7.24E-03–1.55E-02) substitutions per site per year; p=0.017, Mann-Whitney sum rank test), while individuals with early viral set point ≥ 3.0 log10 copies/ml had higher substitution rates in gag (median (IQR) 5.66E-03 (3.45E-03–7.94E-03) vs. 1.78E-03 (4.57E-04–5.15E-03); p=0.028; Mann-Whitney sum rank test).
Conclusions
The results suggest that in primary HIV-1C infection, (1) intra-host evolutionary rates in env gp120 V1C5 are about 3-fold higher than in gag; (2) selection pressure in env is more frequent than in gag; (3) initiation of ART does not change substitution rates in HIV-1C env or gag, at least within the first 3–4 months after starting ART; and (4) intra-host evolutionary rates in gag and env gp120 V1C5 are higher in individuals with elevated levels of early viral set point.
doi:10.1016/j.meegid.2013.02.023
PMCID: PMC3759599  PMID: 23523818
HIV-1; subtype C; evolutionary rates; substitution rates; primary infection
16.  Phylogeny of a relapsing fever Borrelia species transmitted by the hard tick Ixodes scapularis 
The discovery of Borrelia species that were related to the agents of relapsing fever but were transmitted by hard ticks rather than soft ticks challenged previous taxonomies based largely on microbe-host specificities and geographic considerations. One of these newly-identified organisms is the Borrelia miyamotoi sensu lato strain LB-2001 from North America and transmitted by Ixodes scapularis. This or related strains have been identified as the cause of human disease, but comparatively little is known about their biology or genetics. Using recently acquired chromosome sequence of LB-2001 together with database sequences and additional sequences determined here, I carried out comparisons of the several species of Borrelia, including those in the two major clades: the relapsing fever group of species and the Lyme disease group of species. Phylogenetic inference at the species level was based on four data sets: whole chromosomes of ~1 Mb each, and concatenated sequences of 19 ribosomal protein genes, 3 conserved nucleic acid enzymes (rpoC, recC, and dnaE), and 4 contiguous genes for nucleotide salvage on a large plasmid. Analyses using neighbor-joining, maximum likelihood, and Bayesian methods were largely concordant for each of the trees. They showed that LB-2001 and related hard tick-associated organisms, like B. lonestari, are deeply positioned within the RF group of species and that these organisms did not, as some earlier estimations had suggested, constitute a paraphyletic group. The analyses also provided further evidence that major changes in host ranges and life cycles, such as hard to soft ticks or vice versa, may not correlate well with overall sequence differences. The genetic differences between LB-2001 and B. miyamotoi sensu stricto justify provisional use of the “sensu lato” designation for LB-2001.
doi:10.1016/j.meegid.2014.04.022
PMCID: PMC4182126  PMID: 24813576
tick-borne disease; spirochetes; Lyme disease; genomics
17.  Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma dimidiata 
Triatoma dimidiata, one of the major vectors of Chagas disease in Central America, is found in both domestic and peri-domestic habitats. Questions concerning population boundaries, infestation rates, insecticide resistance, and geographic dispersal of triatomine bugs persist and may be resolved using genetic markers such as microsatellites. Microsatellites are short tandem repeats found dispersed throughout a genome and can be useful for genotypic identification. We developed a plasmid library from the genomic DNA isolated from a single T. dimidiata adult collected in Guatamala. Ten thousand clones were screened using a probe consisting of nine microsatellite oligonucleotides. Eight loci appear polymorphic among populations found in Guatemala, Honduras, and Mexico, and thus are potentially useful for population genetic applications.
PMCID: PMC4152310  PMID: 12798021
Chagas disease; Microsatellites; Triatoma
18.  Bioinformatics of varicella-zoster virus: Single nucleotide polymorphisms define clades and attenuated vaccine genotypes 
Varicella zoster virus (VZV) is one of the human herpesviruses. To date, over 40 complete VZV genomes have been sequenced and analyzed. The VZV genome contains around 125,000 base pairs including 70 open reading frames (ORFs). Enumeration of single nucleotide polymorphisms (SNPs) has determined that the following ORFs are the most variable (in descending order): 62, 22, 29, 28, 37, 21, 54, 31, 1 and 55. ORF 62 is the major immediate early regulatory VZV gene. Further SNP analysis across the entire genome has led to the observation that VZV strains can be broadly grouped into clades within a phylogenetic tree. VZV strains collected in Singapore provided important sequence data for construction of the phylogenetic tree. Currently 5 VZV clades are recognized; they have been designated clades 1 through 5. Clades 1 and 3 include European/North American strains; clade 2 includes Asian strains, especially from Japan; and clade 5 includes strains from India. Clade 4 includes some strains from Europe, but its geographic origins need further documentation.. Within clade 1, five variant viruses have been isolated with a missense mutation in the gE (ORF 68) glycoprotein; these strains have an altered increased cell spread phenotype. Bioinformatics analyses of the attenuated vaccine strains have also been performed, with a subsequent discovery of a stop-codon SNP in ORFO as a likely attenuation determinant. Taken together, these VZV bioinformatics analyses have provided enormous insights into VZV phylogenetics as well as VZV SNPs associated with attenuation.
doi:10.1016/j.meegid.2012.11.008
PMCID: PMC3594394  PMID: 23183312
19.  Human papillomavirus type 16 long control region and E6 variants stratified by cervical disease stage 
Highlights
•We sequenced HPV16 LCR–E6 variants in cervical disease samples from England.•98% of variants were of the EUR lineage.•Site-specific entropy identified several variable sites in the LCR and E6.•No single or combination of sites were associated with disease, including E6 T350G.
Objective
Certain intra-type variants of HPV16 have been shown to be associated with an increased risk of developing high grade cervical disease, but their potential association is confounded by apparent geographic and phylogenetic lineage dependency. The objective of this study was to evaluate the relationship between HPV16 sequence variants and cervical disease stage in monospecific infection samples from a single lineage (European, EUR) in England.
Methods
One hundred and twelve women singly infected with HPV16 and displaying normal and abnormal cytology grades were selected. An 1187 bp fragment encompassing the entire LCR and a portion of the E6 open reading frame was sequenced to identify intra-type variants. Intra-type diversity was estimated using Shannon entropy.
Results
Almost all samples (110/112; 98%) were assigned to the EUR lineage, one sample was classified as European-Asian (EAS) and another African (Afr1a). The mean pairwise distance of the EUR sequences in this study was low (0.29%; 95%CI 0.13–0.45%) but there were nevertheless several sites in the LCR (n = 5) and E6 (n = 2) that exhibited a high degree of entropy. None of these sites, however, including the T350G non-synonymous (L83V) substitution in E6, alone or in combination, were found to be associated with cervical disease stage.
Conclusions
Despite using single infection samples and samples from a single variant lineage, intra-type variants of HPV16 were not differentially associated with cervical disease. Monitoring intra-lineage, site-specific variants, such as T350G, is unlikely to be of diagnostic value.
doi:10.1016/j.meegid.2014.05.009
PMCID: PMC4150919  PMID: 24823962
Human papillomavirus; Cervical cancer; HPV16; Variants; LCR; E6
20.  A survey of host range genes in poxvirus genomes 
Poxviruses are widespread pathogens, which display extremely different host ranges. Whereas some poxviruses, including variola virus, display narrow host ranges, others such as cowpox viruses naturally infect a wide range of mammals. The molecular basis for differences in host range are poorly understood but apparently depend on the successful manipulation of the host antiviral response. Some poxvirus genes have been shown to confer host tropism in experimental settings and are thus called host range factors. Identified host range genes include vaccinia virus K1L, K3L, E3L, B5R, C7L and SPI-1, cowpox virus CP77/CHOhr, ectromelia virus p28 and 022, and myxoma virus T2, T4, T5, 11L, 13L, 062R and 063R. These genes encode for ankyrin repeat-containing proteins, tumor necrosis factor receptor II homologs, apoptosis inhibitor T4-related proteins, Bcl-2-related proteins, pyrin domain-containing proteins, cellular serine protease inhibitors (serpins), short complement-like repeats containing proteins, KilA-N/RING domain-containing proteins, as well as inhibitors of the double-stranded RNA-activated protein kinase PKR. We conducted a systematic survey for the presence of known host range genes and closely related family members in poxvirus genomes, classified them into subgroups based on their phylogenetic relationship and correlated their presence with the poxvirus phylogeny. Common themes in the evolution of poxvirus host range genes are lineage-specific duplications and multiple independent inactivation events. Our analyses yield new insights into the evolution of poxvirus host range genes. Implications of our findings for poxvirus host range and virulence are discussed.
doi:10.1016/j.meegid.2012.12.002
PMCID: PMC4080715  PMID: 23268114
Poxvirus; Host range genes; Virus evolution; Host–pathogen interactions
21.  Connecting the study of wild influenza with the potential for pandemic disease 
Continuing outbreaks of pathogenic (H5N1) and pandemic (SOIVH1N1) influenza have underscored the need to understand the origin, characteristics, and evolution of novel influenza A virus (IAV) variants that pose a threat to human health. In the last 4–5 years, focus has been placed on the organization of large-scale surveillance programs to examine the phylogenetics of avian influenza virus (AIV) and host-virus relationships in domestic and wild animals. Here we review the current gaps in wild animal and environmental surveillance and the current understanding of genetic signatures in potentially pandemic strains.
doi:10.1016/j.meegid.2013.02.020
PMCID: PMC3685205  PMID: 23541413
22.  Origins of house reinfestation with Triatoma infestans after insecticide spraying in the Argentine Chaco using wing geometric morphometry 
Identifying the origins of insect vectors collected after community-wide residual insecticide applications is a relevant challenge in the Gran Chaco region where the main vector of Chagas disease Triatoma infestans usually reinfests human dwellings. Wing geometric morphometry was used to compare the right wings of 63 males and 54 females collected at 4 months post-spraying (MPS) with those from 165 males and 111 females collected before full-coverage spraying with pyrethroids in a well-defined rural area in northeastern Argentina. Male and female wing centroid size resulted significantly larger at 4 MPS than before interventions, but no significant changes in shape were detected. Metric disparity (variance of shape) varied significantly in males but not in females. Using shape variables, a relatively large fraction of post-spraying males (70%) and females (54%) could not be differentiated from those collected at the same source house or at the nearest infested house before interventions. Bugs collected at 4 and 8 MPS in a persistently infested house were mainly assigned to the source house. These results support the hypothesis of persistent bug populations that survived the insecticide application at local spatial scales, and are consistent with the occurrence of vector control failures most likely related to moderate pyrethroid resistance. Wing geometric morphometry is a useful tool for identifying sources of reinfestation, but it is limited by the spatial structure found in the reference populations. Combined with field and genetic data, this approach may contribute to the understanding of the reinfestation process and improvement of vector control strategies.
doi:10.1016/j.meegid.2013.03.044
PMCID: PMC3740411  PMID: 23557838
Triatoma infestans; geometric morphometry; reinfestation; pyrethroid insecticide resistance
23.  The SLC4A1 gene is under differential selective pressure in primates infected by Plasmodium falciparum and related parasites 
Malaria is a disease caused by Plasmodium parasites and is responsible for high mortality in humans. This disease is caused by four different species of Plasmodium though the main source of mortality is Plasmodium falciparum. Humans have a number of genetic adaptations that act to combat Plasmodium. One adaptation is a deletion in the SLC4A1 gene that leads to Southeast Asian ovalocytosis (SAO). There is evidence that SAO erythrocytes are resistant to multiple Plasmodium species. Here we analyze SLC4A1 in 23 primates and mammals to test for differential selective pressures among different primate lineages. Because primates are infected with both human Plasmodium parasites and their relatives, this analysis can be used to test which human Plasmodium parasite is the likely target of SAO. A significantly different pattern of molecular evolution was found in humans and African apes, species that are infected by P. falciparum and its relatives. This effect was restricted to the cytosolic domain of the SLC4A1 gene. The evidence is consistent with a different selective regime operating on this gene domain in humans and African apes, when compared to other primates and mammals. Alternatively, this pattern is consistent with a relaxation of selection or weak adaptive evolution operating on a small number of amino acids. The adaptive interpretation of the results is consistent with the SAO allele of the SLC4A1 gene interacting with P. falciparum in humans, rather than other Plasmodium parasites. However, additional investigation of the relationship between SLC4A1 variants and Plasmodium in humans and African apes is required to test whether the different selective regime in humans and African apes is due to natural selection or relaxed constraint.
doi:10.1016/j.meegid.2012.02.019
PMCID: PMC4076009  PMID: 22426110
Malaria; Evolution; Adaptation; African ape; Hominoid; Plasmodium
24.  Evaluation of Sequence Ambiguities of the HIV-1 pol gene as a Method to Identify Recent HIV-1 Infection in Transmitted Drug Resistance Surveys 
Identification of recent HIV infection within populations is a public health priority for accurate estimation of HIV incidence rates and transmitted drug resistance. Determining HIV incidence rates by prospective follow-up of HIV-uninfected individuals is challenging and serological assays have important limitations. HIV diversity within an infected host increases with duration of infection. In this analysis, we explore a simple bioinformatics approach to assess viral diversity by determining the percentage of ambiguous base calls in sequences derived from standard genotyping of HIV-1 protease and reverse transcriptase. Sequences from 691 recently infected (≤1 year) and chronically infected (>1 year) individuals from Sweden, Vietnam and Ethiopia were analyzed for ambiguity. A significant difference (p <0.0001) in the proportion of ambiguous bases was observed between sequences from individuals with recent and chronic infection in both HIV-1 subtype B and non-B infection, consistent with previous studies. In our analysis, a cutoff of <0.47% ambiguous base calls identified recent infection with a sensitivity and specificity of 88.8% and 74.6% respectively. 1,728 protease and reverse transcriptase sequences from 36 surveys of transmitted HIV drug resistance performed following World Health Organization guidance were analyzed for ambiguity. The 0.47% ambiguity cutoff was applied and survey sequences were classified as likely derived from recently or chronically infected individuals. 71% of patients were classified as likely to have been infected within one year of genotyping but results varied considerably amongst surveys. This bioinformatics approach may provide supporting population-level information to identify recent infection but its application is limited by infection with more than one viral variant, decreasing viral diversity in advanced disease and technical aspects of population based sequencing. Standardization of sequencing techniques and base calling and the addition of other parameters such as CD4 cell count may address some of the technical limitations and increase the usefulness of the approach.
doi:10.1016/j.meegid.2013.03.050
PMCID: PMC4066879  PMID: 23583545
HIV; viral diversity; ambiguity; incidence; resistance; bioinformatics
25.  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

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