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We conducted a population genomic study of the crested caracara (Caracara plancus) using samples (n = 290) collected from individuals in Florida, Texas, and Arizona, United States. Crested caracaras are non-migratory raptors ranging from the southern tip of South America to the southern United States, including a federally protected relict population in Florida long thought to have been isolated since the last ice age. Our objectives were to evaluate genetic diversity and population structure of Florida's apparently isolated population and to evaluate taxonomic relationships of crested caracaras at the northern edge of their range. Using DNA purified from blood samples, we conducted double-digest restriction site associated DNA sequencing and sequenced the mitochondrial ND2 gene. Analyses of population structure using over 9,000 SNPs suggest that two major clusters are best supported, one cluster including only Florida individuals and the other cluster including Arizona and Texas individuals. Both SNPs and mitochondrial haplotypes reveal the Florida population to be highly differentiated genetically from Arizona and Texas populations, whereas, Arizona and Texas populations are moderately differentiated from each other. The Florida population's mitochondrial haplotypes form a separate monophyletic group, while Arizona and Texas populations share mitochondrial haplotypes. Results of this study provide substantial genetic evidence that Florida's crested caracaras have experienced long-term isolation from caracaras in Arizona and Texas and thus, represent a distinct evolutionary lineage possibly warranting distinction as an Evolutionarily Significant Unit (ESU) or subspecies. This study will inform conservation strategies focused on long-term survival of Florida's distinct, panmictic population., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genome sequencing can generate findings beyond the initial test indication that may be relevant to a patient or research participant's health. In the decade since the American College of Medical Genetics and Genomics published its recommendations for reporting these findings, consensus regarding terminology has remained elusive and a variety of terms are in use globally. We conducted a scoping review to explore terminology choice and the justifications underlying those choices. Documents were included if they contained a justification for their choice of term(s) related to findings beyond the initial genomic test indication. From 3571 unique documents, 52 were included, just over half of which pertained to the clinical context (n = 29, 56%). We identified four inter-related concepts used to defend or oppose terms: expectedness of the finding, effective communication, relatedness to the original test indication, and how genomic information was generated. A variety of justifications were used to oppose the term "incidental," whereas "secondary" had broader support as a term to describe findings deliberately sought. Terminology choice would benefit from further work to include the views of patients. We contend that clear definitions will improve ethical debate and support communication about genomic findings beyond the initial test indication., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Kudzu ( Pueraria montana var. lobata ), a plant native to Southeastern Asia, has become a major noxious weed covering millions of hectares in the Southern United States. A kudzu patch displaying virus-like symptoms located in Ackerman, northeastern Mississippi (MS), was used as a source for virus isolation and characterization involving mechanical and vector transmission, ultrastructural observation, surveys, Sanger and high-throughput genome sequencing, and sequence analyses. The results revealed the presence of a new potyvirus in infected kudzu, closely related to wisteria vein mosaic virus (WVMV) and provisionally named kudzu chlorotic ring blotch virus (KudCRBV). Genome features and pairwise comparison with six WVMV genomes currently available in GenBank and three additional isolates from MS sequenced in this work suggest that KudCRBV is likely a member of a new species in the genus Potyvirus . Furthermore, under experimental conditions, KudCRBV was successfully transmitted by cotton and potato aphids and mechanically to soybean and beans. A state-wide survey revealed several kudzu patches infected by the virus in northern MS.

Competing Interests: Conflict of Interest Funding and support listed here did not support development of this document unless included in the Acknowledgments section. N.S.A.-H. is an equity holder of 23andMe; serves as a scientific advisory board member for Allelica; received personal fees from Genentech, Allelica, and 23andMe; received research funding from Akcea; and was previously employed by Regeneron Pharmaceuticals. W.K.C. is a member of the scientific advisory board of Regeneron Genetics Center. D.T.M. has received an honorarium from Ambry Genetics. D.R.S. is supported by the Intramural Research Program of the Division of Cancer Epidemiology and Genetics of the National Cancer Institute, Rockville, MD, and also performs contract clinical telehealth services for Genome Medical, Inc., in accordance with relevant National Cancer Institute ethics policies. All other authors declare no conflicts of interest.

The Bioinformation and DNA Data Bank of Japan (DDBJ) Center (https://www.ddbj.nig.ac.jp) maintains database archives that cover a wide range of fields in life sciences. As a founding member of the International Nucleotide Sequence Database Collaboration (INSDC), our primary mission is to collect and distribute nucleotide sequence data, as well as their study and sample information, in collaboration with the National Center for Biotechnology Information in the United States and the European Bioinformatics Institute. In addition to INSDC resources, the Center operates databases for functional genomics (GEA: Genomic Expression Archive), metabolomics (MetaboBank), and human genetic and phenotypic data (JGA: Japanese Genotype-Phenotype Archive). These databases are built on the supercomputer of the National Institute of Genetics, whose remaining computational capacity is actively utilized by domestic researchers for large-scale biological data analyses. Here, we report our recent updates and the activities of our services., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The uptake of exome/genome sequencing has introduced unexpected testing results (incidental findings) that have become a major challenge for both testing laboratories and providers. While the American College of Medical Genetics and Genomics has outlined guidelines for laboratory management of clinically actionable secondary findings, debate remains as to whether incidental findings should be returned to patients, especially those representing pediatric populations., Methods: The Sequencing Analysis and Diagnostic Yield working group in the Clinical Sequencing Evidence-Generating Research Consortium has collected a cohort of pediatric patients found to harbor a genomic sequencing-identified non-ACMG-recommended incidental finding. The incidental variants were not thought to be associated with the indication for testing and were disclosed to patients and families., Results: In total, 23 "non-ACMG-recommended incidental findings were identified in 21 pediatric patients included in the study. These findings span four different research studies/laboratories and demonstrate differences in incidental finding return rate across study sites. We summarize specific cases to highlight core considerations that surround identification and return of incidental findings (uncertainty of disease onset, disease severity, age of onset, clinical actionability, and personal utility), and suggest that interpretation of incidental findings in pediatric patients can be difficult given evolving phenotypes. Furthermore, return of incidental findings can benefit patients and providers, but do present challenges., Conclusions: While there may be considerable benefit to return of incidental genetic findings, these findings can be burdensome to providers and present risk to patients. It is important that laboratories conducting genomic testing establish internal guidelines in anticipation of detection. Moreover, cross-laboratory guidelines may aid in reducing the potential for policy heterogeneity across laboratories as it relates to incidental finding detection and return. However, future discussion is required to determine whether cohesive guidelines or policy statements are warranted., (© 2022. The Author(s).)

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic revealed a worldwide lack of effective molecular surveillance networks at local, state, and national levels, which are essential to identify, monitor, and limit viral community spread. SARS-CoV-2 variants of concern (VOCs) such as Alpha and Omicron, which show increased transmissibility and immune evasion, rapidly became dominant VOCs worldwide. Our objective was to develop an evidenced-based genomic surveillance algorithm, combining reverse transcription polymerase chain reaction (RT-PCR) and sequencing technologies to quickly identify highly contagious VOCs, before cases accumulate exponentially., Methods: Deidentified data were obtained from 508,969 patients tested for coronavirus disease 2019 (COVID-19) with the TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) in four CLIA-certified clinical laboratories in Puerto Rico (n = 86,639) and in three CLIA-certified clinical laboratories in the United States (n = 422,330)., Results: TaqPath data revealed a frequency of S Gene Target Failure (SGTF) > 47% for the last week of March 2021 in both, Puerto Rico and US laboratories. The monthly frequency of SGTF in Puerto Rico steadily increased exponentially from 4% in November 2020 to 47% in March 2021. The weekly SGTF rate in US samples was high (>8%) from late December to early January and then also increased exponentially through April (48%). The exponential increase in SGFT prevalence in Puerto Rico was concurrent with a sharp increase in VOCs among all SARS-CoV-2 sequences from Puerto Rico uploaded to Global Influenza Surveillance and Response System (GISAID) (n = 461). Alpha variant frequency increased from <1% in the last week of January 2021 to 51.5% of viral sequences from Puerto Rico collected in the last week of March 2021., Conclusions: According to the proposed evidence-based algorithm, approximately 50% of all SGTF patients should be managed with VOCs self-quarantine and contact tracing protocols, while WGS confirms their lineage in genomic surveillance laboratories. Our results suggest this workflow is useful for tracking VOCs with SGTF., (© 2022 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome., Competing Interests: Declaration of interests P.-A.K. and F.I.S. are cofounders and shareholders of Dioscure Therapeutics SE. F.I.S. is a consultant and shareholder of IFM Therapeutics. J.R.H. is founder and board member of Isoplexis and PACT Pharma. J.D.G. declares contracted research with Gilead, Lilly, and Regeneron. K.H., J.M.J., and A.J.B. work at Quanterix Corporation. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Variant visualization plays an important role in supporting the viral evolution analysis, extremely valuable during the COVID-19 pandemic. VirusViz is a web-based application for comparing variants of selected viral populations and their sub-populations; it is primarily focused on SARS-CoV-2 variants, although the tool also supports other viral species (SARS-CoV, MERS-CoV, Dengue, Ebola). As input, VirusViz imports results of queries extracting variants and metadata from the large database ViruSurf, which integrates information about most SARS-CoV-2 sequences publicly deposited worldwide. Moreover, VirusViz accepts sequences of new viral populations as multi-FASTA files plus corresponding metadata in CSV format; a bioinformatic pipeline builds a suitable input for VirusViz by extracting the nucleotide and amino acid variants. Pages of VirusViz provide metadata summarization, variant descriptions, and variant visualization with rich options for zooming, highlighting variants or regions of interest, and switching from nucleotides to amino acids; sequences can be grouped, groups can be comparatively analyzed. For SARS-CoV-2, we manually collect mutations with known or predicted levels of severity/virulence, as indicated in linked research articles; such critical mutations are reported when observed in sequences. The system includes light-weight project management for downloading, resuming, and merging data analysis sessions. VirusViz is freely available at http://gmql.eu/virusviz/., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Polyomaviruses are ancient DNA viruses that infect several species of animals. While recognition of the family Polyomaviridae has grown rapidly, there are few studies that consider their potential association with disease. Carnivora are a diverse and widespread order affected by polyomaviruses (PyVs) that have co-evolved with their hosts for millions of years. PyVs have been identified in sea lions, raccoons, badgers, Weddell seals, and dogs. We have discovered a polyomavirus, tentatively named "Ursus americanus polyomavirus 1" (UaPyV1) in black bears (Ursus americanus). UaPyV1 was detectable in various tissues of six out of seven bears submitted for necropsy. Based on viral phylogenetic clustering and detection of the virus in multiple individuals, we suggest that black bears are the natural hosts for UaPyV1. In this albeit small group, there is no clear relationship between UaPyV1 infection and any specific disease.

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real-time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real-time PCR reaction. By analysing all available 678 type 2 PRRSV (PRRSV-2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4-ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine-like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 94.7% (54/57) of Ingelvac PRRS ® MLV (MLV) strain and the MLV-like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real-time PCR assay. Evaluated with 417 PRRSV-2 positive clinical samples, 95% were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay., (© 2020 Wiley-VCH GmbH.)

The goal of the National Cancer Institute's (NCI's) Genomic Data Commons (GDC) is to provide the cancer research community with a data repository of uniformly processed genomic and associated clinical data that enables data sharing and collaborative analysis in the support of precision medicine. The initial GDC dataset include genomic, epigenomic, proteomic, clinical and other data from the NCI TCGA and TARGET programs. Data production for the GDC started in June, 2015 using an OpenStack-based private cloud. By June of 2016, the GDC had analyzed more than 50,000 raw sequencing data inputs, as well as multiple other data types. Using the latest human genome reference build GRCh38, the GDC generated a variety of data types from aligned reads to somatic mutations, gene expression, miRNA expression, DNA methylation status, and copy number variation. In this paper, we describe the pipelines and workflows used to process and harmonize the data in the GDC. The generated data, as well as the original input files from TCGA and TARGET, are available for download and exploratory analysis at the GDC Data Portal and Legacy Archive ( https://gdc.cancer.gov/ ).

The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org/) has been the core infrastructure for collecting and providing nucleotide sequence data and metadata for >30 years. Three partner organizations, the DNA Data Bank of Japan (DDBJ) at the National Institute of Genetics in Mishima, Japan; the European Nucleotide Archive (ENA) at the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI) in Hinxton, UK; and GenBank at National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health in Bethesda, Maryland, USA have been collaboratively maintaining the INSDC for the benefit of not only science but all types of community worldwide., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nodaviruses are small bisegmented RNA viruses belonging to the family Nodaviridae . Nodaviruses have been identified in different hosts, including insects, fishes, shrimps, prawns, dogs, and bats. A novel porcine nodavirus was first identified in the United States by applying next-generation sequencing on brain tissues of pigs with neurological signs, including uncontrollable shaking. RNA1 of the porcine nodavirus had the highest nucleotide identity (51.1%) to the Flock House virus, whereas its RNA2 shared the highest nucleotide identity (48%) with the RNA2 segment of caninovirus (Canine nodavirus). Genetic characterization classified porcine nodavirus as a new species under the genus Alphanodavirus . Further studies are needed to understand the pathogenicity and clinical impacts of this virus.

Full genome sequences are increasingly used to track the geographic spread and transmission dynamics of viral pathogens. Here, with a focus on Israel, we sequence 212 SARS-CoV-2 sequences and use them to perform a comprehensive analysis to trace the origins and spread of the virus. We find that travelers returning from the United States of America significantly contributed to viral spread in Israel, more than their proportion in incoming infected travelers. Using phylodynamic analysis, we estimate that the basic reproduction number of the virus was initially around 2.5, dropping by more than two-thirds following the implementation of social distancing measures. We further report high levels of transmission heterogeneity in SARS-CoV-2 spread, with between 2-10% of infected individuals resulting in 80% of secondary infections. Overall, our findings demonstrate the effectiveness of social distancing measures for reducing viral spread.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first identified in Wuhan, China late in 2019. Nine months later (Sept. 23, 2020), the virus has infected > 31.6 million people around the world and caused > 971.000 (3.07 %) fatalities in 220 countries and territories. Research on the genetics of the SARS-CoV-2 genome, its mutants and their penetrance can aid future defense strategies. By analyzing sequence data deposited between December 2019 and end of May 2020, we have compared nucleotide sequences of 570 SARS-CoV-2 genomes from China, Europe, the US, and India to the sequence of the Wuhan isolate. During worldwide spreading among human populations, at least 10 distinct hotspot mutations had been selected and found in up to > 80 % of viral genomes. Many of these mutations led to amino acid exchanges in replication-relevant viral proteins. Mutations in the SARS-CoV-2 genome would also impinge upon the secondary structure of the viral RNA molecule and its repertoire of interactions with essential cellular and viral proteins. The increasing frequency of SARS-CoV-2 mutation hotspots might select for dangerous viral pathogens. Alternatively, in a 29.900 nucleotide-genome, there might be a limit to the number of mutable and selectable sites which, when exhausted, could prove disadvantageous to viral survival. The speed, at which novel SARS-CoV-2 mutants are selected and dispersed around the world, could pose problems for the development of vaccines and therapeutics., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Morphological and molecular evaluation of tapeworms of the genus Bothriocephalus Rudolphi, 1808 (Cestoda: Bothriocephalidea), based on newly collected and uniformly fixed worms from freshwater fishes in Canada and the United States has revealed unexpected diversity. With a combination of selected morphological features and 4 molecular markers (18S rDNA V8 region, ITS1, ITS2, and COI gene sequences), the following morphotypes and lineages of the Bothriocephalus cuspidatus Cooper, 1917 complex were identified, several of which are specific to their respective fish definitive hosts and may represent separate species: B. cuspidatus sensu stricto from walleye, Sander vitreus (type host), which likely includes a miniature morphotype from Johnny darter, Etheostoma nigrum (both Percidae); Bothriocephalus morphotype from pumpkinseed, Lepomis gibbosus (Centrarchidae); and Bothriocephalus morphotype from rock bass, Ambloplites rupestris (Centrarchidae). The Bothriocephalus morphotype from goldeye, Hiodon alosoides (Hiodontidae), may also represent a separate lineage (possibly Bothriocephalus texomensisSelf, 1954) but requires additional studies. A morphotype from smallmouth bass, Micropterus dolomieu, based on a single specimen, is morphologically and genetically very similar to the morphotype from rock bass. Morphological study of the scolex and strobila of heat-killed and fixed specimens has revealed consistent differences, often subtle, that allowed us to differentiate between these morphotypes., (© American Society of Parasitologists 2020.)

The glycoprotein encoded by the ACKR1 gene expresses the Duffy blood group antigens and is a receptor for malaria parasites. We recently described 18 long-range ACKR1 alleles in an autochthonous population of a malaria endemic region. Extending this work, we sequenced the gene in a 53-sample repository established by the US Food and Drug Administration (FDA) as reference reagents for blood group genotyping. The FDA samples have been characterized for 19 genes; however, long-range haplotype information for these genes, including ACKR1, was lacking. We used a hybrid approach, novel for this type of gene, to characterize ACKR1 by combining two next-generation sequencing technologies, the short-read massively parallel sequencing and the long-read nanopore sequencing. The expedient integration of data from both next-generation sequencing systems were necessary and sufficient to allow determination of all 25 long-range ACKR1 alleles found in the 53 samples accurately. All 25 alleles identified in our current FDA cohort were novel and, unexpectedly, none had been observed among the 18 alleles in our previous study. The alleles will be useful for validation, calibration, and proficiency testing of red cell genotyping. The lack of any overlap between the ACKR1 alleles in the two studies documents differences in mutation rate and recombination frequency among populations. The exact haplotype and their interethnic or interpopulation dissimilarities can influence disease susceptibility and therapy., (Published by Elsevier Inc.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70 %. Improved sensitivity is, therefore, urgently required., Methods: We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances., Results: The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2., Conclusion: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We sequenced the genome of the North American groundhog, Marmota monax , also known as the woodchuck. Our sequencing strategy included a combination of short, high-quality Illumina reads plus long reads generated by both Pacific Biosciences and Oxford Nanopore instruments. Assembly of the combined data produced a genome of 2.74 Gbp in total length, with an N50 contig size of 1,094,236 bp. To annotate the genome, we mapped the genes from another M. monax genome and from the closely related Alpine marmot, Marmota marmota , onto our assembly, resulting in 20,559 annotated protein-coding genes and 28,135 transcripts. The genome assembly and annotation are available in GenBank under BioProject PRJNA587092., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Puiu D et al.)

The psychrophilic (cold-loving) fungus Pseudogymnoascus destructans was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found P. destructans was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family Partitiviridae. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of P. destructans were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus Pseudogymnoascus, as non-pathogenic fungi P. appendiculatus (1 isolate) and P. roseus (6 isolates) tested negative. P. destructans PdPV-1 could be a valuable tool to investigate fungal biogeography and the host-pathogen interactions in bat WNS.

Petunia plants are used for urban landscaping in many parts of the world, including South Korea. In this study, we aimed to investigate the occurrence of petunia vein clearing virus (PVCV) infection in petunia plants in Seoul, South Korea. PVCV was detected from 23 of 79 petunia samples collected from Seoul. We obtained the complete genome sequences of the Korean isolates in this study (called PVCV-Kr, Kr2, and Kr3), which were compared with the genome sequence of the USA isolate of the virus (PVCV-USA). The genomic DNA of the three PVCV isolates was found to comprise 7210-7267 nucleotides (nts), which is 4-15 nts longer than the PVCV-USA genome. The genomes of the Kr and Kr2 isolates encode a large polyprotein of 252 kDa (2180 amino acids (aa)). The genome of the Kr3 isolate encodes a large polyprotein of 255 kDa (2203 aa). The polyprotein has six protein domains: a movement protein (MP; 72 aa), a coiled-coil domain (CC; 33 aa), an RNA-binding domain (RB; 18 aa), a protease (PR; 21 aa), a reverse transcriptase (RT; 196 aa), and an RNase H (RH; 121 aa). The large polyprotein and six domains of the three isolates showed 93.9-100.0% sequence homology with those of PVCV-USA. Furthermore, the polymerase polyprotein gene (PR, RT, and RH) of the four PVCV isolates containing the USA isolate grouped with those of Rice tungro bacilliform virus and Soybean chlorotic mottle virus, which belong to the same family (Caulimoviridae). Our findings suggested that the Korean isolates represent a new isolate of PVCV. To our knowledge, this is the first report of PVCV detection in South Korea.

The Nanoarchaeota are small cells with reduced genomes that are found attached to and dependent on a second archaeal cell for their growth and replication. Initially found in marine hydrothermal environments and subsequently in terrestrial geothermal hot springs, the Nanoarchaeota species that have been described are obligate ectobionts, each with a different host species. However, no viruses had been described that infect the Nanoarchaeota. Here, we identify a virus infecting Nanoarchaeota by the use of a combination of viral metagenomic and bioinformatic approaches. This virus, tentatively named Nanoarchaeota Virus 1 (NAV1), consists of a 35.6-kb circular DNA genome coding for 52 proteins. We further demonstrate that this virus is broadly distributed among Yellowstone National Park hot springs. NAV1 is one of the first examples of a virus infecting a single-celled organism that is itself an ectobiont of another single-celled organism. IMPORTANCE Here, we present evidence of the first virus found to infect Nanoarchaeota, a symbiotic archaean found in acidic hot springs of Yellowstone National Park, USA. Using culture-independent techniques, we provide the genome sequence and identify the archaeal host species of a novel virus, NAV1. NAV1 is the first example of a virus infecting an archaeal species that is itself an obligate symbiont and dependent on a second host organism for growth and cellular replication. On the basis of annotation of the NAV1 genome, we propose that this virus is the founding member of a new viral family, further demonstrating the remarkable genetic diversity of archaeal viruses., (Copyright © 2020 American Society for Microbiology.)

The cytokinin dehydrogenase gene HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1-3 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley., Competing Interests: The authors have declared that no competing interests exist.

Syphilis is a sexually transmitted infection caused by Treponema pallidum, which is highly prevalent in several countries, including Brazil. The use of bioinformatics' tools for the identification of resistance genes is an important practice for the study of microorganisms, such as T. pallidum. In this study, the complete genomes of 43 strains of T. pallidum, isolated from different countries, were analyzed. A total of 41,514 sequences were obtained, and compared against prokaryote resistance gene databases using BLASTn, BLASTx and RGI for gene alignment and prediction. From the alignments, it was possible to identify antibiotic resistance genes for each strain. The genes identified in each comparison were grouped according to the antibiotic category in which they show resistance to. The antibiotic-resistant genes related to drugs used to treat syphilis were grouped separately. The in silico tools used have shown to be effective in identifying resistance genes in genomes of T. pallidum strains. Due to the lack of research and accurate information regarding the antibiotic resistance genes in T. pallidum, this study serves as a basis for studies in molecular biology whose aim is the identification of these genes, besides being a reference to help in the control and treatment of this infection.

To compare overall genome structure and transcription activator-like effector content, we completely sequenced Xanthomonas axonopodis pv. glycines strain 12-2, isolated in 1992 in Thailand, and strain EB08, isolated in 2008 in the United States (Iowa) using PacBio technology. We reassembled the genome sequence for a second US strain, 8ra, derived from a 1980 Iowa isolate, from existing PacBio reads. Despite geographic and temporal separation, the three genomes are highly syntenous, and their transcription activator-like effector repertoires are highly conserved., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

From the severe porcine epidemic diarrhoea (PED) epidemics that struck in 2013 in the United States of America and other countries of North and South America, two types of porcine epidemic diarrhoea virus (PEDV) were isolated, namely the InDel and the non-InDel strains. They are differentiated by insertions/deletions in the S1 nucleotide sequence of the S gene, and differences in virulence were observed from the clinical cases. In 2014, a PED outbreak occurred in a pig farm in France, from which an InDel strain was isolated. This study aimed at comparing, under experimental conditions, the pathogenicity and the direct and indirect transmissions between a non-InDel strain isolated from a PED-affected piglet in 2014 in the USA and the French InDel strain. All infected pigs showed clinical signs with the non-InDel strain although only the inoculated and direct contact pigs showed clinical signs in the InDel strain group. Although viral RNA was detected in air samples with both strains, the indirect contact pigs remained free from infection with the InDel strain in contrast to the non-InDel group in which airborne transmission occurred in the indirect contact pigs. All infected pigs shed virus in faeces regardless of PEDV strain with 9 of 30 pigs showing intermittent faecal shedding. The transmission rate by direct contact was found to be 2.17-fold higher than the non-InDel strain compared with the InDel. In conclusion, the InDel strain was less pathogenic than the non-InDel strain in our experimental conditions. The transmission route differed between the two strains. Direct contact was the main transmission route for the InDel strain, although the non-InDel strain was transmitted through direct contact and indirectly through the air., (© 2018 Blackwell Verlag GmbH.)

The Mojave rattlesnake (Crotalus scutulatus) inhabits deserts and arid grasslands of the western United States and Mexico. Despite considerable interest in its highly toxic venom and the recognition of two subspecies, no molecular studies have characterized range-wide genetic diversity and population structure or tested species limits within C. scutulatus. We used mitochondrial DNA and thousands of nuclear loci from double-digest restriction site associated DNA sequencing to infer population genetic structure throughout the range of C. scutulatus, and to evaluate divergence times and gene flow between populations. We find strong support for several divergent mitochondrial and nuclear clades of C. scutulatus, including splits coincident with two major phylogeographic barriers: the Continental Divide and the elevational increase associated with the Central Mexican Plateau. We apply Bayesian clustering, phylogenetic inference, and coalescent-based species delimitation to our nuclear genetic data to test hypotheses of population structure. We also performed demographic analyses to test hypotheses relating to population divergence and gene flow. Collectively, our results support the existence of four distinct lineages within C. scutulatus, and genetically defined populations do not correspond with currently recognized subspecies ranges. Finally, we use approximate Bayesian computation to test hypotheses of divergence among multiple rattlesnake species groups distributed across the Continental Divide, and find evidence for co-divergence at this boundary during the mid-Pleistocene., (Copyright © 2018 Elsevier Inc. All rights reserved.)

An obstacle to establishing widely useful data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. Earlier versions of EPA Methods 1609 and 1611 for enterococci used cellular reference materials for quantifying enterococci in unknown test samples, however, EPA updates to these fundamentally DNA-based analysis methods have shifted toward the use of DNA standards. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic plasmid DNA standards that have been made available for updated EPA Methods 1609.1 and 1611.1 as well as for EPA Draft Method C for Escherichia coli. To obtain the most accurate concentration estimates possible, part of this effort was to develop a data analysis model for determining the fluorescence thresholds that distinguish positive from negative droplets produced by the ddPCR reactions. Versions of this model are described for applications with individual reactions, multiple reactions within a ddPCR system run, and multiple reactions within and across different system runs. The latter version was applied toward determinations of error in the concentration estimates of the standards from replicate analyses of each standard in multiple ddPCR system runs. Mean concentration estimates for the five standards from the ddPCR analyses were 4.356, 3.381, 2.371, 1.641 and 1.071 log 10 copies/5 μL with associated standard deviations of 0.074, 0.082, 0.108, 0.131 and 0.188, respectively. These estimates contrasted with expected log 10 concentrations of 4.6, 3.6, 2.6, 1.9 and 1.3 copies/5 μL, respectively, based on the yield of the plasmid reported by the vendor and spectrophotometric analysis of the initial stock solution of this material. These results illustrate how the analyses of original stocks may lead to potential bias(es) in the concentration estimates of final DNA standards and subsequently in the estimates of unknown test samples determined from these standards in qPCR analyses., (Published by Elsevier B.V.)

Rotavirus G (RVG) strains have been detected in a variety of avian species, but RVG genomes have been published from only a single pigeon and two chicken strains. Two turkey RVG strains were identified and characterized, one in a hatchery with no reported health issues and the other in a hatchery with high embryo/poult mortality. The two turkey RVG strains shared only an 85.3 % nucleotide sequence identity in the VP7 gene while the other genes possessed high nucleotide identity among them (96.3-99.9 %). Low nucleotide percentage identities (31.6-87.3 %) occurred among the pigeon and chicken RVG strains. Interestingly, potential recombination events were detected between our RVG strains and a human RVB strain, in the VP6 and NSP3 segments. The epidemiology of RVG in avian flocks and the pathogenicity of the two different RVG strains should be further investigated to understand the ecology and impact of RVG in commercial poultry flocks.

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) has been an economically important virus in the USA for over 30 years. However the complete sequence of only one TSWV isolate PA01 characterized from pepper in Pennsylvania is available., Results: The large (L) RNA of a TSWV WA-USA isolate was cloned and sequenced. It consisted of 8914 nucleotides (nt) encoding a single open reading frame of 8640 nts in the viral-complementary sense. The ORF potentially codes for RNA-dependent RNA polymerase (RdRp) of 330.9 kDa. Two untranslated regions of 241 and 33 nucleotides were present at the 5' and 3' termini, respectively that shared conserved tospoviral sequences. Phylogenetic analysis using nucleotide sequences of the complete L RNA showed that TSWV WA-USA isolate clustered with the American and Asian TSWV isolates which formed a distinct clade from Euro-Asiatic Tospoviruses. Phylogeny of the amino acid sequence of all tospoviral RdRps used in this study showed that all the known TSWV isolates including the USA isolate described in this study formed a distinct and a close cluster with that of Impateins necrotic spot virus. Multiple sequence alignment revealed conserved motifs in the RdRp of TSWV. Recombination analysis identified two recombinants including the TSWV WA-USA isolate. Among them, three recombination events were detected in the conserved motifs of the RdRp., Conclusions: Sequence analysis and phylogenetic analysis of the L RNA showed distinct clustering with selected TSWV isolates reported from elsewhere. Conserved motifs in the core polymerase region of the RdRp and recombination events were identified.

Despite the implementation of cystic fibrosis (CF) newborn screening programs across the United States, the identification of CFTR gene variants in nonwhite populations compared with whites remains suboptimal. Our objective was to establish the spectrum of CFTR variants and their frequencies in CF patients in the United States with African, Native American, Asian, East Indian, or Middle Eastern backgrounds. By using direct DNA sequencing, we identified two CFTR variants in 89 of 140 affected nonwhite individuals with uncharacterized genotypes. Seven variants were novel. Multiplex ligation-dependent probe amplification detected 14 rearrangements in the remaining 51 patients, 6 of which were novel. Deletions and duplications accounted for 17% of unidentified alleles. A cross-sectional analysis of genotyping data from the CF Foundation Patient Registry was performed, comparing 3496 nonwhite patients with 22,206 white CF patients. Patients of Hispanic, black, or Asian ancestry were less likely to have two identified CFTR variants (P < 0.0001 for Hispanics and blacks, P = 0.003 for Asians), and more likely to carry no mutations on the commonly used 23 mutation carrier screening panel (P < 0.0001). We analyzed the mutations recorded for each ancestry and summarized the most frequent ones. This research could facilitate equity in mutation detection between white and nonwhite or mixed-ethnicity CF patients, enabling an earlier diagnosis improving their quality of life., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Subalpine forest ecosystems influence global carbon cycling. However, little is known about the compositions of their soil microbial communities and how these may vary with soil environmental conditions. The goal of this study was to characterize the soil microbial communities in a subalpine forest watershed in central Montana (Stringer Creek Watershed within the Tenderfoot Creek Experimental Forest) and to investigate their relationships with environmental conditions and soil carbonaceous gases. As assessed by tagged Illumina sequencing of the 16S rRNA gene, community composition and structure differed significantly among three landscape positions: high upland zones (HUZ), low upland zones (LUZ), and riparian zones (RZ). Soil depth effects on phylogenetic diversity and β-diversity varied across landscape positions, being more evident in RZ than in HUZ. Mantel tests revealed significant correlations between microbial community assembly patterns and the soil environmental factors tested (water content, temperature, oxygen, and pH) and soil carbonaceous gases (carbon dioxide concentration and efflux and methane concentration). With one exception, methanogens were detected only in RZ soils. In contrast, methanotrophs were detected in all three landscape positions. Type I methanotrophs dominated RZ soils, while type II methanotrophs dominated LUZ and HUZ soils. The relative abundances of methanotroph populations correlated positively with soil water content (R = 0.72, P < 0.001) and negatively with soil oxygen (R = -0.53, P = 0.008). Our results suggest the coherence of soil microbial communities within and differences in communities between landscape positions in a subalpine forested watershed that reflect historical and contemporary environmental conditions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Sequencing of the Mycobacterium tuberculosis pncA gene allows for pyrazinamide susceptibility testing. We summarize data on pncA polymorphisms that do not confer resistance at a susceptibility breakpoint of 100 μg/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the U.S. Centers for Disease Control and Prevention., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We report the first complete nucleotide sequence of a tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae) isolate from the United States. The tripartite genome of PA01 consisted of L, M and S RNAs of 8914, 4765 and 2984 nt, respectively. Similarity percentages in nucleotide and amino acid sequence among PA01 and previously characterized TSWV isolates are provided here. Phylogenetic analysis on the RNA-dependent RNA polymerase (RdRp) gene placed PA01 in a different clade from an isolate from Hawaii that was partially characterized previously. Evidence of two putative reassortment events in the M segment, among PA01 and isolates from South Korea, Italy and Brazil, was found by phylogenetic and recombination analysis, further supporting a role for genetic exchange among isolates of different geographic origin in TSWV evolution.

In the late summer and the fall of 2014, the United States experienced an unprecedented outbreak of enterovirus D68 (EV-D68) infections. During the outbreak, we collected nasopharyngeal swab specimens from patients in the Lower Hudson Valley of New York. Here, we conduct a retrospective study on the genomic diversity of EV-D68 strains. We first employ a metagenomic shotgun sequencing protocol on a total of 93 clinical samples, including 21 negative controls, the results of which allow assembly of 20 EV-D68 genomes, six complete and 14 near-complete. We then investigate their genetic relationships, along with additional 20 EV-D68 strains having whole-genome sequences publicly available. Our comparative genomic analysis uncovers that the majority (26/29) of EV-D68 strains circulating in the 2014 outbreak differ significantly from prior ones, have a main feature of three variables, C1817T, C3277A, and A4020G, and belong to a new clade. C3277A causes amino acid substitution T860N in the protease 2A(pro) cleavage site between VP1 and 2A, whereas A4020G causes S1108G in a 3C(pro) cleavage site between 2B and 2C. The two functional mutations may alter the proteases' cleavage efficiency, leading to increased rate of viral replication and transmission. These provide insights into the evolution of epidemic EV-D68 strains.

Background: Hop is an economically important crop for the Pacific Northwest USA as well as other regions of the world. It is a perennial crop with rhizomatous or clonal propagation system for varietal distribution. A big concern for growers as well as brewers is variety purity and questions are regularly posed to public agencies concerning the availability of genotype testing. Current means for genotyping are based upon 25 microsatellites that provides relatively accurate genotyping but cannot always differentiate sister-lines. In addition, numerous PCR runs (25) are required to complete this process and only a few laboratories exist that perform this service. A genotyping protocol based upon SNPs would enable rapid accurate genotyping that can be assayed at any laboratory facility set up for SNP-based genotyping. The results of this study arose from a larger project designed for whole genome association studies upon the USDA-ARS hop germplasm collection consisting of approximately 116 distinct hop varieties and germplasm (female lines) from around the world., Results: The original dataset that arose from partial sequencing of 121 genotypes resulted in the identification of 374,829 SNPs using TASSEL-UNEAK pipeline. After filtering out genotypes with more than 50% missing data (5 genotypes) and SNP markers with more than 20% missing data, 32,206 highly filtered SNP markers across 116 genotypes were identified and considered for this study. Minor allele frequency (MAF) was calculated for each SNP and ranked according to the most informative to least informative. Only those markers without missing data across genotypes as well as 60% or less heterozygous gamete calls were considered for further analysis. Genetic distances among individuals in the study were calculated using the marker with the highest MAF value, then by using a combination of the two markers with highest MAF values and so on. This process was reiterated until a set of markers was identified that allowed for all genotypes in the study to be genetically differentiated from each other. Next, we compared genetic matrices calculated from the minimal marker sets [(Table 2; 6-, 7-, 8-, 10- and 12-marker set matrices] and that of a matrix calculated from a set of markers with no missing data across all 116 samples (1006 SNP markers). The minimum number of markers required to meet both specifications was a set of 7-markers (Table 3). These seven SNPs were then aligned with a genome assembly, and DNA sequence both upstream and downstream were used to identify primer sequences that can be used to develop seven amplicons for high resolution melting curve PCR detection or other SNP-based PCR detection methods., Conclusions: This study identifies a set of 7 SNP markers that may prove useful for the identification and validation of hop varieties and accessions. Variety validation of unknown samples assumes that the variety under question has been included a priori in a discovery panel. These results are based upon in silica studies and markers need to be validated using different SNP marker technology upon a differential set of hop genotypes. The marker sequence data and suggested primer sets provide potential means to fingerprint hop varieties in most genetic laboratories utilizing SNP-marker technology.

Objective: Endothelial dysfunction assessed by brachial artery flow-mediated dilation (FMD) is a marker of early atherosclerosis. Glucocorticoid receptor gene (NR3C1) regulates many biological processes, including stress response, behavioral, cardiometabolic and immunologic functions. Genetic variants in NR3C1 have been associated with atherosclerosis and related risk factors. This study investigated the association of NR3C1 promoter methylation with FMD, independent of genetic and family-level environmental factors., Methods: We studied 84 middle-aged, male-male monozygotic twin pairs recruited from the Vietnam Era Twin Registry. Brachial artery FMD was measured by ultrasound. DNA methylation levels at 22 CpG residues in the NR3C1 exon 1F promoter region were quantified by bisulfite pyrosequencing in genomic DNA isolated from peripheral blood leukocytes. Co-twin control analyses were conducted to examine the association of methylation variation with FMD, adjusting for smoking, physical activity, body mass index, lipids, blood pressure, fasting glucose, and depressive symptoms. Multiple testing was corrected using the false discovery rate., Results: Mean methylation level across the 22 studied CpG sites was 2.02%. Methylation alterations at 12 out of the 22 CpG residues were significantly associated with FMD. On average, a 1% increase in the intra-pair difference in mean DNA methylation was associated with 2.83% increase in the intra-pair difference in FMD (95% CI: 1.46-4.20; P < 0.0001) after adjusting for risk factors and multiple testing., Conclusion: Methylation variation in NR3C1 exon 1F promoter significantly influences subclinical atherosclerosis, independent of genetic, early family environmental and other risk factors., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Massively parallel sequencing (MPS) technology is capable of determining the sizes of short tandem repeat (STR) alleles as well as their individual nucleotide sequences. Thus, single nucleotide polymorphisms (SNPs) within the repeat regions of STRs and variations in the pattern of repeat units in a given repeat motif can be used to differentiate alleles of the same length. In this study, MPS was used to sequence 28 forensically-relevant Y-chromosome STRs in a set of 41 DNA samples from the 3 major U.S. population groups (African Americans, Caucasians, and Hispanics). The resulting sequence data, which were analyzed with STRait Razor v2.0, revealed 37 unique allele sequence variants that have not been previously reported. Of these, 19 sequences were variations of documented sequences resulting from the presence of intra-repeat SNPs or alternative repeat unit patterns. Despite a limited sampling, two of the most frequently-observed variants were found only in African American samples. The remaining 18 variants represented allele sequences for which there were no published data with which to compare. These findings illustrate the great potential of MPS with regard to increasing the resolving power of STR typing and emphasize the need for sample population characterization of STR alleles., (Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.)

Cystic fibrosis (CF) is one of the most common recessive conditions among whites, with an estimated carrier frequency of 1 in 25 in the United States. Population-based CF carrier screening was implemented in the United States in 2001. The number of mutations screened by each laboratory may vary; however, the 23 most common CF mutations recommended for screening by the American College of Medical Genetics and American College of Obstetricians and Gynecologists are included in all platforms. The c.1364C>A (p.A455E) mutation located in exon 10 of the CFTR gene is one of the 23 mutations. Because CFTR exon 10 and its flanking intronic regions are duplicated and transposed onto several other chromosomes of the human genome during evolution and function as unprocessed pseudogenes, variations in the CFTR pseudogenes may confound CF screening results for mutations located in exon 10 of the CFTR gene. We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.

Antiviral drug susceptibility is one of the evaluation criteria of pandemic potential posed by an influenza virus. Influenza A viruses of swine (IAV-S) can play an important role in generating novel variants, yet limited information is available on the drug resistance profiles of IAV-S circulating in the U.S. Phenotypic analysis of the IAV-S isolated in the U.S. (2009-2011) (n=105) revealed normal inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir, zanamivir, and peramivir. Screening NA sequences from IAV-S collected in the U.S. (1930-2014) showed 0.03% (1/3396) sequences with clinically relevant H274Y-NA substitution. Phenotypic analysis of IAV-S isolated in the U.S. (2009-2011) confirmed amantadine resistance caused by the S31N-M2 and revealed an intermediate level of resistance caused by the I27T-M2. The majority (96.7%, 589/609) of IAV-S with the I27T-M2 in the influenza database were isolated from pigs in the U.S. The frequency of amantadine-resistant markers among IAV-S in the U.S. was high (71%), and their distribution was M-lineage dependent. All IAV-S of the Eurasian avian M lineage were amantadine-resistant and possessed either a single S31N-M2 substitution (78%, 585/747) or its combination with the V27A-M2 (22%, 162/747). The I27T-M2 substitution accounted for 43% (429/993) of amantadine resistance in classic swine M lineage. Phylogenetic analysis showed that both S31N-M2 and I27T-M2 emerged stochastically but appeared to be fixed in the U.S. IAV-S population. This study defines a drug-susceptibility profile, identifies the frequency of drug-resistant markers, and establishes a phylogenetic approach for continued antiviral-susceptibility monitoring of IAV-S in the U.S., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The complex and unresolved evolutionary origins of the 2009 H1N1 influenza pandemic exposed major gaps in our knowledge of the global spatial ecology and evolution of influenza A viruses in swine (swIAVs). Here we undertake an expansive phylogenetic analysis of swIAV sequence data and demonstrate that the global live swine trade strongly predicts the spatial dissemination of swIAVs, with Europe and North America acting as sources of viruses in Asian countries. In contrast, China has the world's largest swine population but is not a major exporter of live swine, and is not an important source of swIAVs in neighbouring Asian countries or globally. A meta-population simulation model incorporating trade data predicts that the global ecology of swIAVs is more complex than previously thought, and the United States and China's large swine populations are unlikely to be representative of swIAV diversity in their respective geographic regions, requiring independent surveillance efforts throughout Latin America and Asia.