Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.

Vector communities under global change may exacerbate and redistribute infectious disease risk.

Vector-borne parasites may be transmitted by multiple vector species, resulting in an increased risk of transmission, potentially at larger spatial scales compared to any single vector species. Additionally, the different abilities of patchily distributed vector species to acquire and transmit parasites will lead to varying degrees of transmission risk. Investigation of how vector community composition and parasite transmission change over space due to variation in environmental conditions may help to explain current patterns in diseases but also informs our understanding of how patterns will change under climate and land-use change. We developed a novel statistical approach using a multi-year, spatially extensive case study involving a vector-borne virus affecting white-tailed deer transmitted by Culicoides midges. We characterized the structure of vector communities, established the ecological gradient controlling change in structure, and related the ecology and structure to the amount of disease reporting observed in host populations. We found that vector species largely occur and replace each other as groups, rather than individual species. Moreover, community structure is primarily controlled by temperature ranges, with certain communities being consistently associated with high levels of disease reporting. These communities are essentially composed of species previously undocumented as potential vectors, whereas communities containing putative vector species were largely associated with low levels, or even absence, of disease reporting. We contend that the application of metacommunity ecology to vector-borne infectious disease ecology can greatly aid the identification of transmission hotspots and an understanding of the ecological drivers of parasite transmission risk both now and in the future.

Isolation of Heartland Virus from Lone Star Ticks, Georgia, USA, 2019.

Report of a human death and exposure of white-tailed deer to Heartland virus (HRTV) in Georgia, USA, prompted the sampling of questing ticks during 2018-2019 in 26 sites near where seropositive deer were captured and the residence of the human case-patient. We processed 9,294 Amblyomma americanum ticks in pools by virus isolation in Vero E6 cells and reverse transcription PCR. Positive pools underwent whole-genome sequencing. Three pools were positive for HRTV (minimum infection rate 0.46/1,000 ticks) and none for Bourbon virus. Cell cultures confirmed HRTV presence in 2 pools. Genome sequencing, achieved for the 3 HRTV isolates, showed high similarity among samples but marked differences with previously sequenced HRTV isolates. The isolation and genomic characterization of HRTV from A. americanum ticks in Georgia confirm virus presence in the state. Clinicians and public health professionals should be aware of this emerging tickborne pathogen.

Susceptibility of wild turkeys (Meleagris gallopavo) to experimental West Nile virus infection.

Since the introduction of West Nile virus (WNV) to North America in 1999, WNV is estimated to have contributed to population-level declines in numerous avian species. However, the potential impacts of this virus on many free-ranging upland game bird species, including the wild turkey (Meleagris gallopavo), which is undergoing regional population declines, remain unknown. Herein, two age groups (∼5 to 6 weeks and ∼15 to 16 weeks post-hatch) of juvenile wild turkeys were subcutaneously inoculated with WNV, sampled daily from 1 to 7 days post-inoculation (dpi), and euthanized on 14 dpi. No clinical signs and minimal gross lesions were attributable to WNV infection. Peak viraemia titres were similar between age groups (<101.7 to 104.6 plaque-forming units [PFU]/ml), but the duration of viraemia was longer in the old group (3-4 days) than in the young group (0-3 days). Intermittent oral and/or cloacal viral shedding from 2 to 7 dpi was detected in both age groups. No infectious virus was detected in the heart, brain, kidney, skeletal muscle, spleen, and feathers from WNV-inoculated turkeys euthanized on 14 dpi. All WNV-inoculated birds seroconverted by 14 dpi, as well as two co-housed sham-inoculated birds. The most consistent microscopic lesions among all WNV-inoculated birds were mild lymphoplasmacytic myocarditis and encephalitis. Minimal immunohistochemical labelling was detected in tissues in addition to scant macrophages within the blood, spleen, and bone marrow. These data suggest WNV is unlikely to pose a significant risk to wild turkey populations, although the possibility remains that WNV may indirectly decrease fitness or predispose wild turkeys to other health stressors.RESEARCH HIGHLIGHTS Clinical disease was not observed in wild turkeys experimentally infected with WNV.Pathology attributed to WNV was mild and included brain and heart inflammation.Viraemias suggest WNV-infected wild turkeys do not play a role in WNV transmission.No age-associated differences in WNV clinical disease or pathology were observed.

Indels, structural variation, and recombination drive genomic diversity in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has a great capacity for evolutionary adaptation to evade host immunity and develop drug resistance. Current understanding of parasite evolution is impeded by the fact that a large fraction of the genome is either highly repetitive or highly variable and thus difficult to analyze using short-read sequencing technologies. Here, we describe a resource of deep sequencing data on parents and progeny from genetic crosses, which has enabled us to perform the first genome-wide, integrated analysis of SNP, indel and complex polymorphisms, using Mendelian error rates as an indicator of genotypic accuracy. These data reveal that indels are exceptionally abundant, being more common than SNPs and thus the dominant mode of polymorphism within the core genome. We use the high density of SNP and indel markers to analyze patterns of meiotic recombination, confirming a high rate of crossover events and providing the first estimates for the rate of non-crossover events and the length of conversion tracts. We observe several instances of meiotic recombination within copy number variants associated with drug resistance, demonstrating a mechanism whereby fitness costs associated with resistance mutations could be compensated and greater phenotypic plasticity could be acquired.

Terrestrial Bird Migration and West Nile Virus Circulation, United States.

Host migration and emerging pathogens are strongly associated, especially with regard to zoonotic diseases. West Nile virus (WNV), a mosquitoborne pathogen capable of causing severe, sometimes fatal, neuroinvasive disease in humans, is maintained in highly mobile avian hosts. Using phylogeographic approaches, we investigated the relationship between WNV circulation in the United States and the flight paths of terrestrial birds. We demonstrated southward migration of WNV in the eastern flyway and northward migration in the central flyway, which is consistent with the looped flight paths of many terrestrial birds. We also identified 3 optimal locations for targeted WNV surveillance campaigns in the United States-Illinois, New York, and Texas. These results illustrate the value of multidisciplinary approaches to surveillance of infectious diseases, especially zoonotic diseases.

An interactive genome browser of association results from the UK10K cohorts project.

UNLABELLED: High-throughput sequencing technologies survey genetic variation at genome scale and are increasingly used to study the contribution of rare and low-frequency genetic variants to human traits. As part of the Cohorts arm of the UK10K project, genetic variants called from low-read depth (average 7×) whole genome sequencing of 3621 cohort individuals were analysed for statistical associations with 64 different phenotypic traits of biomedical importance. Here, we describe a novel genome browser based on the Biodalliance platform developed to provide interactive access to the association results of the project. AVAILABILITY AND IMPLEMENTATION: The browser is available at http://www.uk10k.org/dalliance.html. Source code for the Biodalliance platform is available under a BSD license from http://github.com/dasmoth/dalliance, and for the LD-display plugin and backend from http://github.com/dasmoth/ldserv.

K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa.

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.

Association mapping by pooled sequencing identifies TOLL 11 as a protective factor against Plasmodium falciparum in Anopheles gambiae.

BACKGROUND: The genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density. METHODS: We designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine. RESULTS: In the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection. CONCLUSIONS: We present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.

Genetic characterization of epizootic hemorrhagic disease virus strains isolated from cattle in Israel.

Epizootic hemorrhagic disease virus (EHDV), a member of the genus Orbivirus not reported previously in Israel, was isolated from Israeli cattle during a 'bluetongue-like' disease outbreak in 2006. To ascertain the origin of this new virus, three isolates from the outbreak were fully sequenced and compared with available sequences. Whilst the L2 gene segment clustered with the Australian EHDV serotype 7 (EHDV-7) reference strain, most of the other segments were clustered with EHDV isolates of African/Middle East origin, specifically Bahrain, Nigeria and South Africa. The M6 gene had genetic relatedness to the Australian/Asian strains, but with the limited data available the significance of this relationship is unclear. Only one EHDV-7 L2 sequence was available, and as this gene encodes the serotype-specific epitope, the relationship of these EHDV-7 L2 genes to an Australian EHDV-7 reflects the serotype association, not necessarily the origin. The genetic data indicated that the strains affecting Israel in 2006 may have been related to similar outbreaks that occurred in North Africa in the same year. This finding also supports the hypothesis that EHDV entered Israel during 2006 and was not present there before this outbreak.

Effect of Temperature on Replication of Epizootic Hemorrhagic Disease Viruses in Culicoides sonorensis (Diptera: Ceratopogonidae).

Replication of arboviruses, including orbiviruses, within the vector has been shown to be temperature dependent. Cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Previous research with epizootic hemorrhagic disease virus (EHDV) serotype 1 demonstrated that higher temperatures were associated with shorter extrinsic incubation periods in Culicoides sonorensis Wirth & Jones, a confirmed vector of EHDV in North America. To further our understanding of the effect of temperature on replication of EHDV within the vector, C. sonorensis were experimentally infected with one of three EHDV strains representing three serotypes (1, 2, and 7). Midges were fed defibrinated white-tailed deer (Odocoileus virginianus) blood spiked with EHDV (≥10(6.5) TCID(50)/ml) through a parafilm membrane using an artificial feeding device and were then held at 20, 25, or 30°C. In addition to this in vitro method, a white-tailed deer experimentally infected with EHDV-7 was used to provide an infectious bloodmeal to determine if the results were comparable with those from the in vitro feeding method. Whole midges were processed for virus isolation and titration at regular intervals following feeding; midges with ≥10(2.7) TCID(50) were considered potentially competent to transmit virus. The virus recovery rates were high throughout the study and all three viruses replicated within C. sonorensis to high titer (≥ 10(2.7) TCID(50)/midge). Across all virus strains, the time to detection of potentially competent midges decreased with increasing temperature: 12-16 d postfeeding (dpf) at 20°C, 4-6 dpf at 25°C, and 2-4 dpf at 30°C. Significant differences in replication of the three viruses in C. sonorensis were observed, with EHDV-2 replicating to a high titer in a smaller proportion of midges and with lower peak titers. The findings are consistent with previous studies of related orbiviruses, showing that increasing temperature can shorten the apparent extrinsic incubation period for multiple EHDV strains (endemic and exotic) in C. sonorensis.

Long term impacts of combined sewer overflow remediation on water quality and population dynamics of Culex quinquefasciatus, the main urban West Nile virus vector in Atlanta, GA.

BACKGROUND: Combined sewers are a significant source of urban water pollution due to periodic discharges into natural streams. Such events (called combined sewer overflows, or CSOs) contribute to the impairment of natural waterways and are associated with increased mosquito productivity and elevated risk of West Nile virus transmission. OBJECTIVES: We investigated the impact of CSOs on water quality and immature mosquito productivity in the city of Atlanta, Georgia, one year before and four years after CSO facility remediation. METHODS: Water quality (ammonia, phosphate, nitrate and dissolved oxygen concentrations), immature mosquitoes (larvae and pupae), water temperature and rainfall were quantified biweekly between June-October at two urban creeks during 2008-2012. A before-after control-intervention design tested the impact of remediation on mosquito productivity and water quality, whereas generalized linear mixed-effect models quantified the factors explaining the long term impacts of remediation on mosquito productivity. RESULTS: Ammonia and phosphate concentrations and late immature (fourth-instar and pupae) mosquito populations were significantly higher in CSO than in non-CSO creeks, while dissolved oxygen concentrations were lower. Remediation significantly improved water quality estimates (particularly ammonia and dissolved oxygen) and reduced the number of overflows, mosquito productivity and the overall contribution of CSO-affected streams as sources of vectors of West Nile virus. CONCLUSIONS: The quality of water in CSOs provided a suitable habitat for immature mosquitoes. Remediation of the CSO facility through the construction of a deep storage tunnel improved water quality indices and reduced the productivity of mosquito species that can serve as vectors of West Nile virus.

Lymphoproliferative Disease Virus and Reticuloendotheliosis Virus Detection and Disease in Wild Turkeys (Meleagris gallopavo).

Lymphoproliferative disease virus (LPDV) and reticuloendotheliosis virus (REV) are oncogenic retroviruses that can cause disease in wild and domestic fowl. Lymphoproliferative disease virus infections are common and widespread in Wild Turkeys (Meleagris gallopavo) in the US and east-central Canada, while REV has been detected worldwide in numerous avian host species. We tested tissues (spleen, liver, and/or bone marrow, plus neoplastic tissue, if present) from 172 Wild Turkeys that underwent necropsy from December 2018 through October 2021 for both viruses using PCR. We evaluated demographic, geographic, temporal, and seasonal data by chi-square test of independence and logistic regression for turkeys infected with LPDV and/or REV. At least one of these retroviruses was detected in 80.8% (139/172) of Wild Turkeys from 15 US states, with significantly more turkeys being positive for LPDV (72.1%, 124/172) versus REV (43.6%, 75/172; P<0.001). Both viruses (coinfections) were detected in 34.9% (60/172) of turkeys. Among LPDV-infected turkeys (including coinfections), bone marrow had the highest detection rate (38/58, 65.5%), significantly higher than spleen (30/58, 51.7%) and liver (20/58, 34.5%; P<0.001). In REV-infected turkeys, bone marrow had the highest detection rate (24/58, 41.4%). All three tissues (spleen, liver, bone marrow) concurrently tested positive in most (15/25, 60%) REV-infected turkeys. These results suggest LPDV tissue tropism for bone marrow, whereas REV may have broader tissue tropism. Histopathology consistent with lymphoid proliferation and/or neoplasia characteristic of lymphoproliferative disease was evident in 29/172 (16.9%) turkeys assessed, including two REV-only-infected turkeys. Season was significantly associated with LPDV prevalence (highest in winter); year and season were both significantly associated with REV prevalence (highest in 2020 and winter). These data contribute to optimizing diagnostic strategies that may aid in pathogen monitoring and improve detections to increase our understanding of the potential impacts of these viruses on Wild Turkey populations.

Surveillance for Selected Pathogens and Parasites of Northern Bobwhite (Colinus virginianus) from Western Oklahoma, USA, 2018-20.

The Northern Bobwhite (Colinus virginianus) has been undergoing a range-wide population decline. Potential causes for declines across its historic range have been investigated for decades and include habitat loss and fragmentation and a variety of parasitic and infectious diseases. Although there have been studies on bobwhite ecology in Oklahoma, USA, relatively little is known about parasites and pathogens in the region. We evaluated the health of free-ranging bobwhites from nine sites in western Oklahoma. From 2018 to 2020, 206 bobwhites were evaluated for gross and microscopic lesions and tested for selected pathogens. In general, bobwhites were in good nutritional condition with ample muscle mass and fat stores. No significant gross lesions were observed in any bobwhite and no significant histologic lesions were detected in a subset. There was no evidence of infection with or exposure to reticuloendotheliosis virus, West Nile virus, respiratory Mycoplasmataceae species, Pasteurella multocida, intestinal Eimeria spp., or oral Trichomonas spp. Several pathogens of potential concern were detected, including avian adenovirus (8.6%), Toxoplasma gondii (2.3%), and haemosporidians (a Haemoproteus sp. (1.5%), Leucocytozoon schoutedeni (1.5%), and Plasmodium homopolare haplotype 2 [lineage LAIRI01; 3.6%]). Physaloptera sp. (12%) and Sarcocystis sp. (1%) were detected in the breast muscle. Low intraspecific genetic diversity was noted for Physaloptera sp., and sequences were most similar to Physaloptera sequences from bobwhites and grasshoppers (Orthoptera) in Texas. Low intensities of chewing lice, chiggers, and ticks were observed. A subset of bobwhites had evidence of exposure to selected toxicants and heavy metals; a small number had low levels of iron, manganese, zinc, molybdenum, and copper, which were not considered diagnostically relevant. In general, bobwhites from western Oklahoma appeared to be in good health with a low diversity of pathogens detected, but future work is needed to understand potentially changing disease risks for this population.

Storage time and temperature of filter paper strips affect anti-West Nile virus antibody detection in 2 galliform species.

The effects of filter paper strip (FPS) storage time and temperature on antibody detection are poorly understood despite widespread use in wildlife research. We collected sera and FPSs from 23 wild turkeys (Meleagris gallopavo) and 20 northern bobwhites (Colinus virginianus) experimentally infected with West Nile virus (WNV) to compare FPS storage methods with WNV plaque reduction neutralization test (PRNT) sensitivity. FPS storage methods included: immediate elution after drying, and storage at -20°C for 3 mo, -20°C for 6 mo, room temperature (RT) for 3 mo, and RT for 6 mo prior to elution. FPS eluates and sera were co-titrated to determine endpoint antibody titers, which were compared between FPS sera eluted immediately and sera, and among FPS eluates that underwent different storage conditions. Antibody titers were ~4-fold less in FPS sera eluted immediately versus sera, and dropped more frequently below PRNT detection threshold in northern bobwhites, which had ~10-fold lower serum antibody titers than wild turkeys. Antibody titers were lower in FPS samples stored at RT and for 6 mo. WNV serologic surveys may result in falsely low seroprevalence estimates if FPSs are stored at RT for ≥ 3 mo before elution.

Multi-Year Comparison of Community- and Species-Level West Nile Virus Antibody Prevalence in Birds from Atlanta, Georgia and Chicago, Illinois, 2005-2016.

West Nile virus (WNV) is prevalent in the United States but shows considerable variation in transmission intensity. The purpose of this study was to compare patterns of WNV seroprevalence in avian communities sampled in Atlanta, Georgia and Chicago, Illinois during a 12-year period (Atlanta 2010-2016; Chicago 2005-2012) to reveal regional patterns of zoonotic activity of WNV. WNV antibodies were measured in wild bird sera using ELISA and serum neutralization methods, and seroprevalence among species, year, and location of sampling within each city were compared using binomial-distributed generalized linear mixed-effects models. Seroprevalence was highest in year-round and summer-resident species compared with migrants regardless of region; species explained more variance in seroprevalence within each city. Northern cardinals were the species most likely to test positive for WNV in each city, whereas all other species, on average, tested positive for WNV in proportion to their sample size. Despite similar patterns of seroprevalence among species, overall seroprevalence was higher in Atlanta (13.7%) than in Chicago (5%). Location and year of sampling had minor effects, with location explaining more variation in Atlanta and year explaining more variation in Chicago. Our findings highlight the nature and magnitude of regional differences in WNV urban ecology.

Putting hornets on the genomic map.

Hornets are the largest of the social wasps, and are important regulators of insect populations in their native ranges. Hornets are also very successful as invasive species, with often devastating economic, ecological and societal effects. Understanding why these wasps are such successful invaders is critical to managing future introductions and minimising impact on native biodiversity. Critical to the management toolkit is a comprehensive genomic resource for these insects. Here we provide the annotated genomes for two hornets, Vespa crabro and Vespa velutina. We compare their genomes with those of other social Hymenoptera, including the northern giant hornet Vespa mandarinia. The three hornet genomes show evidence of selection pressure on genes associated with reproduction, which might facilitate the transition into invasive ranges. Vespa crabro has experienced positive selection on the highest number of genes, including those putatively associated with molecular binding and olfactory systems. Caste-specific brain transcriptomic analysis also revealed 133 differentially expressed genes, some of which are associated with olfactory functions. This report provides a spring-board for advancing our understanding of the evolution and ecology of hornets, and opens up opportunities for using molecular methods in the future management of both native and invasive populations of these over-looked insects.

A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. "Hillquist").

Blackberries (Rubus spp.) are the fourth most economically important berry crop worldwide. Genome assemblies and annotations have been developed for Rubus species in subgenus Idaeobatus, including black raspberry (R. occidentalis), red raspberry (R. idaeus), and R. chingii, but very few genomic resources exist for blackberries and their relatives in subgenus Rubus. Here we present a chromosome-length assembly and annotation of the diploid blackberry germplasm accession "Hillquist" (R. argutus). "Hillquist" is the only known source of primocane-fruiting (annual-fruiting) in tetraploid fresh-market blackberry breeding programs and is represented in the pedigree of many important cultivars worldwide. The "Hillquist" assembly, generated using Pacific Biosciences long reads scaffolded with high-throughput chromosome conformation capture sequencing, consisted of 298 Mb, of which 270 Mb (90%) was placed on 7 chromosome-length scaffolds with an average length of 38.6 Mb. Approximately 52.8% of the genome was composed of repetitive elements. The genome sequence was highly collinear with a novel maternal haplotype-resolved linkage map of the tetraploid blackberry selection A-2551TN and genome assemblies of R. chingii and red raspberry. A total of 38,503 protein-coding genes were predicted, of which 72% were functionally annotated. Eighteen flowering gene homologs within a previously mapped locus aligning to an 11.2 Mb region on chromosome Ra02 were identified as potential candidate genes for primocane-fruiting. The utility of the "Hillquist" genome has been demonstrated here by the development of the first genotyping-by-sequencing-based linkage map of tetraploid blackberry and the identification of possible candidate genes for primocane-fruiting. This chromosome-length assembly will facilitate future studies in Rubus biology, genetics, and genomics and strengthen applied breeding programs.

Culex flavivirus and West Nile virus in Culex quinquefasciatus populations in the southeastern United States.

Little is known of the interactions between insect-only flaviviruses and other arboviruses in their mosquito hosts, or the potential public health significance of these associations. The specific aims of this study were to describe the geographic distribution, prevalence, and seasonal infection rates of Culex flavivirus (CxFV) and West Nile virus (WNV) in Culex quinquefasciatus Say in the Southeastern United States, investigate the potential association between CxFV and WNV prevalence in Cx. quinquefasciatus and describe the phylogenetic relationship among CxFV and WNV isolates from the Southeastern United States and around the world. Using ArboNET records, 11 locations were selected across Georgia, Mississippi, and Louisiana that represented a range of WNV human case incidence levels. Cx. quinquefasciatus were trapped weekly throughout the summer of 2009 and pools were screened for flavivirus RNA by reverse transcriptase polymerase chain reaction. Cx. quinquefasciatus from Georgia had significantly higher CxFV infection rates than either Mississippi or Louisiana. CxFV was not detected in Mississippi after July, and no CxFV was detected in Cx. quinquefasciatus in Louisiana. In Georgia, CxFV infection rates were variable between and within counties and over time. WNV infection rates were not significantly different across states or months, and WNV sequences from all three states were identical to each other in the envelope and NS5 gene regions. Phylogenetically, NS5 and E gene sequences from Georgia CxFV isolates clustered with CxFV from Japan, Iowa, and Texas. Multiple CxFV genetic variants were found circulating simultaneously in Georgia. No evidence was found supporting an association between WNV and CxFV infection prevalence in Cx. quinquefasciatus.