Novel clades of tick-borne pathogenic nairoviruses in Europe.

Members of the Orthonairovirus genus (family Nairoviridae) include many tick-borne viruses of significant human and animal health impact, with several recently-documented pathogenic viruses lacking sufficient epidemiological information. We screened 215 adult ticks of seven species collected in Bulgaria, Georgia, Latvia and Poland for orthonairoviruses, followed by nanopore sequencing (NS) for genome characterization. Initial generic amplification revealed Sulina virus (SULV, Orthonairovirus sulinaense), for which an updated amplification assay was used, revealing an overall prevalence of 2.7% in Ixodes ricinus ticks from Latvia. Three complete and additional partial SULV genomes were generated, that consistently formed a separate, distinct clade with further intragroup divergence in the maximum likelihood analyses. Comparisons with previously described viruses from Romania exhibited similar genome topologies, albeit with divergent motifs and cleavage sites on the glycoprotein precursor. Preliminary evidence of recombination involving the S segment was documented, in addition to variations in predicted viral glycoproteins. Generic screening further identified Tacheng tick virus 1 (TCTV1, Orthonairovirus tachengense), with documented human infections, in Dermacentor reticulatus ticks from Poland, with a prevalence of 0.9%. Subsequent NS and assembly provided the first complete TCTV1 genome outside of China, where it was originally described. Phylogenetic analysis of virus genome segments revealed TCTV1-Poland as a discrete taxon within the TCTV1 cluster in the Orthonairovirus genus, representing a geographically segregated clade. Comparable genome topology with TCTV1 from China was observed, aside from minor variations in the M segment. Similar to SULV, TCTV1 exhibited several mismatches on previously described screening primer binding sites, likely to prevent amplification. These findings indicate presence of novel TCTV1 and SULV clades in Eastern Europe, confirming the expansion of orthonairoviruses with pathogenic potential.

Data release: targeted systematic literature search for tick and tick-borne pathogen distributions in six countries in sub-Saharan Africa from 1901 to 2020.

BACKGROUND: Surveillance data documenting tick and tick-borne disease (TBD) prevalence is needed to develop risk assessments and implement control strategies. Despite extensive research in Africa, there is no standardized, comprehensive review. METHODS: Here we tackle this knowledge gap, by producing a comprehensive review of research articles on ticks and TBD between 1901 and 2020 in Chad, Djibouti, Ethiopia, Kenya, Tanzania, and Uganda. Over 8356 English language articles were recovered. Our search strategy included 19 related MeSH terms. Articles were reviewed, and 331 met inclusion criteria. Articles containing mappable data were compiled into a standardized data schema, georeferenced, and uploaded to VectorMap. RESULTS: Tick and pathogen matrixes were created, providing information on vector distributions and tick-pathogen associations within the six selected African countries. CONCLUSIONS: These results provide a digital, mappable database of current and historical tick and TBD distributions across six countries in Africa, which can inform specific risk modeling, determine surveillance gaps, and guide future surveillance priorities.

Nanopore-based metagenomics reveal a new Rickettsia in Europe.

Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum.

The African mosquito-borne diseasosome: geographical patterns, range expansion and future disease emergence.

Mosquito-borne diseases (MBDs) threaten public health and food security globally. We provide the first biogeographic description of the African mosquito fauna (677 species) and the 151 mosquito-borne pathogens (MBPs) they transmit. While mosquito species richness agrees with expectations based on Africa's land surface, African arboviruses and mammalian plasmodia are more speciose than expected. Species assemblages of mosquitoes and MBPs similarly separate sub-Saharan Africa from North Africa, and those in West and Central Africa from eastern and southern Africa. Similarities between mosquitoes and MBPs in diversity and range size suggest that mosquitoes are key in delimiting the range of MBPs. With approximately 25% endemicity, approximately 50% occupying one to three countries and less than 5% occupying greater than 25 countries, the ranges of mosquitoes and MBPs are surprisingly small, suggesting that most MBPs are transmitted by a single mosquito species. Exceptionally widespread mosquito species feed on people and livestock, and most are high-altitude-windborne migrants. Likewise, widespread MBPs are transmitted among people or livestock by widespread mosquitoes, suggesting that adapting to people or livestock and to widespread mosquito species promote range expansion in MBPs. Range size may predict range expansion and emergence risk. We highlight key knowledge gaps that impede prediction and mitigation of future emergence of local and global MBDs.

The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Phylogenomics reveals the history of host use in mosquitoes.

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188-250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Impact of nanopore-based metagenome sequencing on tick-borne virus detection.

Introduction: We evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays. Methods: Forty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach. Results: Eleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted segment read counts was observed between PCR-positive and PCR-negative samples. NS further enabled the initial description of Quaranjavirus sequences in ticks, where human and avian pathogenicity of particular isolates had been previously documented. Discussion: NS was observed to surpass broad-range and nested amplification in detection and to generate sufficient genome-wide data for investigating virus diversity. It can be employed for monitoring pathogens in tick vectors or human/animal clinical samples in hot-spot regions for examining zoonotic spillover.

High Levels of Diversity in Anopheles Subgenus Kerteszia Revealed by Species Delimitation Analyses.

The Anopheles subgenus Kerteszia is a poorly understood group of mosquitoes that includes several species of medical importance. Although there are currently twelve recognized species in the subgenus, previous studies have shown that this is likely to be an underestimate of species diversity. Here, we undertake a baseline study of species delimitation using the barcode region of the mtDNA COI gene to explore species diversity among a geographically and taxonomically diverse range of Kerteszia specimens. Beginning with 10 of 12 morphologically identified Kerteszia species spanning eight countries, species delimitation analyses indicated a high degree of cryptic diversity. Overall, our analyses found support for at least 28 species clusters within the subgenus Kerteszia. The most diverse taxon was Anopheles neivai, a known malaria vector, with eight species clusters. Five other species taxa showed strong signatures of species complex structure, among them Anopheles bellator, which is also considered a malaria vector. There was some evidence for species structure within An. homunculus, although the results were equivocal across delimitation analyses. The current study, therefore, suggests that species diversity within the subgenus Kerteszia has been grossly underestimated. Further work will be required to build on this molecular characterization of species diversity and will rely on genomic level approaches and additional morphological data to test these species hypotheses.

Global Distribution of Aedes aegypti and Aedes albopictus in a Climate Change Scenario of Regional Rivalry.

Arboviral mosquito vectors are key targets for the surveillance and control of vector-borne diseases worldwide. In recent years, changes to the global distributions of these species have been a major research focus, aimed at predicting outbreaks of arboviral diseases. In this study, we analyzed a global scenario of climate change under regional rivalry to predict changes to these species' distributions over the next century. Using occurrence data from VectorMap and environmental variables (temperature and precipitation) from WorldClim v. 2.1, we first built fundamental niche models for both species with the boosted regression tree modelling approach. A scenario of climate change on their fundamental niche was then analyzed. The shared socioeconomic pathway scenario 3 (regional rivalry) and the global climate model Geophysical Fluid Dynamics Laboratory Earth System Model v. 4.1 (GFDL-ESM4.1; gfdl.noaa.gov) were utilized for all analyses, in the following time periods: 2021-2040, 2041-2060, 2061-2080, and 2081-2100. Outcomes from these analyses showed that future climate change will affect Ae. aegypti and Ae. albopictus distributions in different ways across the globe. The Northern Hemisphere will have extended Ae. aegypti and Ae. albopictus distributions in future climate change scenarios, whereas the Southern Hemisphere will have the opposite outcomes. Europe will become more suitable for both species and their related vector-borne diseases. Loss of suitability in the Brazilian Amazon region further indicated that this tropical rainforest biome will have lower levels of precipitation to support these species in the future. Our models provide possible future scenarios to help identify locations for resource allocation and surveillance efforts before a significant threat to human health emerges.

A Novel Coronavirus and a Broad Range of Viruses in Kenyan Cave Bats.

BACKGROUND AND METHODS: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing. RESULTS: Viruses belonging to the Astroviridae, Circoviridae, Coronaviridae, Dicistroviridae, Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis, Myotis ricketti, Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified. CONCLUSIONS: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.

Metagenomic Investigation of Ticks From Kenyan Wildlife Reveals Diverse Microbial Pathogens and New Country Pathogen Records.

Focusing on the utility of ticks as xenosurveillance sentinels to expose circulating pathogens in Kenyan drylands, host-feeding ticks collected from wild ungulates [buffaloes, elephants, giraffes, hartebeest, impala, rhinoceros (black and white), zebras (Grévy's and plains)], carnivores (leopards, lions, spotted hyenas, wild dogs), as well as regular domestic and Boran cattle were screened for pathogens using metagenomics. A total of 75 host-feeding ticks [Rhipicephalus (97.3%) and Amblyomma (2.7%)] collected from 15 vertebrate taxa were sequenced in 46 pools. Fifty-six pathogenic bacterial species were detected in 35 pools analyzed for pathogens and relative abundances of major phyla. The most frequently observed species was Escherichia coli (62.8%), followed by Proteus mirabilis (48.5%) and Coxiella burnetii (45.7%). Francisella tularemia and Jingmen tick virus (JMTV) were detected in 14.2 and 13% of the pools, respectively, in ticks collected from wild animals and cattle. This is one of the first reports of JMTV in Kenya, and phylogenetic reconstruction revealed significant divergence from previously known isolates and related viruses. Eight fungal species with human pathogenicity were detected in 5 pools (10.8%). The vector-borne filarial pathogens (Brugia malayi, Dirofilaria immitis, Loa loa), protozoa (Plasmodium spp., Trypanosoma cruzi), and environmental and water-/food-borne pathogens (Entamoeba histolytica, Encephalitozoon intestinalis, Naegleria fowleri, Schistosoma spp., Toxoplasma gondii, and Trichinella spiralis) were detected. Documented viruses included human mastadenovirus C, Epstein-Barr virus and bovine herpesvirus 5, Trinbago virus, and Guarapuava tymovirus-like virus 1. Our findings confirmed that host-feeding ticks are an efficient sentinel for xenosurveillance and demonstrate clear potential for wildlife-livestock-human pathogen transfer in the Kenyan landscape.

Ticks (Acari: Ixodidae) and Associated Pathoge Collected From Domestic Animals and Vegetation in Stann Creek District, Southeastern Belize, Central America.

Data on the prevalence and distribution of ticks and tick-borne diseases in Belize are lacking. Ticks (n = 564) collected from dogs, horses, and vegetation in two villages in Stann Creek District in southeastern Belize in 2018, were molecularly identified and screened for tick-borne nonviral human pathogens. The identity of 417 ticks was molecularly confirmed by DNA barcoding as Rhipicephalus sanguineus (Latreille) (66.43%), Amblyomma ovale Koch (15.59%), Dermacentor nitens Neumann (11.51%), Amblyomma sp. ADB0528 (3.6%), and the remainder being small records (2.87%) of Amblyomma coelebs Neumann, Amblyomma imitator Kohls, Amblyomma tapirellum Dunn, Amblyomma auricularium Conil, and Amblyomma maculatum Koch. Individual tick extracts were screened for the presence of Rickettsia spp., Babesia spp., Babesia microti, Borrelia spp., Ehrlichia spp., and Anaplasma spp. using available conventional polymerase chain reaction (PCR) assays. Rickettsia parkeri strain Atlantic Rainforest was identified in five specimens of A. ovale, and one other unidentified tick, all collected from dogs. Another unidentified tick-also collected from a dog-tested positive for an undefined but previously detected Ehrlichia sp. With the exception of D. nitens, all eight other tick species identified in this study were collected on dogs, suggesting that dogs could be usefully employed as sentinel animals for tick surveillance in Belize.

Diversity, composition, altitude, and seasonality of high-altitude windborne migrating mosquitoes in the Sahel: Implications for disease transmission.

Recent studies have reported Anopheles mosquitoes captured at high-altitude (40-290 m above ground) in the Sahel. Here, we describe this migration modality across genera and species of African Culicidae and examine its implications for disease transmission and control. As well as Anopheles, six other genera-Culex, Aedes, Mansonia, Mimomyia, Lutzia, and Eretmapodites comprised 90% of the 2,340 mosquitoes captured at altitude. Of the 50 molecularly confirmed species (N = 2,107), 33 species represented by multiple specimens were conservatively considered high-altitude windborne migrants, suggesting it is a common migration modality in mosquitoes (31-47% of the known species in Mali), and especially in Culex (45-59%). Overall species abundance varied between 2 and 710 specimens/species (in Ae. vittatus and Cx. perexiguus, respectively). At altitude, females outnumbered males 6:1, and 93% of the females have taken at least one blood meal on a vertebrate host prior to their departure. Most taxa were more common at higher sampling altitudes, indicating that total abundance and diversity are underestimated. High-altitude flight activity was concentrated between June and November coinciding with availability of surface waters and peak disease transmission by mosquitoes. These hallmarks of windborne mosquito migration bolster their role as carriers of mosquito-borne pathogens (MBPs). Screening 921 mosquitoes using pan-Plasmodium assays revealed that thoracic infection rate in these high-altitude migrants was 2.4%, providing a proof of concept that vertebrate pathogens are transported by windborne mosquitoes at altitude. Fourteen of the 33 windborne mosquito species had been reported as vectors to 25 MBPs in West Africa, which represent 32% of the MBPs known in that region and include those that inflict the heaviest burden on human and animal health, such as malaria, yellow fever, dengue, and Rift Valley fever. We highlight five arboviruses that are most likely affected by windborne mosquitoes in West Africa: Rift Valley fever, O'nyong'nyong, Ngari, Pangola, and Ndumu. We conclude that the study of windborne spread of diseases by migrating insects and the development of surveillance to map the sources, routes, and destinations of vectors and pathogens is key to understand, predict, and mitigate existing and new threats of public health.

Phylogenetic analysis of the Neotropical Albitarsis Complex based on mitogenome data.

BACKGROUND: Some of the most important malaria vectors in South America belong to the Albitarsis Complex (Culicidae; Anophelinae; Anopheles). Understanding the origin, nature, and geographical distribution of species diversity in this important complex has important implications for vector incrimination, control, and management, and for modelling future responses to climate change, deforestation, and human population expansion. This study attempts to further explore species diversity and evolutionary history in the Albitarsis Complex by undertaking a characterization and phylogenetic analysis of the mitogenome of all 10 putative taxa in the Albitarsis Complex. METHODS: Mitogenome assembly and annotation allowed for feature comparison among Albitarsis Complex and Anopheles species. Selection analysis was conducted across all 13 protein-coding genes. Maximum likelihood and Bayesian inference methods were used to construct gene and species trees, respectively. Bayesian methods were also used to jointly estimate species delimitation and species trees. RESULTS: Gene composition and order were conserved across species within the complex. Unique signatures of positive selection were detected in two species-Anopheles janconnae and An. albitarsis G-which may have played a role in the recent and rapid diversification of the complex. The COI gene phylogeny does not fully recover the mitogenome phylogeny, and a multispecies coalescent-based phylogeny shows that considerable uncertainty exists through much of the mitogenome species tree. The origin of divergence in the complex dates to the Pliocene/Pleistocene boundary, and divergence within the distinct northern South American clade is estimated at approximately 1 million years ago. Neither the phylogenetic trees nor the delimitation approach rejected the 10-species hypothesis, although the analyses could not exclude the possibility that four putative species with scant a priori support (An. albitarsis G, An. albitarsis H, An. albitarsis I, and An. albitarsis J), represent population-level, rather than species-level, splits. CONCLUSION: The lack of resolution in much of the species tree and the limitations of the delimitation analysis warrant future studies on the complex using genome-wide data and the inclusion of additional specimens, particularly from two putative species, An. albitarsis I and An. albitarsis J.

Mosquito species identification using convolutional neural networks with a multitiered ensemble model for novel species detection.

With over 3500 mosquito species described, accurate species identification of the few implicated in disease transmission is critical to mosquito borne disease mitigation. Yet this task is hindered by limited global taxonomic expertise and specimen damage consistent across common capture methods. Convolutional neural networks (CNNs) are promising with limited sets of species, but image database requirements restrict practical implementation. Using an image database of 2696 specimens from 67 mosquito species, we address the practical open-set problem with a detection algorithm for novel species. Closed-set classification of 16 known species achieved 97.04 ± 0.87% accuracy independently, and 89.07 ± 5.58% when cascaded with novelty detection. Closed-set classification of 39 species produces a macro F1-score of 86.07 ± 1.81%. This demonstrates an accurate, scalable, and practical computer vision solution to identify wild-caught mosquitoes for implementation in biosurveillance and targeted vector control programs, without the need for extensive image database development for each new target region.

Towards an ecosystem model of infectious disease.

Increasingly intimate associations between human society and the natural environment are driving the emergence of novel pathogens, with devastating consequences for humans and animals alike. Prior to emergence, these pathogens exist within complex ecological systems that are characterized by trophic interactions between parasites, their hosts and the environment. Predicting how disturbance to these ecological systems places people and animals at risk from emerging pathogens-and the best ways to manage this-remains a significant challenge. Predictive systems ecology models are powerful tools for the reconstruction of ecosystem function but have yet to be considered for modelling infectious disease. Part of this stems from a mistaken tendency to forget about the role that pathogens play in structuring the abundance and interactions of the free-living species favoured by systems ecologists. Here, we explore how developing and applying these more complete systems ecology models at a landscape scale would greatly enhance our understanding of the reciprocal interactions between parasites, pathogens and the environment, placing zoonoses in an ecological context, while identifying key variables and simplifying assumptions that underly pathogen host switching and animal-to-human spillover risk. As well as transforming our understanding of disease ecology, this would also allow us to better direct resources in preparation for future pandemics.

From e-voucher to genomic data: Preserving archive specimens as demonstrated with medically important mosquitoes (Diptera: Culicidae) and kissing bugs (Hemiptera: Reduviidae).

Scientific collections such as the U.S. National Museum (USNM) are critical to filling knowledge gaps in molecular systematics studies. The global taxonomic impediment has resulted in a reduction of expert taxonomists generating new collections of rare or understudied taxa and these large historic collections may be the only reliable source of material for some taxa. Integrated systematics studies using both morphological examinations and DNA sequencing are often required for resolving many taxonomic issues but as DNA methods often require partial or complete destruction of a sample, there are many factors to consider before implementing destructive sampling of specimens within scientific collections. We present a methodology for the use of archive specimens that includes two crucial phases: 1) thoroughly documenting specimens destined for destructive sampling-a process called electronic vouchering, and 2) the pipeline used for whole genome sequencing of archived specimens, from extraction of genomic DNA to assembly of putative genomes with basic annotation. The process is presented for eleven specimens from two different insect subfamilies of medical importance to humans: Anophelinae (Diptera: Culicidae)-mosquitoes and Triatominae (Hemiptera: Reduviidae)-kissing bugs. Assembly of whole mitochondrial genome sequences of all 11 specimens along with the results of an ortholog search and BLAST against the NCBI nucleotide database are also presented.