An aggregated dataset of serial morbidity and titer measurements from influenza A virus-infected ferrets.

Data from influenza A virus (IAV) infected ferrets provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard model can recapitulate many clinical signs of infection present in IAV-infected humans, support virus replication of human, avian, swine, and other zoonotic strains without prior adaptation, and permit evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible. Here, we describe a dataset of 728 ferrets inoculated with 126 unique IAV, conducted by a single research group under a uniform experimental protocol. This collection of morbidity, mortality, and viral titer data represents the largest publicly available dataset to date of in vivo-generated IAV infection outcomes on a per-ferret level.

Key considerations to improve the normalization, interpretation and reproducibility of morbidity data in mammalian models of viral disease.

Viral pathogenesis and therapeutic screening studies that utilize small mammalian models rely on the accurate quantification and interpretation of morbidity measurements, such as weight and body temperature, which can vary depending on the model, agent and/or experimental design used. As a result, morbidity-related data are frequently normalized within and across screening studies to aid with their interpretation. However, such data normalization can be performed in a variety of ways, leading to differences in conclusions drawn and making comparisons between studies challenging. Here, we discuss variability in the normalization, interpretation, and presentation of morbidity measurements for four model species frequently used to study a diverse range of human viral pathogens - mice, hamsters, guinea pigs and ferrets. We also analyze findings aggregated from influenza A virus-infected ferrets to contextualize this discussion. We focus on serially collected weight and temperature data to illustrate how the conclusions drawn from this information can vary depending on how raw data are collected, normalized and measured. Taken together, this work supports continued efforts in understanding how normalization affects the interpretation of morbidity data and highlights best practices to improve the interpretation and utility of these findings for extrapolation to public health contexts.

Highly pathogenic avian influenza A(H5N1) virus of clade 2.3.4.4b isolated from a human case in Chile causes fatal disease and transmits between co-housed ferrets.

Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses have caused large outbreaks within avian populations on five continents, with concurrent spillover into a variety of mammalian species. Mutations associated with mammalian adaptation have been sporadically identified in avian isolates, and more frequently among mammalian isolates following infection. Reports of human infection with A(H5N1) viruses following contact with infected wildlife have been reported on multiple continents, highlighting the need for pandemic risk assessment of these viruses. In this study, the pathogenicity and transmissibility of A/Chile/25945/2023 HPAI A(H5N1) virus, a novel reassortment with four gene segments (PB1, PB2, NP, MP) from North America lineage, isolated from a severe human case in Chile, was evaluated in vitro and using the ferret model. This virus possessed a high capacity to cause fatal disease, characterized by high morbidity and extrapulmonary spread in virus-inoculated ferrets. The virus was capable of transmission to naïve contacts in a direct contact setting, with contact animals similarly exhibiting severe disease, but did not exhibit productive transmission in respiratory droplet or fomite transmission models. Our results indicate that the virus would need to acquire an airborne transmissible phenotype in mammals to potentially cause a pandemic. Nonetheless, this work warrants continuous monitoring of mammalian adaptations in avian viruses, especially in strains isolated from humans, to aid pandemic preparedness efforts.

Exploring associations between viral titer measurements and disease outcomes in ferrets inoculated with 125 contemporary influenza A viruses.

As use of the ferret model to study influenza A virus (IAV) pathogenicity increases, periodic assessment of data generated in this model is warranted, to identify features associated with virus replication throughout the respiratory tract and to refine future analyses. However, protocol-specific differences present between independent laboratories limit easy aggregation of virological data. We compiled viral titer and clinical data from >1,000 ferrets inoculated with 125 contemporary IAV under a consistent experimental protocol (including high- and low-pathogenicity avian, swine-origin, and human viruses, spanning H1, H2, H3, H5, H7, and H9 subtypes) and examined which meaningful and statistically supported associations were present among numerous quantitative measurements. Viral titers correlated positively between ferret nasal turbinate tissue, lung tissue, and nasal wash specimens, though the strength of the associations varied, notably regarding the particular nasal wash summary measure employed and properties of the virus itself. Use of correlation coefficients and mediation analyses further supported the interconnectedness of viral titer measurements taken at different sites throughout the respiratory tract. IAV possessing mammalian host adaptation markers in the HA and PB2 exhibited more rapid growth in the ferret upper respiratory tract early after infection, supported by quantities derived from infectious titer data to capture infection progression, compared with viruses bearing hallmarks of avian IAV. Collectively, this work identifies summary metrics most closely linked with virological and phenotypic outcomes in ferrets, supporting continued refinement of data analyzed from in vivo experimentation, notably from studies conducted to evaluate the public health risk posed by novel and emerging IAV.IMPORTANCEFerrets are frequently employed to study the pandemic potential of novel and emerging influenza A viruses. However, systematic retrospective analyses of data generated from these experiments are rarely performed, limiting our ability to identify trends in this data and explore how analyses can be refined. Using logarithmic viral titer and clinical data aggregated from one research group over 20 years, we assessed which meaningful and statistically supported associations were present among numerous quantitative measurements obtained from influenza A virus (IAV)-infected ferrets, including those capturing viral titers, infection progression, and disease severity. We identified numerous linear correlations between parameters assessing virus replication at discrete sites in vivo, including parameters capturing infection progression not frequently employed in the field, and sought to investigate the interconnected nature of these associations. This work supports continued refinement of data analyzed from in vivo experimentation, notably from studies which evaluate the public health risk posed by IAV.

A naturally occurring HA-stabilizing amino acid (HA1-Y17) in an A(H9N2) low-pathogenic influenza virus contributes to airborne transmission.

IMPORTANCE: Despite the accumulation of evidence showing that airborne transmissible influenza A virus (IAV) typically has a lower pH threshold for hemagglutinin (HA) fusion activation, the underlying mechanism for such a link remains unclear. In our study, by using a pair of isogenic recombinant A(H9N2) viruses with a phenotypical difference in virus airborne transmission in a ferret model due to an acid-destabilizing mutation (HA1-Y17H) in the HA, we demonstrate that an acid-stable A(H9N2) virus possesses a multitude of advantages over its less stable counterpart, including better fitness in the ferret respiratory tract, more effective aerosol emission from infected animals, and improved host susceptibility. Our study provides supporting evidence for the requirement of acid stability in efficient airborne transmission of IAV and sheds light on fundamental mechanisms for virus airborne transmission.

Influenza A Virus Multicycle Replication Yields Comparable Viral Population Emergence in Human Respiratory and Ocular Cell Types.

While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.

Enhanced fitness of SARS-CoV-2 B.1.617.2 Delta variant in ferrets.

Competition assays were conducted in vitro and in vivo to examine how the Delta (B.1.617.2) variant displaced the prototype Washington/1/2020 (WA/1) strain. While WA/1 virus exhibited a moderately increased proportion compared to that in the inoculum following co-infection in human respiratory cells, Delta variant possessed a substantial in vivo fitness advantage as this virus becoming predominant in both inoculated and contact animals. This work identifies critical traits of the Delta variant that likely played a role in it becoming a dominant variant and highlights the necessities of employing multiple model systems to assess the fitness of newly emerged SARS-CoV-2 variants.

Utility of Human In Vitro Data in Risk Assessments of Influenza A Virus Using the Ferret Model.

As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone.

Detection of Airborne Influenza A and SARS-CoV-2 Virus Shedding following Ocular Inoculation of Ferrets.

Despite reports of confirmed human infection following ocular exposure with both influenza A virus (IAV) and SARS-CoV-2, the dynamics of virus spread throughout oculonasal tissues and the relative capacity of virus transmission following ocular inoculation remain poorly understood. Furthermore, the impact of exposure route on subsequent release of airborne viral particles into the air has not been examined previously. To assess this, ferrets were inoculated by the ocular route with A(H1N1)pdm09 and A(H7N9) IAVs and two SARS-CoV-2 (early pandemic Washington/1 and Delta variant) viruses. Virus replication was assessed in both respiratory and ocular specimens, and transmission was evaluated in direct contact or respiratory droplet settings. Viral RNA in aerosols shed by inoculated ferrets was quantified with a two-stage cyclone aerosol sampler (National Institute for Occupational Safety and Health [NIOSH]). All IAV and SARS-CoV-2 viruses mounted a productive and transmissible infection in ferrets following ocular inoculation, with peak viral titers and release of virus-laden aerosols from ferrets indistinguishable from those from ferrets inoculated by previously characterized intranasal inoculation methods. Viral RNA was detected in ferret conjunctival washes from all viruses examined, though infectious virus in this specimen was recovered only following IAV inoculation. Low-dose ocular-only aerosol exposure or inhalation aerosol exposure of ferrets to IAV similarly led to productive infection of ferrets and shedding of aerosolized virus. Viral evolution during infection was comparable between all inoculation routes examined. These data support that both IAV and SARS-CoV-2 can establish a high-titer mammalian infection following ocular exposure that is associated with rapid detection of virus-laden aerosols shed by inoculated animals. IMPORTANCE Documented human infection with influenza viruses and SARS-CoV-2 has been reported among individuals wearing respiratory protection in the absence of eye protection, highlighting the capacity of these respiratory tract-tropic viruses to exploit nonrespiratory routes of exposure to initiate productive infection. However, comprehensive evaluations of how ocular exposure may modulate virus pathogenicity and transmissibility in mammals relative to respiratory exposure are limited and have not investigated multiple virus families side by side. Using the ferret model, we show that ocular exposure with multiple strains of either coronaviruses or influenza A viruses leads to an infection that results in shedding of detectable aerosolized virus from inoculated animals, contributing toward onward transmission of both viruses to susceptible contacts. Collectively, these studies support that the ocular surface represents a susceptible mucosal surface that, if exposed to a sufficient quantity of either virus, permits establishment of an infection which is similarly transmissible as that following respiratory exposure.

Comparative Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Variants in the Ferret Model.

The continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans necessitates evaluation of variants for enhanced virulence and transmission. We used the ferret model to perform a comparative analysis of four SARS-CoV-2 strains, including an early pandemic isolate from the United States (WA1), and representatives of the Alpha, Beta, and Delta lineages. While Beta virus was not capable of pronounced replication in ferrets, WA1, Alpha, and Delta viruses productively replicated in the ferret upper respiratory tract, despite causing only mild disease with no overt histopathological changes. Strain-specific transmissibility was observed; WA1 and Delta viruses transmitted in a direct contact setting, whereas Delta virus was also capable of limited airborne transmission. Viral RNA was shed in exhaled air particles from all inoculated animals but was highest for Delta virus. Prior infection with SARS-CoV-2 offered varied protection against reinfection with either homologous or heterologous variants. Notable genomic variants in the spike protein were most frequently detected following WA1 and Delta virus infection. IMPORTANCE Continued surveillance and risk assessment of emerging SARS-CoV-2 variants are critical for pandemic response and preparedness. As such, in vivo evaluations are indispensable for early detection of variants with enhanced virulence and transmission. Here, we used the ferret model to compare the pathogenicity and transmissibility of an original SARS-CoV-2 isolate (USA-WA1/2020 [WA1]) to those of a panel of Alpha, Beta, and Delta variants, as well as to evaluate protection from homologous and heterologous reinfection. We observed strain-specific differences in replication kinetics in the ferret respiratory tract and virus load emitted into the air, revealing enhanced transmissibility of the Delta virus relative to previously detected strains. Prior infection with SARS-CoV-2 provided varied levels of protection from reinfection, with the Beta strain eliciting the lowest level of protection. Overall, we found that ferrets represent a useful model for comparative assessments of SARS-CoV-2 infection, transmission, and reinfection.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Unveiling the Gut Microbiota and Resistome of Wild Cotton Mice, Peromyscus gossypinus, from Heavy Metal- and Radionuclide-Contaminated Sites in the Southeastern United States.

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment.

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

Ultraconserved element bait set for trypanosomatida target enrichment and phylogenetics.

Lack of knowledge of taxonomic biodiversity and reliable genetic markers in Trypanosomatidae limit our understanding of their phylogenetic relationships. Ultraconserved elements (UCEs) have improved phylogenetic analyses and inferences in many vertebrate and invertebrate taxa. However, it is unknown whether protozoans have these markers, their abundance, and if these could be reliably used for phylogenetics. In this study I design a target enrichment bait set for UCE loci for this group. In silico testing showed good loci recovery rates across 63 taxa and produced consistent, highly supported phylogenetic trees. This bait set adds a new resource of useful genetic markers for Trypanosomatidae phylogenetics.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

How microclimatic variables and blood meal sources influence Rhodnius prolixus abundance and Trypanosoma cruzi infection in Attalea butyracea and Elaeis guineensis palms?

Chagas disease is a zoonosis that affects several million people and is caused by the parasite Trypanosoma cruzi, which is mainly transmitted through the feces of triatomine bugs. Within triatomines, several Rhodnius species have been found inhabiting palms, and certain factors such as palm species and location have been related to the abundance and T. cruzi infection of those insects in palms. In this study, the main goal was to determine if R. prolixus abundances and infection rates in Attalea butyracea and Elaeis guineensis palms are related to ecological factors such as palm species, crown microclimate, and available blood meal sources. Triatomine sampling was performed in two municipalities of Casanare, Colombia, specifically in the intersection of riparian forests and oil palm plantations. For R. prolixus abundance per palm, the predictors showing more relationship were palm species and blood meal species identified in the palm, and for T. cruzi infection per triatomine, they were palm species and nymphal stage. Palm microclimate was very similar in both palm species and did not show a relationship with triatomine abundance. Comparing palm species, A. butyracea showed more blood meal species, including more refractory host species, than E. guineensis, but lower T. cruzi infection rate and parasitaemia. Interestingly, non-arboreal blood meal species were frequently found in the analyzed nymphs, indicating that the blood source for R. prolixus in palms corresponded to all the fauna located in the surrounded landscape and not only in the palm. These results could expose a new ecological scenario to interpret the T. cruzi transmission in sylvatic environments.

Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.

Population genetics of two chromatic morphs of the Chagas disease vector Rhodnius pallescens Barber, 1932 in Panamá.

Rhodnius pallescens is the principal vector of Chagas disease in Panama. Recently a dark chromatic morph has been discovered in the highlands of Veraguas Province. Limited genetic studies have been conducted with regards to the population structure and dispersal potential of Triatominae vectors, particularly in R. pallescens. Next generation sequencing methods such as RADseq and complete mitochondrial DNA (mtDNA) genome sequencing have great potential for examining vector biology across space and time. Here we utilize a RADseq method (3RAD), along with complete mtDNA sequencing, to examine the population structure of the two chromatic morpho types of R. pallescens in Panama. We sequenced 105 R. pallescens samples from five localities in Panama. We generated a 2216 SNP dataset and 6 complete mtDNA genomes. RADseq showed significant differentiation among the five localities (FCT = 0.695; P = .004), but most of this was between localities with the dark vs. light chromatic morphs (Veraguas vs. Panama Oeste). The mtDNA genomes showed a 97-98% similarity between dark and light chromatic morphs across all genes and a 502 bp insert in light morphs. Thus, both the RADseq and mtDNA data showed highly differentiated clades with essentially no gene flow between the dark and light chromatic morphs from Veraguas and central Panama respectively. We discuss the growing evidence showing clear distinctions between these two morpho types with the possibility that these are separate species, an area of research that requires further investigation. Finally, we discuss the cost-effectiveness of 3RAD which is a third of the cost compared to other RADseq methods used recently in Chagas disease vector research.

Phylogeny of the North-Central American clade of blood-sucking reduviid bugs of the tribe Triatomini (Hemiptera: Triatominae) based on the mitochondrial genome.

Triatominae is a subfamily of blood-sucking reduviid hemipterans of public health importance primarily in tropical and sub-tropical regions of the Americas, whose members possess various morphological adaptations closely associated to hematophagy. Despite their medical importance, the systematics of the subfamily is far from resolved, particularly within the tribe Triatomini. Here we employed mitochondrial genome DNA sequences to reconstruct the phylogenetic relationships among 19 species of the North-Central American (NCA) clade of Triatomini and to estimate the times of origin and diversification of its main clades. Twenty-nine mitogenomes were examined for representative specimens of 25 species, including the outgroup. Phylogenetic informativeness estimated for each protein-coding gene showed that cox1, cox2 and atp6 were the most informative markers, whereas atp8 and nad4 had high saturation levels. Phylogenetic analyses excluding the latter two protein-coding genes recovered an almost fully resolved topology. The NCA clade apparently originated shortly after emergence of an initial land bridge of the Panama Isthmus, ca. 15.05-20.05 Mya. An Asian/pantropical subclade with Linshcosteus costalis, Triatoma rubrofasciata and T. migrans was nested within the NCA clade, from which it diverged ca. 12.42-17.3Mya. Uncorrected cox1 and 13 protein-coding gene distances suggest the existence of additional species within the dimidiata complex. In contrast, T. phyllosoma, T. mazzottii and T. longipennis, from the phyllosoma complex, have considerably low cox1 and 13 PCG distances among them, suggesting mitochondrial introgression or conspecificity. Our study yielded a robust phylogeny for the group, which could be tested with further phylogenetic hypotheses based on nuclear genome-wide markers.

Co-occurrence of antibiotic, biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site.

Contaminants such as heavy metals may contribute to the dissemination of antimicrobial resistance (AMR) by enriching resistance gene determinants via co-selection mechanisms. In the present study, a survey was performed on soils collected from four areas at the Savannah River Site (SRS), South Carolina, USA, with varying contaminant profiles: relatively pristine (Upper Three Runs), heavy metals (Ash Basins), radionuclides (Pond B) and heavy metal and radionuclides (Tim's Branch). Using 16S rRNA gene amplicon sequencing, we explored the structure and diversity of soil bacterial communities. Sites with legacies of metal and/or radionuclide contamination displayed significantly lower bacterial diversity compared to the reference site. Metagenomic analysis indicated that multidrug and vancomycin antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) including those associated with copper, arsenic, iron, nickel and zinc were prominent in all soils including the reference site. However, significant differences were found in the relative abundance and diversity of certain ARGs and MRGs in soils with metal/radionuclide contaminated soils compared to the reference site. Co-occurrence patterns revealed significant ARG/MRG subtypes in predominant soil taxa including Acidobacteriaceae, Bradyrhizobium, Mycobacterium, Streptomyces, Verrumicrobium, Actinomadura and Solirubacterales. Overall, the study emphasizes the potential risk of human activities on the dissemination of AMR in the environment.