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Infection with either Rickettsia prowazekii or Orientia tsutsugamushi is common, yet diagnostic capabilities are limited due to the short window for positive identification. Until now, although targeted enrichment had been applied to increase sensitivity of sequencing-based detection for various microorganisms, it had not been applied to sequencing of R. prowazekii in clinical samples. Additionally, hybridization-based targeted enrichment strategies had only scarcely been applied to qPCR of any pathogens in clinical samples. Therefore, we tested a targeted enrichment technique as a proof of concept and found that it dramatically reduced the limits of detection of these organisms by both qPCR and high throughput sequencing. The enrichment methodology was first tested in contrived clinical samples with known spiked-in concentrations of R. prowazekii and O. tsutsugamushi DNA. This method was also evaluated using clinical samples, resulting in the simultaneous identification and characterization of O. tsutsugamushi directly from clinical specimens taken from sepsis patients. We demonstrated that the targeted enrichment technique is helpful by lowering the limit of detection, not only when applied to sequencing, but also when applied to qPCR, suggesting the technique could be applied more broadly to include other assays and/or microbes for which there are limited diagnostic or detection modalities., Competing Interests: AP and LV were employed by Leidos. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Paskey, Schully, Voegtly, Arnold, Cer, Frey, Blair, Clark, Ge, Richards, Farris and Bishop-Lilly.)

We investigated the virome of agaonid fig wasps (Ceratosolen spp.) inside syconia ("fruits") of various Ficus trees fed upon by frugivores such as pteropodid bats in Sub-Saharan Africa. This virome includes representatives of viral families spanning four realms and includes near-complete genome sequences of three novel viruses and fragments of five additional potentially novel viruses evolutionarily associated with insects, fungi, plants, and vertebrates. Our study provides evidence that frugivorous animals are exposed to a plethora of viruses by coincidental consumption of fig wasps, which are obligate pollinators of figs worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

The importance of genomic surveillance on emerging diseases continues to be highlighted with the ongoing SARS-CoV-2 pandemic. Here, we present an analysis of a new bat-borne mumps virus (MuV) in a captive colony of lesser dawn bats ( Eonycteris spelaea ). This report describes an investigation of MuV-specific data originally collected as part of a longitudinal virome study of apparently healthy, captive lesser dawn bats in Southeast Asia (BioProject ID PRJNA561193) which was the first report of a MuV-like virus, named dawn bat paramyxovirus (DbPV), in bats outside of Africa. More in-depth analysis of these original RNA sequences in the current report reveals that the new DbPV genome shares only 86% amino acid identity with the RNA-dependent RNA polymerase of its closest relative, the African bat-borne mumps virus (AbMuV). While there is no obvious immediate cause for concern, it is important to continue investigating and monitoring bat-borne MuVs to determine the risk of human infection.

Since 1814, when rubella was first described, the origins of the disease and its causative agent, rubella virus (Matonaviridae: Rubivirus), have remained unclear 1 . Here we describe ruhugu virus and rustrela virus in Africa and Europe, respectively, which are, to our knowledge, the first known relatives of rubella virus. Ruhugu virus, which is the closest relative of rubella virus, was found in apparently healthy cyclops leaf-nosed bats (Hipposideros cyclops) in Uganda. Rustrela virus, which is an outgroup to the clade that comprises rubella and ruhugu viruses, was found in acutely encephalitic placental and marsupial animals at a zoo in Germany and in wild yellow-necked field mice (Apodemus flavicollis) at and near the zoo. Ruhugu and rustrela viruses share an identical genomic architecture with rubella virus 2,3 . The amino acid sequences of four putative B cell epitopes in the fusion (E1) protein of the rubella, ruhugu and rustrela viruses and two putative T cell epitopes in the capsid protein of the rubella and ruhugu viruses are moderately to highly conserved 4-6 . Modelling of E1 homotrimers in the post-fusion state predicts that ruhugu and rubella viruses have a similar capacity for fusion with the host-cell membrane 5 . Together, these findings show that some members of the family Matonaviridae can cross substantial barriers between host species and that rubella virus probably has a zoonotic origin. Our findings raise concerns about future zoonotic transmission of rubella-like viruses, but will facilitate comparative studies and animal models of rubella and congenital rubella syndrome.

Obligate hematophagous ectoparasitic flies of the superfamily Hippoboscoidea are distributed worldwide, but their role as vectors and reservoirs of viruses remains understudied. We examined hippoboscoid bat flies (family Nycteribiidae) parasitizing Angolan soft-furred fruit bats ( Lissonycteris angolensis ruwenzorii ) from Bundibugyo District, Uganda. Using metagenomic methods, we detected 21 variants of the rhabdovirid genus Ledantevirus , which contains medically important "bat-associated" viruses. These 21 viruses, representing at least two divergent viral lineages, infected 26 bat flies from 8 bats in a single roost. Cophylogenetic analyses of viruses and bat flies resulted in strong evidence of virus-host codivergence, indicating vertical transmission of bat fly ledanteviruses. Examination of oral swabs from bats revealed ledantevirus RNA in the saliva of 1 out of 11 bats, with no evidence of insect genetic material in the mouth of this bat. These data demonstrate that bat flies can harbor diverse ledanteviruses even in a single roost and that the predominant mode of transmission is likely vertical (among bat flies), but that bats can become infected and shed viruses orally. In conclusion, bat flies may serve as ectoparasitic reservoirs of "bat-associated" viruses that only transiently or sporadically infect bats., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

The virosphere is largely unexplored and the majority of viruses are yet to be represented in public sequence databases. Bats are rich reservoirs of viruses, including several zoonoses. In this study, high throughput sequencing (HTS) of viral RNA extracted from swabs of four body sites per bat per timepoint is used to characterize the virome through a longitudinal study of a captive colony of fruit nectar bats, species Eonycteris spelaea in Singapore. Through unbiased shotgun and target enrichment sequencing, we identify both known and previously unknown viruses of zoonotic relevance and define the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. To our knowledge, this is the first study that combines probe-based viral enrichment with HTS to create a viral profile from multiple swab sites on individual bats and their cohort. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Given the known risk for bat-human zoonoses, a more complete understanding of the viral dynamics in South-eastern Asian bats has significant implications for disease prevention and control. The findings of this study will be of interest to U.S. Department of Defense personnel stationed in the Asia-Pacific region and regional public health laboratories engaged in emerging infectious disease surveillance efforts., (Published by Oxford University Press 2020. This work is written by US Government employees and is in the public domain in the US.)

Background: Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing., Results: This study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the U.S. Department of Defense and various international public health agencies. The newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. Target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with Ct values suggesting that amplicon sequencing would be likely to fail. Increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage., Conclusions: Hybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. As the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA -positive plasmid, pC02. Prior characterization of qacA -containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis . We screened additional qacA -positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA -harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA -containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.