Selective deforestation and exposure of African wildlife to bat-borne viruses.

Proposed mechanisms of zoonotic virus spillover often posit that wildlife transmission and amplification precede human outbreaks. Between 2006 and 2012, the palm Raphia farinifera, a rich source of dietary minerals for wildlife, was nearly extirpated from Budongo Forest, Uganda. Since then, chimpanzees, black-and-white colobus, and red duiker were observed feeding on bat guano, a behavior not previously observed. Here we show that guano consumption may be a response to dietary mineral scarcity and may expose wildlife to bat-borne viruses. Videos from 2017-2019 recorded 839 instances of guano consumption by the aforementioned species. Nutritional analysis of the guano revealed high concentrations of sodium, potassium, magnesium and phosphorus. Metagenomic analyses of the guano identified 27 eukaryotic viruses, including a novel betacoronavirus. Our findings illustrate how "upstream" drivers such as socioeconomics and resource extraction can initiate elaborate chains of causation, ultimately increasing virus spillover risk.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Characterization of the bacterial microbiome of non-hematophagous bats and associated ectoparasites from Brazil.

Introduction: Bats, along with their ectoparasites, harbor a wide diversity of symbiotic and potential pathogenic bacteria. Despite the enormous diversity of bats (181 species), few studies aimed to investigate the bacterial microbiome of Brazilian chiropterans and associated ectoparasites. This study aimed to characterize the bacterial microbiome of non-hematophagous bats and associated Streblidae flies and Macronyssidae and Spinturnicidae mites in the state of Mato Grosso do Sul, midwestern Brazil. Methods: Oral and rectal swabs were collected from 30 bats (Artibeus lituratus [n = 13], Artibeus planirostris [n = 9], Eptesicus furinalis [n = 5], Carollia perspicillata [n = 2], and Platyrrhinus lineatus [n = 1]). In addition, a total of 58 mites (15 Macronyssidae and 43 Spinturnicidae) and 48 Streblidae bat flies were collected from the captured bats. After DNA extraction and purification, each sample's bacterial composition was analyzed with metagenomic sequencing. Results: The microbiome composition of both oral and rectal bat swab samples showed that Gammaproteobacteria was the most abundant bacterial class. Spiroplasma, Wolbachia and Bartonella represented the most abundant genera in Streblidae flies. While Wolbachia (Alphaproteobacteria) was the most abundant genus found in Spinturnicidae, Arsenophonus (Gammaproteobacteria) was found in high abundance in Macronyssidae mites. In addition to characterizing the microbiome of each sample at the class and genus taxonomic levels, we identified medically significant bacteria able to infect both animals and humans in oral (Streptococcus and Anaplasma) and rectal swabs (Enterobacter, Klebsiella, Escherichia, Enterococcus, Streptococcus), Macronyssidae (Anaplasma, Bartonella, Ehrlichia) and Spinturnicidae (Anaplasma, Bartonella) mites as well as Streblidae flies (Spiroplasma, Bartonella). Discussion and conclusion: Besides expanding the knowledge on the bacterial microbiome of non-hematophagous bats and Streblidae flies from Brazil, the present work showed, for the first time, the bacterial community of bat-associated Macronyssidae and Spinturnicidae mites.

Genomic Surveillance of Rabies Virus in Georgian Canines.

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).

Genomic Characterization of a Relative of Mumps Virus in Lesser Dawn Bats of Southeast Asia.

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.

SARS-CoV-2 Infections in Vaccinated and Unvaccinated Populations in Camp Lemonnier, Djibouti, from April 2020 to January 2022.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the disparity between developed and developing countries for infectious disease surveillance and the sequencing of pathogen genomes. The majority of SARS-CoV-2 sequences published are from Europe, North America, and Asia. Between April 2020 and January 2022, 795 SARS-CoV-2-positive nares swabs from individuals in the U.S. Navy installation Camp Lemonnier, Djibouti, were collected, sequenced, and analyzed. In this study, we described the results of genomic sequencing and analysis for 589 samples, the first published viral sequences for Djibouti, including 196 cases of vaccine breakthrough infections. This study contributes to the knowledge base of circulating SARS-CoV-2 lineages in the under-sampled country of Djibouti, where only 716 total genome sequences are available at time of publication. Our analysis resulted in the detection of circulating variants of concern, mutations of interest in lineages in which those mutations are not common, and emerging spike mutations.

SARS-CoV-2 Outbreak Dynamics in an Isolated US Military Recruit Training Center With Rigorous Prevention Measures.

BACKGROUND: Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort. METHODS: Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens. RESULTS: Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks. CONCLUSIONS: Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Immunological and Genetic Investigation of SARS-CoV-2 Reinfection in an Otherwise Healthy, Young Marine Recruit.

We used epidemiologic and viral genetic information to identify a case of likely reinfection in an otherwise healthy, young Marine recruit enrolled in the prospective, longitudinal COVID-19 Health Action Response for Marines (CHARM) study, and we paired these findings with serological studies. This participant had a positive RT-PCR to SARS-CoV-2 upon routine sampling on study day 7, although he was asymptomatic at that time. He cleared the infection within seven days. On study day 46, he had developed symptoms consistent with COVID-19 and tested positive by RT-PCR for SARS-CoV-2 again. Viral whole genome sequencing was conducted from nares swabs at multiple time points. The day 7 sample was determined to be lineage B.1.340, whereas both the day 46 and day 49 samples were B.1.1. The first positive result for anti-SARS-CoV-2 IgM serology was collected on day 49 and for IgG on day 91. This case appears most consistent with a reinfection event. Our investigation into this case is unique in that we compared sequence data from more than just paired specimens, and we also assayed for immune response after both the initial infection and the later reinfection. These data demonstrate that individuals who have experienced an infection with SARS-CoV-2 may fail to generate effective or long-lasting immunity, similar to endemic human beta coronaviruses.

Manual Annotation Studio (MAS): a collaborative platform for manual functional annotation of viral and microbial genomes.

BACKGROUND: Functional genome annotation is the process of labelling functional genomic regions with descriptive information. Manual curation can produce higher quality genome annotations than fully automated methods. Manual annotation efforts are time-consuming and complex; however, software can help reduce these drawbacks. RESULTS: We created Manual Annotation Studio (MAS) to improve the efficiency of the process of manual functional annotation prokaryotic and viral genomes. MAS allows users to upload unannotated genomes, provides an interface to edit and upload annotations, tracks annotation history and progress, and saves data to a relational database. MAS provides users with pertinent information through a simple point and click interface to execute and visualize results for multiple homology search tools (blastp, rpsblast, and HHsearch) against multiple databases (Swiss-Prot, nr, CDD, PDB, and an internally generated database). MAS was designed to accept connections over the local area network (LAN) of a lab or organization so multiple users can access it simultaneously. MAS can take advantage of high-performance computing (HPC) clusters by interfacing with SGE or SLURM and data can be exported from MAS in a variety of formats (FASTA, GenBank, GFF, and excel). CONCLUSIONS: MAS streamlines and provides structure to manual functional annotation projects. MAS enhances the ability of users to generate, interpret, and compare results from multiple tools. The structure that MAS provides can improve project organization and reduce annotation errors. MAS is ideal for team-based annotation projects because it facilitates collaboration.

Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel.

The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency's (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence.

Corrigendum: Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

[This corrects the article DOI: 10.3389/fmicb.2018.02664.].

Detection of Recombinant Rousettus Bat Coronavirus GCCDC1 in Lesser Dawn Bats (Eonycteris spelaea) in Singapore.

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 temporal RNA virome patterns of a lesser dawn bat (Eonycteris spelaea) colony revealed by deep sequencing.

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.

Genomic epidemiology of MRSA infection and colonization isolates among military trainees with skin and soft tissue infection.

PURPOSE: Individuals with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection (SSTI) can be simultaneously colonized with MRSA on multiple body sites. Using whole genome sequencing (WGS), the intrahost relatedness of MRSA colonization and infection isolates was investigated. METHODS: In the context of a prospective case-control study of SSTI, we analyzed colonization and infection isolates from US Army Infantry trainees with purulent infection due to MRSA. At the time of clinical presentation for SSTI, culture swabs were obtained from the infection site, as well as from the patient's nasal, oral, inguinal, and perianal regions. S. aureus culture and susceptibility was performed by standard methods. DNA from MRSA isolates was extracted and libraries were produced. Sequences were generated on an Illumina MiSeq, sequence reads were assembled, and single nucleotide variant (SNV) data were analyzed. RESULTS: Of 74 trainees with MRSA SSTI, 19 (25.7%) were colonized with MRSA. Ten (52.6%) were colonized on more than one body site. Colonization frequency by anatomic site was as follows: inguinal region (33%), nasal region (30%), perianal region (22%), and oral region (14%). A total of 36 MRSA colonization isolates were characterized. The intrahost median number of SNVs between infection and colonization isolates was 17. Among trainees with recurrent MRSA SSTI, limited intrahost diversity suggests that persistent colonization is a major contributor to recurrence risk. CONCLUSIONS: Among military trainees with MRSA SSTI, genomic characterization of infection and colonization isolates revealed a high degree of strain relatedness. Single acquisition events may account for MRSA colonization and infection in this population.

High-Quality Draft Genome Sequence of Pseudomonas aeruginosa 268 Isolated from a Patient with a Left Ventricular Assist Device.

Pseudomonas aeruginosa is known to cause persistent bloodstream infections associated with left ventricular assist devices (LVAD). Here, we present the high-quality draft genome assembly for a clinical isolate, P. aeruginosa 268. The genome sequence is available in GenBank under accession number CP032761.

Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

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.

Characterizing Phage Genomes for Therapeutic Applications.

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.

Genomic Characterization of USA300 Methicillin-Resistant Staphylococcus aureus (MRSA) to Evaluate Intraclass Transmission and Recurrence of Skin and Soft Tissue Infection (SSTI) Among High-Risk Military Trainees.

BACKGROUND: Military trainees are at increased risk for methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection (SSTI). Whole genome sequencing (WGS) can refine our understanding of MRSA transmission and microevolution in congregate settings. METHODS: We conducted a prospective case-control study of SSTI among US Army infantry trainees at Fort Benning, Georgia, from July 2012 to December 2014. We identified clusters of USA300 MRSA SSTI within select training classes and performed WGS on clinical isolates. We then linked genomic, phylogenetic, epidemiologic, and clinical data in order to evaluate intra- and interclass disease transmission. Furthermore, among cases of recurrent MRSA SSTI, we evaluated the intrahost relatedness of infecting strains. RESULTS: Nine training classes with ≥5 cases of USA300 MRSA SSTI were selected. Eighty USA300 MRSA clinical isolates from 74 trainees, 6 (8.1%) of whom had recurrent infection, were subjected to WGS. We identified 2719 single nucleotide variants (SNVs). The overall median (range) SNV difference between isolates was 173 (1-339). Intraclass median SNV differences ranged from 23 to 245. Two phylogenetic clusters were suggestive of interclass MRSA transmission. One of these clusters stemmed from 2 classes that were separated by a 13-month period but housed in the same barracks. Among trainees with recurrent MRSA SSTI, the intrahost median SNV difference was 7.5 (1-48). CONCLUSIONS: Application of WGS revealed intra- and interclass transmission of MRSA among military trainees. An interclass cluster between 2 noncontemporaneous classes suggests a long-term reservoir for MRSA in this setting.