Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic.

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Seafood Wholesale Market. Here, we analyze environmental qPCR and sequencing data collected in the Huanan market in early 2020. We demonstrate that market-linked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity is consistent with market emergence and find increased SARS-CoV-2 positivity near and within a wildlife stall. We identify wildlife DNA in all SARS-CoV-2-positive samples from this stall, including species such as civets, bamboo rats, and raccoon dogs, previously identified as possible intermediate hosts. We also detect animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them with those from farms and other markets. This analysis provides the genetic basis for a shortlist of potential intermediate hosts of SARS-CoV-2 to prioritize for serological and viral sampling.

Temporal and Spatial Changes in the Microbiome Following Pediatric Severe Traumatic Brain Injury.

OBJECTIVES: The microbiome may be affected by trauma and critical illness. Many studies of the microbiome in critical illness are restricted to a single body site or time point and confounded by preexisting conditions. We report temporal and spatial alterations in the microbiome of previously healthy children with severe traumatic brain injury (TBI). DESIGN: We collected oral, rectal, and skin swabs within 72 hours of admission and then twice weekly until ICU discharge. Samples were analyzed by 16S rRNA gene amplicon sequencing. Children undergoing elective outpatient surgery served as controls. Alpha and beta diversity comparisons were performed with Phyloseq, and differentially abundant taxa were predicted using Analysis of Composition of Microbiomes. SETTING: Five quaternary-care PICUs. PATIENTS: Patients less than 18 years with severe TBI requiring placement of an intracranial pressure monitor. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Three hundred twenty-seven samples were analyzed from 23 children with severe TBI and 35 controls. The community composition of initial oral (F = 3.2756, R2 = 0.0535, p = 0.012) and rectal (F = 3.0702, R2 = 0.0649, p = 0.007) samples differed between TBI and control patients. Rectal samples were depleted of commensal bacteria from Ruminococcaceae, Bacteroidaceae, and Lachnospiraceae families and enriched in Staphylococcaceae after TBI (p < 0.05). In exploratory analyses, antibiotic exposure, presence of an endotracheal tube, and occurrence of an infection were associated with greater differences of the rectal and oral microbiomes between TBI patients and healthy controls, whereas enteral nutrition was associated with smaller differences (p < 0.05). CONCLUSIONS: The microbiome of children with severe TBI is characterized by early depletion of commensal bacteria, loss of site specificity, and an enrichment of potential pathogens. Additional studies are needed to determine the impact of these changes on clinical outcomes.

Time-Dependent Displacement of Commensal Skin Microbes by Pathogens at the Site of Colorectal Surgery.

BACKGROUND: Although the healthy human skin microbiome has been the subject of recent studies, it is not known whether alterations among commensal microbes contribute to surgical site infections (SSIs). Our objective in this study was to characterize temporal and spatial variation in the skin microbiota of patients undergoing colorectal surgery and determine if dysbiosis contributes to SSIs. METHODS: Sixty one adults scheduled to undergo elective colon or rectal resection were identified by convenience sampling. By analyzing bacterial 16S rRNA gene sequences isolated from clinical samples, we used a culture-independent strategy to monitor perioperative changes in microbial diversity of fecal samples and the skin. RESULTS: A total of 990 samples from 61 patients were analyzed. Alpha diversity on the skin decreased after surgery but later recovered at the postoperative clinic visit. In most patients, we observed a transient postoperative loss of skin commensals (Corynebacterium and Propionibacterium) at the surgical site, which were replaced by potential pathogens and intestinal anaerobes (eg, Enterobacteriaceae). These changes were not observed on skin that was uninvolved in the surgical incision (chest wall). One patient developed a wound infection. Incisional skin swabs from this patient demonstrated a sharp postoperative increase in the abundance of Enterococcus, which was also cultured from wound drainage. CONCLUSIONS: We observed reproducible perioperative changes in the skin microbiome following surgery. The low incidence of SSIs in this cohort precluded analysis of associations between dysbiosis and infection. We postulate that real-time monitoring of the skin microbiome could provide actionable findings about the pathogenesis of SSIs.

Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia.

The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954-1955) and between 2008-2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms.

Evolution and Cryo-electron Microscopy Capsid Structure of a North American Bat Adenovirus and Its Relationship to Other Mastadenoviruses.

Since the first description of adenoviruses in bats in 2006, a number of micro- and megabat species in Europe, Africa, and Asia have been shown to carry a wide diversity of adenoviruses. Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV) recovered from a Rafinesque's big-eared bat (Corynorhinus rafinesquii) in Kentucky, USA, which is the first adenovirus isolated from North American bats. This virus (BtAdV 250-A) exhibits a close phylogenetic relationship with Canine mastadenovirus A (CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy reconstructions of the BtAdV 250-A capsid and also analyzed the in vitro host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV. IMPORTANCE: Although many adenoviruses are host specific and likely codiverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to Canine mastadenovirus A (CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged. Although most adenoviruses tend to cause limited disease in their natural hosts, CAdV A is unusual in that it may cause high morbidity and sometimes fatal infections in immunocompetent hosts and is thus an important pathogen of carnivores. Here, we performed a comparative evolutionary and structural study of representative bat and canine adenoviruses to better understand the relationship between these two viral groups.

Peroxisome Proliferator-activated Receptor-γ Coactivator 1-α (PGC1α) Protects against Experimental Murine Colitis.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is the primary regulator of mitochondrial biogenesis and was recently found to be highly expressed within the intestinal epithelium. PGC1α is decreased in the intestinal epithelium of patients with inflammatory bowel disease, but its role in pathogenesis is uncertain. We now hypothesize that PGC1α protects against the development of colitis and helps to maintain the integrity of the intestinal barrier. We selectively deleted PGC1α from the intestinal epithelium of mice by breeding a PGC1α(loxP/loxP) mouse with a villin-cre mouse. Their progeny (PGC1α(ΔIEC) mice) were subjected to 2% dextran sodium sulfate (DSS) colitis for 7 days. The SIRT1 agonist SRT1720 was used to enhance PGC1α activation in wild-type mice during DSS exposure. Mice lacking PGC1α within the intestinal epithelium were more susceptible to DSS colitis than their wild-type littermates. Pharmacologic activation of PGC1α successfully ameliorated disease and restored mitochondrial integrity. These findings suggest that a depletion of PGC1α in the intestinal epithelium contributes to inflammatory changes through a failure of mitochondrial structure and function as well as a breakdown of the intestinal barrier, which leads to increased bacterial translocation. PGC1α induction helps to maintain mitochondrial integrity, enhance intestinal barrier function, and decrease inflammation.

Dietary Supplementation With Nonfermentable Fiber Alters the Gut Microbiota and Confers Protection in Murine Models of Sepsis.

OBJECTIVES: Links between microbial alterations and systemic inflammation have been demonstrated in chronic disease, but little is known about these interactions during acute inflammation. This study investigates the effect of dietary supplementation with cellulose, a nonfermentable fiber, on the gut microbiota, inflammatory markers, and survival in two murine models of sepsis. DESIGN: Prospective experimental study. SETTING: University laboratory. SUBJECTS: Six-week-old male C57BL/6 wild-type mice. INTERVENTIONS: Mice were assigned to low-fiber, normal-fiber, or high-fiber diets with or without antibiotics for 2 weeks and then subjected to sepsis by cecal ligation and puncture or endotoxin injection. Fecal samples were collected for microbiota analyses before and after dietary interventions. MEASUREMENTS AND MAIN RESULTS: Mice that received a high-fiber diet demonstrated increased survival after cecal ligation and puncture relative to mice receiving low-fiber or normal-fiber diets. The survival benefit was associated with decreased serum concentration of pro-inflammatory cytokines, reduced neutrophil infiltration in the lungs, and diminished hepatic inflammation. The high-fiber diet also increased survival after endotoxin injection. Bacterial 16S ribosomal RNA gene sequences from each sample were amplified, sequenced, and analyzed. Fiber supplementation yielded an increase in relative abundance of the genera Akkermansia and Lachnospiraceae, taxa commonly associated with metabolic health. Administration of antibiotics to mice on the high-fiber diet negated the enrichment of Akkermansia species and the survival benefit after cecal ligation and puncture. CONCLUSION: Dietary supplementation with cellulose offers a microbe-mediated survival advantage in murine models of sepsis. Improved understanding of the link between diet, the microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis.

Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population.

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.

Acute Appendicitis in Children Is Associated With a Local Expansion of Fusobacteria.

BACKGROUND: Lumenal obstruction has typically been regarded as the cause of acute appendicitis (AA). Recent evidence including data from "antibiotics first" trials suggests that this disease may result from invasion of the appendix by specific pathogens. Small studies have identified an abundance of bacteria from the genus Fusobacterium in appendixes from patients with AA. We aimed to validate these findings in a larger cohort of children with appendicitis in addition to profiling the appendiceal microbiota in a population of children without appendicitis. METHODS: Appendix swabs were collected from children undergoing appendectomy for AA (n = 60), incidental appendectomy for reasons other than appendicitis (n = 18), or ileocecectomy for inflammatory bowel disease (n = 7), in addition to samples from other sites. Bacterial 16S ribosomal RNA gene sequences from each sample were amplified, sequenced, and analyzed with the UPARSE and QIIME programs. RESULTS: We found that the normal human appendix harbors populations of Fusobacteria that are generally absent in fecal samples from healthy adults and children. In patients with AA, Fusobacteria populations proliferate and often persist despite several weeks of broad-spectrum antibiotics prior to surgery. Relative to non-AA samples, AA samples were depleted of sequences from the genus Bacteroides Phylogenetic analysis of sequence data indicates that F. nucleatum, F. necrophorum, and F. varium are the species of Fusobacterium observed in AA samples. CONCLUSIONS: These results indicate that the appendiceal niche harbors distinct microbial populations that likely contribute to the pathogenesis of appendicitis, which may one day be leveraged to improve the diagnosis and/or treatment of patients with AA.

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus.

UNLABELLED: Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. IMPORTANCE: The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

Topographic diversity of the respiratory tract mycobiome and alteration in HIV and lung disease.

RATIONALE: Microbiome studies typically focus on bacteria, but fungal species are common in many body sites and can have profound effects on the host. Wide gaps exist in the understanding of the fungal microbiome (mycobiome) and its relationship to lung disease. OBJECTIVES: To characterize the mycobiome at different respiratory tract levels in persons with and without HIV infection and in HIV-infected individuals with chronic obstructive pulmonary disease (COPD). METHODS: Oral washes (OW), induced sputa (IS), and bronchoalveolar lavages (BAL) were collected from 56 participants. We performed 18S and internal transcribed spacer sequencing and used the neutral model to identify fungal species that are likely residents of the lung. We used ubiquity-ubiquity plots, random forest, logistic regression, and metastats to compare fungal communities by HIV status and presence of COPD. MEASUREMENTS AND MAIN RESULTS: Mycobiomes of OW, IS, and BAL shared common organisms, but each also had distinct members. Candida was dominant in OW and IS, but BAL had 39 fungal species that were disproportionately more abundant than in the OW. Fungal communities in BAL differed significantly by HIV status and by COPD, with Pneumocystis jirovecii significantly overrepresented in both groups. Other fungal species were also identified as differing in HIV and COPD. CONCLUSIONS: This study systematically examined the respiratory tract mycobiome in a relatively large group. By identifying Pneumocystis and other fungal species as overrepresented in the lung in HIV and in COPD, it is the first to determine alterations in fungal communities associated with lung dysfunction and/or HIV, highlighting the clinical relevance of these findings. Clinical trial registered with www.clinicaltrials.gov (NCT00870857).

Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania.

Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.

Comparative analysis of the complete genome sequence of the California MSW strain of myxoma virus reveals potential host adaptations.

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.

Genome scale evolution of myxoma virus reveals host-pathogen adaptation and rapid geographic spread.

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.

GeneDB--an annotation database for pathogens.

GeneDB (http://www.genedb.org) is a genome database for prokaryotic and eukaryotic pathogens and closely related organisms. The resource provides a portal to genome sequence and annotation data, which is primarily generated by the Pathogen Genomics group at the Wellcome Trust Sanger Institute. It combines data from completed and ongoing genome projects with curated annotation, which is readily accessible from a web based resource. The development of the database in recent years has focused on providing database-driven annotation tools and pipelines, as well as catering for increasingly frequent assembly updates. The website has been significantly redesigned to take advantage of current web technologies, and improve usability. The current release stores 41 data sets, of which 17 are manually curated and maintained by biologists, who review and incorporate data from the scientific literature, as well as other sources. GeneDB is primarily a production and annotation database for the genomes of predominantly pathogenic organisms.

The salivary microbiota of patients with acute lower respiratory tract infection-A multicenter cohort study.

The human microbiome contributes to health and disease, but the oral microbiota is understudied relative to the gut microbiota. The salivary microbiota is easily accessible, underexplored, and may provide insight into response to infections. We sought to determine the composition, association with clinical features, and heterogeneity of the salivary microbiota in patients with acute lower respiratory tract infection (LRTI). We conducted a multicenter prospective cohort study of 147 adults with acute LRTI presenting to the emergency department of seven hospitals in three states (Pennsylvania, Michigan, and Ohio) between May 2017 and November 2018. Salivary samples were collected in the emergency department, at days 2-5 if hospitalized, and at day 30, as well as fecal samples if patients were willing. We compared salivary microbiota profiles from patients to those of healthy adult volunteers by sequencing and analyzing bacterial 16-rRNA. Compared to healthy volunteers, the salivary microbiota of patients with LRTI was highly distinct and strongly enriched with intestinal anaerobes such as Bacteroidaceae, Ruminococcaceae, and Lachnospiraceae (e.g., mean 10% relative abundance of Bacteroides vs < 1% in healthy volunteers). Within the LRTI population, COPD exacerbation was associated with altered salivary microbiota composition compared to other LRTI conditions. The largest determinant of microbiota variation within the LRTI population was geography (city in which the hospital was located).

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea.

Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae.

Epithelial NAD+ depletion drives mitochondrial dysfunction and contributes to intestinal inflammation.

Introduction: We have previously demonstrated that a pathologic downregulation of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1α) within the intestinal epithelium contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanism underlying downregulation of PGC1α expression and activity during IBD is not yet clear. Methods: Mice (male; C57Bl/6, Villincre/+;Pgc1afl/fl mice, and Pgc1afl/fl) were subjected to experimental colitis and treated with nicotinamide riboside. Western blot, high-resolution respirometry, nicotinamide adenine dinucleotide (NAD+) quantification, and immunoprecipitation were used to in this study. Results: We demonstrate a significant depletion in the NAD+ levels within the intestinal epithelium of mice undergoing experimental colitis, as well as humans with ulcerative colitis. While we found no decrease in the levels of NAD+-synthesizing enzymes within the intestinal epithelium of mice undergoing experimental colitis, we did find an increase in the mRNA level, as well as the enzymatic activity, of the NAD+-consuming enzyme poly(ADP-ribose) polymerase-1 (PARP1). Treatment of mice undergoing experimental colitis with an NAD+ precursor reduced the severity of colitis, restored mitochondrial function, and increased active PGC1α levels; however, NAD+ repletion did not benefit transgenic mice that lack PGC1α within the intestinal epithelium, suggesting that the therapeutic effects require an intact PGC1α axis. Discussion: Our results emphasize the importance of PGC1α expression to both mitochondrial health and homeostasis within the intestinal epithelium and suggest a novel therapeutic approach for disease management. These findings also provide a mechanistic basis for clinical trials of nicotinamide riboside in IBD patients.

Previous infection with seasonal coronaviruses does not protect male Syrian hamsters from challenge with SARS-CoV-2.

SARS-CoV-2 variants and seasonal coronaviruses continue to cause disease and coronaviruses in the animal reservoir pose a constant spillover threat. Importantly, understanding of how previous infection may influence future exposures, especially in the context of seasonal coronaviruses and SARS-CoV-2 variants, is still limited. Here we adopted a step-wise experimental approach to examine the primary immune response and subsequent immune recall toward antigenically distinct coronaviruses using male Syrian hamsters. Hamsters were initially inoculated with seasonal coronaviruses (HCoV-NL63, HCoV-229E, or HCoV-OC43), or SARS-CoV-2 pango B lineage virus, then challenged with SARS-CoV-2 pango B lineage virus, or SARS-CoV-2 variants Beta or Omicron. Although infection with seasonal coronaviruses offered little protection against SARS-CoV-2 challenge, HCoV-NL63-infected animals had an increase of the previously elicited HCoV-NL63-specific neutralizing antibodies during challenge with SARS-CoV-2. On the other hand, primary infection with HCoV-OC43 induced distinct T cell gene signatures. Gene expression profiling indicated interferon responses and germinal center reactions to be induced during more similar primary infection-challenge combinations while signatures of increased inflammation as well as suppression of the antiviral response were observed following antigenically distant viral challenges. This work characterizes and analyzes seasonal coronaviruses effect on SARS-CoV-2 secondary infection and the findings are important for pan-coronavirus vaccine design.

Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic.

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Wholesale Seafood Market, the site with the most reported wildlife vendors in the city of Wuhan, China. Here, we analyze publicly available qPCR and sequencing data from environmental samples collected in the Huanan market in early 2020. We demonstrate that the SARS-CoV-2 genetic diversity linked to this market is consistent with market emergence, and find increased SARS-CoV-2 positivity near and within a particular wildlife stall. We identify wildlife DNA in all SARS-CoV-2 positive samples from this stall. This includes species such as civets, bamboo rats, porcupines, hedgehogs, and one species, raccoon dogs, known to be capable of SARS-CoV-2 transmission. We also detect other animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them to those from other markets. This analysis provides the genetic basis for a short list of potential intermediate hosts of SARS-CoV-2 to prioritize for retrospective serological testing and viral sampling.