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We find ourselves ten months into one of the most catastrophic global health events of our lifetime and, disturbingly, we still do not know how it began. What’s even more troubling is that despite the critical importance of this question, efforts to investigate the origins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and of the associated disease, coronavirus disease 2019 (COVID-19), have become mired in politics, poorly supported assumptions and assertions, and incomplete information. To avoid or mitigate the dire consequences of this and future pandemics (here, people in PPE bury a victim in Delhi, India in June), unraveling the origins of SARS-CoV-2 and COVID-19 will be essential—even though a definitive answer may be elusive, and an objective analysis means broaching some uncomfortable possibilities. Image credit: Shutterstock/PradeepGaurs. SARS-CoV-2 is a betacoronavirus whose apparent closest relatives, RaTG13 and RmYN02, are reported to have been collected from bats in 2013 and 2019, respectively, in Yunnan Province, China (1). COVID-19 was first reported in December 2019 more than 1,000 miles away in Wuhan City, Hubei Province, China. Beyond these facts, the “origin story” is missing many key details, including a plausible and suitably detailed recent evolutionary history of the virus, the identity and provenance of its most recent ancestors, and surprisingly, the place, time, and mechanism of transmission of the first human infection. Even though a definitive answer may not be forthcoming, and even though an objective analysis requires addressing some uncomfortable possibilities, it is crucial that we pursue this question. Preventing the next pandemic depends on understanding the origins of this one. There are several potential origin scenarios. First, SARS-CoV-2 may … [↵][1]1Email: relman{at}stanford.edu. [1]: #xref-corresp-1-1

Reciprocal, intimate relationships between the human microbiome and the host immune system are shaped by past microbial encounters and prepare the host for future ones. Antibiotics and other antimicrobials leave their mark on both the microbiome and host immunity. Antimicrobials alter the structure of the microbiota, expand the host-specific pool of antimicrobial-resistance genes and organisms, degrade the protective effects of the microbiota against invasion by pathogens, and may impair vaccine efficacy. Through these effects on the microbiome they may affect immune responses. Vaccines that exert protective or therapeutic effects against pathogens may reduce the use of antimicrobials, the development and spread of antimicrobial resistance, and the harmful impacts of these drugs on the microbiome. Other strategies involving manipulation of the microbiome to deplete antibiotic-resistant organisms or to enhance immune responses to vaccines may prove valuable in addressing antimicrobial resistance as well. This article describes the intersections of immunity, microbiome and antimicrobial exposure, and the use of vaccines and other alternative strategies for the control and management of antimicrobial resistance.

We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1-2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2(-) mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2(+) mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2(+) and Fut2(-) mice with Bacteroides thetaiotaomicron, a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2(-) mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241-4247, an operon responsive to terminal β-galactose, the precursor that accumulates in the Fut2(-) mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241-4247 operon was also seen in humanized Fut2(-) mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.

The human oropharynx is a reservoir for many potential pathogens, including streptococcal species that cause endocarditis. Although oropharyngeal microbes have been well described, viral communities are essentially uncharacterized. We conducted a metagenomic study to determine the composition of oropharyngeal DNA viral communities (both phage and eukaryotic viruses) in healthy individuals and to evaluate oropharyngeal swabs as a rapid method for viral detection. Viral DNA was extracted from 19 pooled oropharyngeal swabs and sequenced. Viral communities consisted almost exclusively of phage, and complete genomes of several phage were recovered, including Escherichia coli phage T3, Propionibacterium acnes phage PA6, and Streptococcus mitis phage SM1. Phage relative abundances changed dramatically depending on whether samples were chloroform treated or filtered to remove microbial contamination. pblA and pblB genes of phage SM1 were detected in the metagenomes. pblA and pblB mediate the attachment of S. mitis to platelets and play a significant role in S. mitis virulence in the endocardium, but have never previously been detected in the oral cavity. These genes were also identified in salivary metagenomes from three individuals at three time points and in individual saliva samples by PCR. Additionally, we demonstrate that phage SM1 can be induced by commonly ingested substances. Our results indicate that the oral cavity is a reservoir for pblA and pblB genes and for phage SM1 itself. Further studies will determine the association between pblA and pblB genes in the oral cavity and the risk of endocarditis.

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.

We thank Keelan and Payne (1) for their interest in our work (2). Their letter emphasizes intrauterine infection as a potential mechanistic link between the vaginal microbiota and preterm birth. Although we share their view of ascending infection as a possible mechanism, we don’t consider it to be the only possible one. For this and other reasons we discuss below, we are hesitant to adopt the view—which we believe would risk introducing investigator biases—that documented intra-amniotic infection is a necessary accompanying condition for establishing a role of the microbiota in preterm birth.

Smallpox has played an unparalleled role in human history and remains a significant potential threat to public health. Despite the historical significance of this disease, we know little about the underlying pathophysiology or the virulence mechanisms of the causative agent, variola virus. To improve our understanding of variola pathogenesis and variola-host interactions, we examined the molecular and cellular features of hemorrhagic smallpox in cynomolgus macaques. We used cDNA microarrays to analyze host gene expression patterns in sequential blood samples from each of 22 infected animals. Variola infection elicited striking and temporally coordinated patterns of gene expression in peripheral blood. Of particular interest were features that appear to represent an IFN response, cell proliferation, immunoglobulin gene expression, viral dose-dependent gene expression patterns, and viral modulation of the host immune response. The virtual absence of a tumor necrosis factor α/NF-κB-activated transcriptional program in the face of an overwhelming systemic infection suggests that variola gene products may ablate this response. These results provide a detailed picture of the host transcriptional response during smallpox infection, and may help guide the development of diagnostic, therapeutic, and prophylactic strategies.

Smallpox virus (variola) poses a significant threat as an agent of bioterrorism. To mitigate this risk, antiviral drugs and an improved vaccine are urgently needed. Satisfactory demonstration of protective efficacy against authentic variola will require development of an animal model in which variola produces a disease course with features consistent with human smallpox. Toward this end, cynomolgus macaques were exposed to several variola strains through aerosol and/or i.v. routes. Two strains, Harper and India 7124, produced uniform acute lethality when inoculated i.v. in high doses (10 9 plaque-forming units). Lower doses resulted in less fulminant, systemic disease and lower mortality. Animals that died had profound leukocytosis, thrombocytopenia, and elevated serum creatinine levels. After inoculation, variola was disseminated by means of a monocytic cell-associated viremia. Distribution of viral antigens by immunohistochemistry correlated with the presence of replicating viral particles demonstrated by electron microscopy and pathology in the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver. These particles resembled those seen in human smallpox. High viral burdens in target tissues were associated with organ dysfunction and multisystem failure. Evidence of coagulation cascade activation (D dimers) corroborated histologic evidence of hemorrhagic diathesis. Depletion of T cell-dependent areas of lymphoid tissues occurred, probably as a consequence of bystander apoptotic mechanisms initiated by infected macrophages. Elaboration of cytokines, including IL-6 and IFN-γ, contribute to a cytokine storm formerly known as “toxemia.” A more precise understanding of disease pathogenesis should provide targets for therapeutic intervention, to be used alone or in combination with inhibitors of variola virus replication.

Archaea have been isolated from the human colon, vagina, and oral cavity, but have not been established as causes of human disease. In this study, we reveal a relationship between the severity of periodontal disease and the relative abundance of archaeal small subunit ribosomal RNA genes (SSU rDNA) in the subgingival crevice by using quantitative PCR. Furthermore, the relative abundance of archaeal small subunit rDNA decreased at treated sites in association with clinical improvement. Archaea were harbored by 36% of periodontitis patients and were restricted to subgingival sites with periodontal disease. The presence of archaeal cells at these sites was confirmed by fluorescent in situ hybridization. The archaeal community at diseased sites was dominated by a Methanobrevibacter oralis -like phylotype and a distinct Methanobrevibacter subpopulation related to archaea that inhabit the gut of numerous animals. We hypothesize that methanogens participate in syntrophic relationships in the subgingival crevice that promote colonization by secondary fermenters during periodontitis. Because they are potential alternative syntrophic partners, our finding of larger Treponema populations sites without archaea provides further support for this hypothesis.

The innate immune response is crucial for defense against microbial pathogens. To investigate the molecular choreography of this response, we carried out a systematic examination of the gene expression program in human peripheral blood mononuclear cells responding to bacteria and bacterial products. We found a remarkably stereotyped program of gene expression induced by bacterial lipopolysaccharide and diverse killed bacteria. An intricately choreographed expression program devoted to communication between cells was a prominent feature of the response. Other features suggested a molecular program for commitment of antigen-presenting cells to antigens captured in the context of bacterial infection. Despite the striking similarities, there were qualitative and quantitative differences in the responses to different bacteria. Modulation of this host-response program by bacterial virulence mechanisms was an important source of variation in the response to different bacteria.

Bordetella pertussis, the causative agent of whooping cough, has many well-studied virulence factors and a characteristic clinical presentation. Despite this information, it is not clear howB. pertussisinteraction with host cells leads to disease. In this study, we examined the interaction ofB. pertussiswith a human bronchial epithelial cell line (BEAS-2B) and measured host transcriptional profiles by using high-density DNA microarrays. The early transcriptional response to this pathogen is dominated by altered expression of cytokines, DNA-binding proteins, and NFκB-regulated genes. This previously unrecognized response toB. pertussiswas modified in similar but nonidentical fashions by the antiinflammatory agents dexamethasone and sodium salicylate. Cytokine protein expression was confirmed, as was neutrophil chemoattraction. We show thatB. pertussisinduces mucin gene transcription by BEAS-2B cells then counters this defense by using mucin as a binding substrate. A set of genes is described for which the catalytic activity of pertussis toxin is both necessary and sufficient to regulate transcription. Host genomic transcriptional profiling, in combination with functional assays to evaluate subsequent biological events, provides insight into the complex interaction of host and pathogen.

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were

Filamentous hemagglutinin is a surface-associated adherence protein of Bordetella pertussis, which is a component of some new acellular pertussis vaccines. The nucleotide sequence of an open reading frame that encompasses the filamentous hemagglutinin structural gene, fhaB, suggests that proteolytic processing is necessary to generate the mature 220-kDa filamentous hemagglutinin product. An Arg-Gly-Asp (RGD) tripeptide is found within filamentous hemagglutinin that may be involved in its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B. pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell binding and infection.