Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

A TLR4 agonist liposome formulation effectively stimulates innate immunity and enhances protection from bacterial infection.

Stimulation of innate immunity can protect against infectious insult and could be used in combination with other therapies. Since antibiotic resistance is an increasing concern, strategies to reduce the dose or eliminate the need for these drugs are warranted. Lipo-CRX is a formulation in which the TLR4 agonist CRX-527 is incorporated into lipid membranes in liposomes. Lipo-CRX is less inflammatory than either CRX-527 or LPS, but retains unique capacity to enhance host defense responses. We compared lipo-CRX to other agonists in vitro using mammalian cells and in vivo in mice, and assessed indicators of innate immune responses and protection from bacterial infection. Lipo-CRX is similar to E. coli LPS in its capacity to activate bovine γδ T cells and to recruit neutrophils into mouse lungs, but with less reactivity in the LAL assay. However, lipo-CRX uniquely induced the production of systemic innate immune cytokines. In the mouse model of brucellosis, delivery of lipo-CRX to the lungs reduced the dissemination of B. abortus. While lipo-CRX or the antibiotic ampicillin alone did not alter B. abortus burdens in the lung, the combination had a synergistic beneficial effect. Our data suggest that stimulating the innate immune system with lipo-CRX, either alone or when combined with antibiotics, can enhance bacterial clearance in the mouse model of brucellosis.

Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection, with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly, SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Pathophysiology of Influenza D Virus Infection in Specific-Pathogen-Free Lambs with or without Prior Mycoplasma ovipneumoniae Exposure.

Polymicrobial pneumonias occur frequently in cattle, swine, and sheep, resulting in major economic losses. Individual pathogens comprising these complex infections may be mild on their own but can instead exhibit synergism or increase host susceptibility. Two examples of such pathogens, Mycoplasma ovipneumoniae (M. ovipneumoniae) and influenza D viruses (IDVs), naturally infect domestic sheep. In sheep, the role of M. ovipneumoniae in chronic nonprogressive pneumonia is well-established, but the pathogenesis of IDV infection has not previously been studied. We utilized a specific-pathogen-free sheep flock to study the clinical response to IDV infection in naïve vs. M. ovipneumoniae-exposed lambs. Lambs were inoculated intranasally with M. ovipneumoniae or mock infection, followed after four weeks by infection with IDV. Pathogen shedding was tracked, and immunological responses were evaluated by measuring acute phase response and IDV-neutralizing antibody titers. While lamb health statuses remained subclinical, M. ovipneumoniae-exposed lambs had significantly elevated body temperatures during IDV infection compared to M. ovipneumoniae-naïve, IDV-infected lambs. Moreover, we found a positive correlation between prior M. ovipneumoniae burden, early-infection IDV shedding, and IDV-neutralizing antibody response. Our findings suggest that IDV infection may not induce clinical symptoms in domestic sheep, but previous M. ovipneumoniae exposure may promote mild IDV-associated inflammation.

An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model.

Angiotensin Converting Enzyme 2 (ACE2) is the primary cell entry receptor for SARS-CoV and SARS-CoV-2 viruses. A disintegrin and metalloproteinase 17 (ADAM17) is a protease that cleaves ectodomains of transmembrane proteins, including that of ACE2 and the proinflammatory cytokine TNF-α, from cell surfaces upon cellular activation. We hypothesized that blockade of ADAM17 activity would alter COVID-19 pathogenesis. To assess this pathway, we blocked the function of ADAM17 using the monoclonal antibody MEDI3622 in the K18-hACE2 transgenic mouse model of COVID-19. Antibody-treated mice were healthier, less moribund, and had significantly lower lung pathology than saline-treated mice. However, the viral burden in the lungs of MEDI3622-treated mice was significantly increased. Thus, ADAM17 appears to have a critical anti-viral role, but also may promote inflammatory damage. Since the inflammatory cascade is ultimately the reason for adverse outcomes in COVID-19 patients, there may be a therapeutic application for the MEDI3622 antibody.

Severe Acute Respiratory Syndrome Coronavirus 2 Is Detected in the Gastrointestinal Tract of Asymptomatic Endoscopy Patients but Is Unlikely to Pose a Significant Risk to Healthcare Personnel.

Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

Experimental infection of specific-pathogen-free domestic lambs with Mycoplasma ovipneumoniae causes asymptomatic colonization of the upper airways that is resistant to antibiotic treatment.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a respiratory pathogen associated with mild to moderate respiratory disease in domestic lambs and severe pneumonia outbreaks in wild ruminants such as bighorn sheep. However, whether M. ovipneumoniae by itself causes clinical respiratory disease in domestic sheep in the absence of secondary bacterial pathogens is still unclear. The goal of our study was to better understand the role of M. ovipneumoniae as a respiratory pathogen in domestic sheep and to explore potential antibiotic treatment approaches. Therefore, we inoculated four 4-month-old, specific-pathogen-free lambs with fresh nasal wash fluids from M. ovipneumoniae-infected sheep. The lambs were monitored for M. ovipneumoniae colonization, M. ovipneumoniae-specific antibodies, clinical signs, and cellular and molecular correlates of lung inflammation for eight weeks. All lambs then were treated with gamithromycin and observed for an additional four weeks. M. ovipneumoniae inoculation resulted in stable colonization of the upper respiratory tract in all M. ovipneumoniae-inoculated, but in none of the four mock-infected control lambs. All M. ovipneumoniae-infected lambs developed a robust antibody response to M. ovipneumoniae within 2 weeks. However, we did not observe significant signs of respiratory disease, evidence of lung damage or inflammation in any of the infected lambs. Interestingly, treatment with gamithromycin, which blocked growth of the M. ovipneumoniae in vitro, failed to reduce M. ovipneumoniae colonization. These observations indicate that, in the absence of co-infections, M. ovipneumoniae caused asymptomatic colonization of the upper respiratory tract that was resistant to clearance by the host immune response and by gamithromycin treatment.

A Synthetic Hydrogel, VitroGel® ORGANOID-3, Improves Immune Cell-Epithelial Interactions in a Tissue Chip Co-Culture Model of Human Gastric Organoids and Dendritic Cells.

Immunosurveillance of the gastrointestinal epithelium by mononuclear phagocytes (MNPs) is essential for maintaining gut health. However, studying the complex interplay between the human gastrointestinal epithelium and MNPs such as dendritic cells (DCs) is difficult, since traditional cell culture systems lack complexity, and animal models may not adequately represent human tissues. Microphysiological systems, or tissue chips, are an attractive alternative for these investigations, because they model functional features of specific tissues or organs using microscale culture platforms that recreate physiological tissue microenvironments. However, successful integration of multiple of tissue types on a tissue chip platform to reproduce physiological cell-cell interactions remains a challenge. We previously developed a tissue chip system, the gut organoid flow chip (GOFlowChip), for long term culture of 3-D pluripotent stem cell-derived human intestinal organoids. Here, we optimized the GOFlowChip platform to build a complex microphysiological immune-cell-epithelial cell co-culture model in order to study DC-epithelial interactions in human stomach. We first tested different tubing materials and chip configurations to optimize DC loading onto the GOFlowChip and demonstrated that DC culture on the GOFlowChip for up to 20 h did not impact DC activation status or viability. However, Transwell chemotaxis assays and live confocal imaging revealed that Matrigel, the extracellular matrix (ECM) material commonly used for organoid culture, prevented DC migration towards the organoids and the establishment of direct MNP-epithelial contacts. Therefore, we next evaluated DC chemotaxis through alternative ECM materials including Matrigel-collagen mixtures and synthetic hydrogels. A polysaccharide-based synthetic hydrogel, VitroGel®-ORGANOID-3 (V-ORG-3), enabled significantly increased DC chemotaxis through the matrix, supported organoid survival and growth, and did not significantly alter DC activation or viability. On the GOFlowChip, DCs that were flowed into the chip migrated rapidly through the V-ORG matrix and reached organoids embedded deep within the chip, with increased interactions between DCs and gastric organoids. The successful integration of DCs and V-ORG-3 embedded gastric organoids into the GOFlowChip platform now permits real-time imaging of MNP-epithelial interactions and other investigations of the complex interplay between gastrointestinal MNPs and epithelial cells in their response to pathogens, candidate drugs and mucosal vaccines.

Titers, Prevalence, and Duration of SARS-CoV-2 Antibodies in a Local COVID-19 Outbreak and Following Vaccination.

Information concerning the development of neutralizing antibodies and their duration will be critical to establishing herd immunity for COVID-19. We sought to evaluate SARS-CoV-2 spike protein receptor-binding domain (RBD)-specific antibodies, their duration, and capacity for SARS-CoV-2 neutralization in volunteers while the pandemic spread within our community starting in March 2020. Those participants with the highest starting titers had the longest-lasting response, up to 12 months post-diagnosis. SARS-CoV-2 neutralization capacity was correlated with anti-RBD antibody levels. The majority of our participants with confirmed COVID-19 diagnosis had very mild or asymptomatic infections. We also detected low and largely non-neutralizing anti-RBD IgG titers in a few participants with no known COVID-19 diagnosis. Finally, we found that antibody responses induced by vaccination were significantly higher than those induced by natural infection. Thus, our study suggests that vaccination is still critical even for those naturally infected or diagnosed with COVID-19.

Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.

SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression.

Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7aΔ115) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response.

Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure.

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

SARS-CoV-2 genomic surveillance identifies naturally occurring truncations of ORF7a that limit immune suppression.

Over 200,000 whole genome sequences of SARS-CoV-2 have been determined for viruses isolated from around the world. These sequences have been critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We have isolated one of these mutant viruses from a patient sample and used viral challenge experiments to demonstrate that Δ115 mutation results in a growth defect. ORF7a has been implicated in immune modulation, and we show that the C-terminal truncation results in distinct changes in interferon stimulated gene expression. Collectively, this work indicates that ORF7a mutations occur frequently and that these changes affect viral mechanisms responsible for suppressing the immune response.

Complete Genome Sequence of Brucella abortus Phage EF4, Determined Using Long-Read Sequencing.

Brucellaphage EF4 was isolated from elk feces. The 38,321-bp double-stranded DNA genome is predicted to contain 72 coding regions, 38 of which have been assigned predicted functions. This phage displays nucleotide similarity to other brucellaphages of the genus Perisivirus.

Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus Is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin.

Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

Getting "Inside" Type I IFNs: Type I IFNs in Intracellular Bacterial Infections.

Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial "sensing" mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

Therapeutic Potential of Polyphenols from Epilobium Angustifolium (Fireweed).

Epilobium angustifolium is a medicinal plant used around the world in traditional medicine for the treatment of many disorders and ailments. Experimental studies have demonstrated that Epilobium extracts possess a broad range of pharmacological and therapeutic effects, including antioxidant, anti-proliferative, anti-inflammatory, antibacterial, and anti-aging properties. Flavonoids and ellagitannins, such as oenothein B, are among the compounds considered to be the primary biologically active components in Epilobium extracts. In this review, we focus on the biological properties and the potential clinical usefulness of oenothein B, flavonoids, and other polyphenols derived from E. angustifolium. Understanding the biochemical properties and therapeutic effects of polyphenols present in E. angustifolium extracts will benefit further development of therapeutic treatments from this plant. Copyright © 2016 John Wiley & Sons, Ltd.

Type I Interferon Counters or Promotes Coxiella burnetii Replication Dependent on Tissue.

Coxiella burnetii is an intracellular pathogen and the cause of Q fever. Gamma interferon (IFN-γ) is critical for host protection from infection, but a role for type I IFN in C. burnetii infection has not been determined. Type I IFN supports host protection from a related pathogen, Legionella pneumophila, and we hypothesized that it would be similarly protective in C. burnetii infection. In contrast to our prediction, IFN-α receptor-deficient (IFNAR(-/-)) mice were protected from C. burnetii-induced infection. Therefore, the role of type I IFN in C. burnetii infection was distinct from that in L. pneumophila Mice treated with a double-stranded-RNA mimetic were protected from C. burnetii-induced weight loss through an IFNAR-independent pathway. We next treated mice with recombinant IFN-α (rIFN-α). When rIFN-α was injected by the intraperitoneal route during infection, disease-induced weight loss was exacerbated. Mice that received rIFN-α by this route had dampened interleukin 1ß (IL-1ß) expression in bronchoalveolar lavage fluids. However, when rIFN-α was delivered to the lung, bacterial replication was decreased in all tissues. Thus, the presence of type I IFN in the lung protected from infection, but when delivered to the periphery, type I IFN enhanced disease, potentially by dampening inflammatory cytokines. To better characterize the capacity for type I IFN induction by C. burnetii, we assessed expression of IFN-ß transcripts by human macrophages following stimulation with lipopolysaccharide (LPS) from C. burnetii Understanding innate responses in C. burnetii infection will support the discovery of novel therapies that may be alternative or complementary to the current antibiotic treatment.

Adjuvant materials that enhance bovine γδ T cell responses.

Despite the availability of vaccines and antibiotics, viral, bacterial and parasite-induced intestinal and pulmonary diseases still cause significant losses to the livestock industry. Excepting improvements in calf survival due to predation, there have been only modest improvements in bovine calf survival since 1991. Strikingly, digestive and respiratory diseases still account for almost half of the non-predator deaths in calves. The innate immune system has evolved to rapidly recognize and respond to invasive microbial threats. Augmentation of innate immunity is a broad-spectrum, potent and non-specific alternative approach to effectively counter a microbial invasion. In recent years we have focused our research efforts on the development of effective and inexpensive adjuvant therapies for cattle that can be used to help mitigate infection. Unique in our approach to the development of the potential new treatments, is our focus on bovine γδ T cells, which are important lymphocytes of the innate immune system and of particular importance to ruminant immunological health. This review focuses on recent results obtained using two such adjuvant materials.