Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown. Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury. Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence. Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

Background: The major urinary proteins (MUPs) of Mus musculus domesticus are deposited in urine in large quantities, where they bind and release pheromones and also provide an individual 'recognition signal' via their phenotypic polymorphism. Whilst important information about MUP functionality has been gained in recent years, the gene cluster is poorly studied in terms of structure, genic polymorphism and evolution. Results: We combine targeted sequencing, manual genome annotation and phylogenetic analysis to compare the Mup clusters of C57BL/6J and 129 strains of mice. We describe organizational heterogeneity within both clusters: a central array of cassettes containing Mup genes highly similar at the protein level, flanked by regions containing Mup genes displaying significantly elevated divergence. Observed genomic rearrangements in all regions have likely been mediated by endogenous retroviral elements. Mup loci with coding sequences that differ between the strains are identified - including a gene/pseudogene pair - suggesting that these inbred lineages exhibit variation that exists in wild populations. We have characterized the distinct MUP profiles in the urine of both strains by mass spectrometry. The total MUP phenotype data is reconciled with our genomic sequence data, matching all proteins identified in urine to annotated genes. Conclusion: Our observations indicate that the MUP phenotypic polymorphism observed in wild populations results from a combination of Mup gene turnover coupled with currently unidentified mechanisms regulating gene expression patterns. We propose that the structural heterogeneity described within the cluster reflects functional divergence within the Mup gene family.

Immunopathology occurs in the lung and spleen in fatal coronavirus disease (COVID-19), involving monocytes/macrophages and plasma cells. Antiinflammatory therapy reduces mortality, but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 postmortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21, and prothrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated and associated with inflammation severity. Unsupervised clustering identified “early viral” and “late inflammatory” clusters with distinct protein abundance profiles, and differences in illness duration before death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and proapoptotic factors were increased. B-cell receptor signaling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a subset of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3, and CSF-1) was provided by overlap between 1) differential abundance in spleen and lung tissue; 2) meta-analysis of existing datasets; and 3) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue antiviral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation, and lymphocyte depletion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

Background: Tissue inflammation is associated with organ dysfunction and death in Covid-19. The efficacy of dexamethasone in preventing mortality in critical Covid-19 suggests that inflammation has a causal role in death. Whether this deleterious inflammation is a direct response to the presence of SARS-CoV-2, or an independent immuno-pathologic process, is unknown. Methods: Tissue was acquired from detailed post-mortem examinations conducted on 11 well characterised hospitalised patients with fatal Covid-19. SARS-CoV-2 organotropism was mapped at an organ level by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike (S) protein detection. Histological evidence of inflammation and organ injury was systematically examined, and the pulmonary immune response characterized with multiplex immunofluorescence. Findings: SARS-CoV-2 was detected across a wide variety of organs, most frequently in the respiratory tract but also in numerous extra-pulmonary sites. Minimal histological evidence of inflammation was identified in non-pulmonary organs despite frequent detection of viral RNA and protein. At a cellular level, viral protein was identified without adjacent inflammation in the intestine, liver and kidney. Severe inflammatory change was restricted to the lung and reticulo-endothelial system. Diffuse alveolar damage, pulmonary thrombi and a monocyte/myeloid-predominant vasculitis were the predominant pulmonary findings, though there was not a consistent association between viral presence and either the presence or nature of the inflammatory response within the lung. Immunophenotyping revealed an influx of macrophages, monocytes and T cells into pulmonary parenchyma. Bone marrow examination revealed plasmacytosis, erythroid dysplasia and iron-laden macrophages. Plasma cell excess was also present in lymph node, spleen and lung. These stereotyped reticulo-endothelial responses occurred largely independently of the presence of virus in lymphoid tissues. Conclusions: Tissue inflammation and organ dysfunction in fatal Covid-19 do not map to the tissue and cellular distribution of SARS-CoV-2, demonstrating tissue-specific tolerance. We conclude that death in Covid-19 is primarily a consequence of immune-mediated, rather than pathogen-mediated, organ inflammation and injury. Funding: The Chief Scientist Office, LifeArc, Medical Research Scotland, UKRI (MRC).

Arboviruses such as bluetongue virus (BTV) replicate in arthropod vectors involved in their transmission between susceptible vertebrate-hosts. The “classical” BTV strains infect and replicate effectively in cells of their insect-vectors (Culicoides biting-midges), as well as in those of their mammalian-hosts (ruminants). However, in the last decade, some “atypical” BTV strains, belonging to additional serotypes (e.g., BTV-26), have been found to replicate efficiently only in mammalian cells, while their replication is severely restricted in Culicoides cells. Importantly, there is evidence that these atypical BTV are transmitted by direct-contact between their mammalian hosts. Here, the viral determinants and mechanisms restricting viral replication in Culicoides were investigated using a classical BTV-1, an “atypical” BTV-26 and a BTV-1/BTV-26 reassortant virus, derived by reverse genetics. Viruses containing the capsid of BTV-26 showed a reduced ability to attach to Culicoides cells, blocking early steps of the replication cycle, while attachment and replication in mammalian cells was not restricted. The replication of BTV-26 was also severely reduced in other arthropod cells, derived from mosquitoes or ticks. The data presented identifies mechanisms and potential barriers to infection and transmission by the newly emerged “atypical” BTV strains in Culicoides.