How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2−infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

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A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.

Rotaviruses (RVs) are responsible for more than 600,000 child deaths each year. The worldwide introduction of two life oral vaccines RotaTeq and Rotarix is believed to reduce this number significantly. Before the licensing of both vaccines, two new genotypes, G9 and G12, emerged in the human population and were able to spread across the entire globe in a very short time span. To quantify the VP7 mutation rates of these G9 and G12 genotypes and to estimate their most recent common ancestors, we used a Bayesian Markov chain Monte Carlo framework. Based on 356 sequences for G9 and 140 sequences for G12, we estimated mutation rates (nt substitutions/site/year) of 1.87 × 10(-3) (1.45-2.27 × 10(-3)) for G9 and 1.66 × 10(-3) (1.13-2.32 × 10(-3)) for G12. For both the G9 and G12 strains, one particular (sub) lineage was able to disseminate and cause disease across the world. The most recent common ancestors of these particular lineages were dated back to 1989 (1986-1992) and 1995 (1992-1998) for the G9 and G12 genotypes, respectively. These estimates suggest that a single novel RV (e.g., a vaccine escape mutant) can spread worldwide in little more than a decade. These results re-emphasize the need for thorough and continued RV surveillance in order to detect such potential spreading events at an early stage.

Group A rotaviruses (RVAs) are a primary cause of gastroenteritis in children under 5 years of age and are associated with over 500,000 deaths annually, of which the majority occur in developing countries (42, 43). RVAs belong to the family Reoviridae, and the infectious RVA virion is a triple-layered icosahedral particle that contains 11 segments of double-stranded RNA (18). The outer capsid layer is composed of the spike protease-sensitive attachment protein VP4 (P) and the glycoprotein VP7 (G). The nucleotide sequences of the VP7-and VP4-encoding segments form the basis of a dual classification system that defines the G and P genotypes of RVAs, respectively. Although 27 G genotypes and 35 P genotypes have been identified to date (31), only a few RVA G and P genotype combinations contribute substantially to the burden of human disease (29,30,32,53 (33,35). An increase in prevalence of G12 RVAs, mainly associated with P[8] or P [6] and to a lesser extent with P[4] or P[9] VP4s, was observed around the turn of the century. The G12 RVA strains are now considered the sixth common global genotype (29,33,35,49).Two live attenuated oral RVA vaccines, Rotarix (GlaxoSmithKline Biologicals, Belgium) and RotaTeq (Merck & Co., Inc., United States), have been licensed for use in many countries around the world. Rotarix is derived from the attenuated human G1P[8] RVA strain 89-12, which was isolated in Cincinnati, OH, in 1988 (65). RotaTeq contains five human-bovine reassortant RVA strains (WI79-9, SC2-9, WI78-9, BrB-9, and WI79-4, referred to as G1, G2, G3, G4, and P1 reassortants, respectively, for simplicity). The G1 to G4 reassortants each express one of the VP7 proteins of the human RVA parental strains WI79 (G1), SC2 (G2), WI78 (G3), and BrB (G4) and the VP4 protein of the bovine RVA strain WC3 (P7[5]), whereas the P1 reassortant expresses the VP4 protein of the human RVA strain WI79 (P1A [8]) and the VP7 protein of the bovine RVA strain WC3 (G6) (9, 34). Human RVA SC-2 was isolated in 1981 at St. Christopher's Hospital of Philadelphia, and the WI79 and WI78 RVAs were isolated in 1983 at the Children's Hospital of Philadelphia, while the BrB (originally Bricout B) RVA strain was isolated in 1984 at L'Hôpital Armand Trousseau (Paris, France) (34). Both RVA vaccines have been proven to be safe and efficacious in large-scale clinical trials (9,51,59,61,62). The mechanisms by which the vaccines induce immunologic protection in infants have not been clearly elucidated. It likely includes the induction of serum and intestinal serotypespecific neutralizing antibodies directed against VP7 and VP4 and virus-specific cytotoxic T lymphocytes (20,63,64). However, proteins other than VP7 and VP4 may be involved in immune protection as well.

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