Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19) from mild to severe stages including fatality, with pro-in ammatory macrophages as one of the main mediators of lung hyper-in ammation. Therefore, there is an urgent need to better understand the interactions among SARS-CoV-2 permissive cells, macrophage, and the SARS-CoV-2 virus, thereby offering important insights into new therapeutic strategies. Here, we used directed differentiation of human pluripotent stem cells (hPSCs) to establish a lung and macrophage co-culture system and model the host-pathogen interaction and immune response caused by SARS-CoV-2 infection. Among the hPSCderived lung cells, alveolar type II and ciliated cells are the major cell populations expressing the viral receptor ACE2 and co-effector TMPRSS2, and both were highly permissive to viral infection. We found that alternatively polarized macrophages (M2) and classically polarized macrophages (M1) had similar inhibitory effects on SARS-CoV-2 infection. However, only M1 macrophages signi cantly up-regulated in ammatory factors including IL-6 and IL-18, inhibiting growth and enhancing apoptosis of lung cells. Inhibiting viral entry into target cells using an ACE2 blocking antibody enhanced the activity of M2 macrophages, resulting in nearly complete clearance of virus and protection of lung cells. These results suggest a potential therapeutic strategy, in that by blocking viral entrance to target cells while boosting anti-in ammatory action of macrophages at an early stage of infection, M2 macrophages can eliminate SARS-CoV-2, while sparing lung cells and suppressing the dysfunctional hyperin ammatory response mediated by M1 macrophages.

Highlights d Generation of a transcriptional atlas of SARS-CoV-2 infection in hamsters d Infection and transmission can be initiated by respiratory or ocular exposure d Systemic inflammation occurs despite little productive replication in distal tissues d Intranasal IFN-I administered pre-or post-virus challenge reduces disease burden

The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.

SUMMARY Low pathogenic H7N9 influenza has recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in prolonged pathologies collectively referred to as post-acute sequalae of COVID-19 (PASC) or long COVID. To better understand the mechanism underlying long COVID biology, we compared the short- and long-term systemic responses in the golden hamster following either SARS-CoV-2 or influenza A virus (IAV) infection. Results demonstrated that SARS-CoV-2 exceeded IAV in its capacity to cause permanent injury to the lung and kidney and uniquely impacted the olfactory bulb (OB) and epithelium (OE). Despite a lack of detectable infectious virus, the OB and OE demonstrated myeloid and T cell activation, proinflammatory cytokine production, and an interferon response that correlated with behavioral changes extending a month post viral clearance. These sustained transcriptional changes could also be corroborated from tissue isolated from individuals who recovered from COVID-19. These data highlight a molecular mechanism for persistent COVID-19 symptomology and provide a small animal model to explore future therapeutics.

c Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X. PA-X was found to be expressed from the PA segment of influenza A viruses as an alternative product (1). It consists of an N-terminal endonuclease domain of PA (191 amino acids) and a C-terminal PA-X-specific domain (61 amino acids), which is encoded by an overlapping open reading frame generated by a ribosomal frameshift (1, 2). PA-X decreases host and viral mRNA accumulation via its shutoff activity, which is dependent on its endonuclease activity (1, 3). Although PA-X is not essential for viral replication in vitro, it modulates host responses in vivo (1,(4)(5)(6)(7).PA has lower shutoff activity than PA-X, although it has the N-terminal endonuclease domain (1,3,(8)(9)(10)(11)(12), suggesting that the C-terminal PA-X-specific region is also important for the shutoff activity. To examine this possibility, we constructed two C-terminal deletion mutant forms of PA [A/WSN/33(H1N1)] (Fig. 1A). PA, PA-X, PA_191, or PA_252 was expressed in 293 cells together with Renilla luciferase by means of plasmid transfection with Trans IT-293 (Mirus). Renilla luciferase activities were measured with the Renilla-Glo luciferase assay system (Promega) at 24 h posttransfection (Fig. 1B). Renilla luciferase activities were suppressed by PA (0.6-log-fold), PA-X (2.0-log-fold), PA_191 (1.3-log-fold), and PA_252 (0.7-log-fold). To confirm these results, we analyzed the levels of expression of Renilla luciferase and the PA-X and PA variants by Western blotting with an anti-Renilla luciferase polyclonal antibody (Promega) (Fig. 1C) and an anti-PA monoclonal antibody, clone 55/2 (13) (Fig. 1D). Renilla luciferase was detected in the mock lane but not in the PA-X lane. In the PA, PA_191, and PA_252 lanes, the Renilla luciferase expression levels were lower than in the mock lane (43.6, 53.7, and 52.7%, respectively). PA-X was barely detected because of self-suppression, whereas PA, PA_191, and PA_252 were expressed to similar extents. These results indicate that the shutoff activity of PA_191 and PA_252 is lower than that of wild-type PA-X, suggesting that the C-terminal PA-X-specific region is important for PA-X shutoff activity.To pinpoint the amino acid residues in the C-terminal PA-Xspecific region that are critical for PA-X activity, we prepared a series of C-terminal deletion mutant forms of PA-X ( Fig. 2A) and examined their shutoff activities in the luciferase assay (Fig. 2B). Compared with wild-type PA-X (which exhibited 1.8-log-fold less than the mock sample), PA-X_X10, PA-X_X5, and PA_191 showed significantly lower shutoff activity (1.6-, 1.1-, a...

Many broadly reactive human monoclonal antibodies against the hemagglutinin (HA) stem of influenza A virus have been developed for therapeutic applications. These antibodies typically inhibit viral entry steps, especially the HA conformational change that is required for membrane fusion. To better understand the mechanisms by which such antibodies inhibit viral replication, we established broadly reactive human anti-HA stem antibodies and determined the properties of these antibodies by examining their reactivity with 18 subtypes of HA, evaluating their in vivo protective efficacy, identifying their epitopes, and characterizing their inhibitory mechanisms. Among the eight human monoclonal antibodies we generated, which recognized at least 3 subtypes of the soluble HA antigens tested, clone S9-1-10/5-1 reacted with 18 subtypes of HA and protected mice from lethal infection with H1N1pdm09, H3N2, H5N1, and H7N9 viruses. This antibody recognized the HA2 helix A in the HA stem, and inhibited virus particle release from infected cells but did not block viral entry completely. These results show that broadly reactive human anti-HA stem antibodies can exhibit protective efficacy by inhibiting virus particle release. These findings expand our knowledge of the mechanisms by which broadly reactive stem-targeting antibodies inhibit viral replication and provide valuable information for universal vaccine development.

N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of influenza virus PA-X. The shutoff activity of PA-X was suppressed in NatB-deficient cells, and PA-X mutants that are not acetylated by NatB showed reduced shutoff activities. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells. Moreover, mutant PAs that are not acetylated by NatB lost their function in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation is required for the shutoff activity of PA-X and for viral polymerase activity.