To determine if a hypersensitive-type lung pathology might occur when mice were given an inactivated MERS-CoV vaccine and challenged with infectious virus as was seen with SARS-CoV vaccines, we prepared and vaccinated mice with an inactivated MERS-CoV vaccine. Neutralizing antibody was induced by vaccine with and without adjuvant and lung virus was reduced in vaccinated mice after challenge. Lung mononuclear infiltrates occurred in all groups after virus challenge but with increased infiltrates that contained eosinophils and increases in the eosinophil promoting IL-5 and IL-13 cytokines only in the vaccine groups. Inactivated MERS-CoV vaccine appears to carry a hypersensitive-type lung pathology risk from MERS-CoV infection that is similar to that found with inactivated SARS-CoV vaccines from SARS-CoV infection.

Animal models for severe acute respiratory syndrome (SARS) coronavirus infection of humans are needed to elucidate SARS pathogenesis and develop vaccines and antivirals. We developed transgenic mice expressing human angiotensin-converting enzyme 2, a functional receptor for the virus, under the regulation of a global promoter. A transgenic lineage, designated AC70, was among the best characterized against SARS coronavirus infection, showing weight loss and other clinical manifestations before reaching 100% mortality within 8 days after intranasal infection. High virus titers were detected in the lungs and brains of transgene-positive (Tg⁺) mice on days 1 and 3 after infection. Inflammatory mediators were also detected in these tissues, coinciding with high levels of virus replication. Lower virus titers were also detected in other tissues, including blood. In contrast, infected transgene-negative (Tg⁻) mice survived without showing any clinical illness. Pathologic examination suggests that the extensive involvement of the central nervous system likely contributed to the death of Tg⁺mice, even though viral pneumonia was present. Preliminary studies with mice of a second lineage, AC63, in which the transgene expression was considerably less abundant than that in the AC70 line, revealed that virus replication was largely restricted to the lungs but not the brain. Importantly, despite significant weight loss, infected Tg⁺AC63 mice eventually recovered from the illness without any mortality. The severity of the disease that developed in these transgenic mice—AC70 in particular—makes these mouse models valuable not only for evaluating the efficacy of antivirals and vaccines, but also for studying SARS coronavirus pathogenesis.

Severe acute respiratory syndrome (SARS), which is caused by a novel coronavirus (CoV), is a highly communicable disease with the lungs as the major pathological target. Although SARS likely stems from overexuberant host inflammatory responses, the exact mechanism leading to the detrimental outcome in patients remains unknown. Pulmonary macrophages (Mφ), airway epithelium, and dendritic cells (DC) are key cellular elements of the host innate defenses against respiratory infections. While pulmonary Mφ are situated at the luminal epithelial surface, DC reside abundantly underneath the epithelium. Such strategic locations of these cells within the airways make it relevant to investigate their likely impact on SARS pathogenesis subsequent to their interaction with infected lung epithelial cells. To study this, we established highly polarized human lung epithelial Calu-3 cells by using the Transwell culture system. Here we report that supernatants harvested from the apical and basolateral domains of infected Calu-3 cells are potent in modulating the intrinsic functions of Mφ and DC, respectively. They prompted the production of cytokines by both Mφ and DC and selectively induced CD40 and CD86 expression only on DC. However, they compromised the abilities of the DC and Mφ in priming naïve T cells and phagocytosis, respectively. We also identified interleukin-6 (IL-6) and IL-8 as key SARS-CoV-induced epithelial cytokines capable of inhibiting the T-cell-priming ability of DC. Taken together, our results provide insights into the molecular and cellular bases of the host antiviral innate immunity within the lungs that eventually lead to an exacerbated inflammatory cascades and severe tissue damage in SARS patients.

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC₅₀0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.

The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ∼40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days postinfection. High infectious virus titers were recovered primarily from the lungs and brains of mice at 2 and 4 days postinfection, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg⁺mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg⁺mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection.IMPORTANCESmall and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small-animal species for this purpose because of availability and the existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters, and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.

Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NF)κB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-β, -λs, inflammatory mediators, and many IFN-stimulated genes (ISGs). We also showed, for the first time, that IFN-β and IFN-λs were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.