A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients. We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested. Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10⁻⁴-2000 TCID₅₀/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests. The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b. Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal–human interface. Significance Middle East respiratory syndrome (MERS) is a zoonotic disease of global health concern, and dromedary camels are the source of human infection. Although Africa has the largest number of dromedary camels, and MERS-coronavirus (MERS-CoV) is endemic in these camels, locally acquired zoonotic MERS is not reported from Africa. However, little is known of the genetic or phenotypic characterization of MERS-CoV from Africa. In this study we characterize MERS-CoV from Burkina Faso, Nigeria, Morocco, and Ethiopia. We demonstrate viral genetic and phenotypic differences in viruses from West Africa, which may be relevant to differences in zoonotic potential, highlighting the need for studies of MERS-CoV at the animal–human interface.

Significance: Acute lung injury, including impaired alveolar fluid clearance, is a life-threatening complication of severe respiratory virus infection, and effective treatment is lacking. Understanding the mechanism of this complication may suggest novel therapies. Here, we found that, in vitro, influenza A/H5N1 infection impaired alveolar fluid clearance more than did seasonal virus, mimicking its greater severity in patients. We demonstrated that this impairment is mediated by the release of soluble factors from infected cells, leading to down-regulation of alveolar sodium and chloride transporters. Mesenchymal stromal cells prevented or reduced this effect in vitro and in vivo in A/H5N1-infected mice. These cells provide a potentially effective treatment for acute lung injury in severe influenza.

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-α, IFN-β, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease. Here we show that TNF-α, IFN-β, and IFN-λ1 are the key mediators directly induced by the H5N1 virus in primary human macrophages. In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3). IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-β, IFN-λ1, and MCP-1 but only to a partial reduction of TNF-α. IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus. We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-β, IFN-λ1, and MCP-1 but only partly control TNF-α induction. A more precise identification of the differences in the regulation of TNF-α and IFN-β could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.