ABSTRACT The Middle East respiratory syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hypersensitize cells to infection when an S-cleaving protease(s) was present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans. IMPORTANCE MERS is a frequently fatal disease that is caused by a zoonotic CoV. The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals and found that human, camel, and horse receptors sensitized cells to MERS-CoV infection more robustly than goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell surface lung protease greatly increases susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.
ABSTRACT Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized 2 . Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo , knowledge that could help inform strategies to combat infection by emerging CoV.
ABSTRACT The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. IMPORTANCE Infection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD, NTD and S2. Different infected individuals generate vastly different amounts of anti-spike antibodies. By contrast, as we show here, there is a remarkable similarity in the properties of the antibodies produced. Different individuals generate the same proportions of antibodies against each domain of the spike protein. Furthermore, the relationship between the amount of anti-spike antibodies produced and their neutralization efficacy of SARS-CoV-2 is highly conserved. Therefore, the observed variation in the neutralizing activity of the antibody response in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or relative antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.
RNA transcripts of cloned cDNA of the genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm.
Abstract Real-time, reverse transcriptase PCR assays are a pervasive technology used for diagnosis of SARS-CoV-2 infection. These assays produce a cycle threshold value (Ct) corresponding to the first amplification cycle in which reliable amplification is detected. (1)Such Ct values have been used by clinicians and in public health settings to guide treatment, monitor disease progression, assess prognosis, and inform isolation practices. To understanding the risk of reporting out uncalibrated Ct values and potential for instead reporting out calibrated viral load values, we performed a multi-institutional study to benchmark major clinical platforms against a calibrated standard. We found that for any given Ct value, corresponding viral loads varied up to 1000-fold among the different tests. In contrast, when these different assays were calibrated against a common standard and then used to test unknown de-identified specimens at several dilutions, viral load values showed high precision between methods (standard deviation and range of 0.36 and 1.1 log10 genome copies) and high accuracy compared with droplet digital PCR (ddPCR) determinations (difference between mean CDC N2 and Sarbeco E ddPCR determinations and mean determinations by calibrated RT-PCR assays examined in our study of 0.044 log10 genome copies). We, therefore, find strong support for calibration of SARS-CoV-2 RT-PCR tests to allow conversion of cycle thresholds to accurate and precise viral load values that are reproducible across major clinical systems. Implementation of calibrated assays will provide more reliable information for clinical decision making and allow more rigorous interpretation of SARS-CoV-2 laboratory data in clinical and laboratory investigation.
Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from uterine carcinoma and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT medium. After 3 passages the cultures were morphologically homogeneous. They retained an epithelial morphology. Electron microscopic and morphometric studies indicate that these hybrids at the 6th passage differed from parental cells. They exhibited the smallest cellular and cytoplasmic size. The number of mitochondria was similar to that of murine fibroblasts but their size was more homogeneously distributed; the mitochondria matrix was more electron dense than in the parental cells. These data suggest that these hybrids have a different metabolism from that of the parental cells; they contained modified mitochondria.
