Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard-to-grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well-differentiated human airway epithelial cell cultures at air-liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses-specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-and provide a protocol for the detection of double-stranded RNA or viral antigen-positive cells by immunofluorescence microscopy. These techniques, together with a post-imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air-liquid interface (HAE-ALI) Basic Protocol 2: Viral inoculation of HAE-ALI Basic Protocol 3: Immunofluorescence (IF)-based detection of infected cells in HAE-ALI.

Comparison between nucleic acid amplification tests, antigen immunofluorescence assay, and in vitro infectivity in SARS-CoV-2 diagnosis.

The number of SARS-CoV-2 detection tests requested to the laboratories has dramatically increased together with an urgent need to release reliable responses in a very short time. The two options taken into consideration and analyzed in the current study were the point-of-care test (POCT) based on the nucleic acid amplification test (NAAT) and the Antigen (Ag) rapid test. The POCT-NAAT-based assay was compared with a rapid antigen test of nasopharyngeal swab samples. If the specimen tested positive, it was followed by viral load quantification and by the functional assessment of the residual infectivity. When the initial cycle threshold (Ct) was below 20 (100%), and in the range of 20-25 (92%) and of 25-30 (88%), a great concordance between the POCT-NAAT and the Ag test was observed. Moreover, the positivity of the antigen test was well correlated to a successful infection in vitro (78%), with greater concordance when the initial Ct below 20 or above 35 (100%) and in the range 20-25 (83%). Our findings showed that most of the swabs which tested positive using the antigen test were able to infect the cells in vitro, suggesting that probably only these samples hold residual infectivity and therefore an increased risk of virus transmission at the moment of being tested.

Immunofluorescent Antibody Techniques in the Diagnosis of SARS-CoV-2 Infection in Humans.

Immunofluorescence (IF) is an important technique used in the diagnosis of many infectious diseases. In virology, it has proven to be particularly suited to detecting antibody directed against newly emerging viruses able to be cultivated in cell culture. It permits visualization of antibody and allows for antibody class to be determined which is critical to understanding the timing of infection. The procedure used to determine IgG, IgA, and IgM antibody directed against SARS-CoV-2 in humans is described in this chapter. These methods were developed for routine diagnosis of SARS-CoV-2 infection in Australia at the start of the global pandemic in 2020.

Viral culture and immunofluorescence for the detection of SARS-CoV-2 infectivity in RT-PCR positive respiratory samples.

BACKGROUND: Knowing how long SARS-CoV-2-positive individuals can remain infective is crucial for the design of infection prevention and control strategies. Viral culture is the gold standard for detecting an active-replicative virus and evaluating its infectious potential. OBJECTIVE: To assess the correlation of SARS-CoV-2 infectivity with the number of days from symptom onset and the Ct value, using culture as a reference method. Also, to describe a detailed protocol for SARS-CoV-2 culture and immunofluorescence confirmation based on our experience with other respiratory viruses. STUDY DESIGN: 100 consecutive respiratory samples positive for SARS-CoV-2 by RT-PCR from different subjects were inoculated into VERO E6 cells. RESULTS: Viral isolation was successful in 58% of samples. The median number of days from symptom onset for culture-positive samples was 2, and 15 for culture-negative samples. Six positive cultures were obtained in patients ≥14 days after symptom onset, all of whom were immunocompromised or with severe COVID-19. The mean Ct value was 12.64 units higher in culture-negative than in culture-positive samples. The probability of successfully isolating SARS-CoV-2 in samples with a Ct value <22 was 100%, decreasing to 3.1% when >27. CONCLUSIONS: Our findings show a significant positive correlation between the probability of isolating SARS-CoV-2 in culture, fewer days of symptoms and a lower RT-PCR Ct value. SARS-CoV-2 infectivity lasts no more than 14 days from symptom onset in immunocompetent individuals. In contrast, in immunocompromised patients or those with severe COVID-19 infectivity may remain after 14 days. Ct value <22 always indicates infectivity.

Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.

Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection.

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 µM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 µM of HCQ and 104 U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP.

Development of an immunofluorescence assay for detection of SARS-CoV-2.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.

Pulmonary vascular inflammation with fatal coronavirus disease 2019 (COVID-19): possible role for the NLRP3 inflammasome.

BACKGROUND: Pulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs. METHODS: Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1. RESULTS: In lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs. CONCLUSION: Vascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

Dexamethasone sensitizes to ferroptosis by glucocorticoid receptor-induced dipeptidase-1 expression and glutathione depletion.

Dexamethasone is widely used as an immunosuppressive therapy and recently as COVID-19 treatment. Here, we demonstrate that dexamethasone sensitizes to ferroptosis, a form of iron-catalyzed necrosis, previously suggested to contribute to diseases such as acute kidney injury, myocardial infarction, and stroke, all of which are triggered by glutathione (GSH) depletion. GSH levels were significantly decreased by dexamethasone. Mechanistically, we identified that dexamethasone up-regulated the GSH metabolism regulating protein dipeptidase-1 (DPEP1) in a glucocorticoid receptor (GR)-dependent manner. DPEP1 knockdown reversed the phenotype of dexamethasone-induced ferroptosis sensitization. Ferroptosis inhibitors, the DPEP1 inhibitor cilastatin, or genetic DPEP1 inactivation reversed the dexamethasone-induced increase in tubular necrosis in freshly isolated renal tubules. Our data indicate that dexamethasone sensitizes to ferroptosis by a GR-mediated increase in DPEP1 expression and GSH depletion. Together, we identified a previously unknown mechanism of glucocorticoid-mediated sensitization to ferroptosis bearing clinical and therapeutic implications.

Measurement of antibody levels in patients with COVID-19 over time by immunofluorescence assay: a longitudinal observational study.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, antibody screening is a critical tool to assess anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity. We examined variation in antibody titers associated with age and sex among patients with confirmed COVID-19. METHODS: Blood IgG levels were tested in 1081 patients with positive SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (RT-qPCR) tests between 1 September and 31 December 2020. Patients who did not experience reinfection were identified. Serum IgG levels were measured by immunofluorescence assay. Antibody positivity and antibody titers were analyzed according to time since infection, sex, and age. RESULTS: The mean (standard deviation) age was 41.2 (14.2) years and 41.2% of patients were women. The lowest antibody positivity rate between the first and ninth month post-infection was detected in the sixth month. The lowest antibody titers among patients aged 20 to 80 years occurred in those aged 30 to 39 years. The IgG titer was positively correlated with age in years (r = 0.125) and decades (r = 0.126). CONCLUSIONS: Six months after infection, anti-SARS-CoV-2 antibody titers increased. Anti-SARS-CoV-2 antibody titers also increased with age. Immunity and pathogenicity should be investigated in addition to antibody positivity rates and antibody titers.

SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1, and NRP-1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems.

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor-binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule-1 (Kim-1), and neuropilin-1 (NRP-1). In this study, we examined the entry tropism of SARS-CoV-2 and SARS-CoV using S protein-based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim-1 and NRP-1 recombinant proteins. Furthermore, cells with robust SARS-CoV-2 pseudovirus infection had strong expression of either ACE2 or Kim-1 and NRP-1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS-CoV-2 or SARS-CoV pseudovirus with the level of glycosylated ACE2 (gly-ACE2). These results reveal that SARS-CoV-2 cell entry could be mediated by either an ACE2-dependent or -independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types.

Seroprevalence of SARS-CoV-2 antibodies among rural healthcare workers.

The objective of this longitudinal cohort study was to determine the seroprevalence of antibodies to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in healthcare workers employed at healthcare settings in three rural counties in eastern South Dakota and western Minnesota from May 13, 2020, through December 22, 2020. Three blood draws were performed at five clinical sites and tested for the presence of antibodies against the SARS-CoV-2. Serum samples were tested for the presence of antibodies using a fluorescent microsphere immunoassay (FMIA), neutralization of SARS-CoV-2 spike-pseudotyped particles (SARS-CoV-2pp) assay, and serum virus neutralization (SVN) assay. The seroprevalence was determined to be 1/336 (0.29%) for samples collected from 5/13/20 to 7/13/20, 5/260 (1.92%) for samples collected from 8/13/20 to 9/25/20, and 35/235 (14.89%) for samples collected from 10/16/20 to 12/22/20. Eight of the 35 (22.8%) seropositive individuals identified in the final draw did not report a previous diagnosis with COVID-19. There was a high correlation (>90%) between the FMIA and virus neutralization assays. Each clinical site's seroprevalence was higher than the cumulative incidence for the general public in the respective county as reported by state public health agencies. As of December 2020, there was a high percentage (85%) of seronegative individuals in the study population.

[Preparation of different fragments of SARS-CoV-2 N protein and its application in fluorescence chromatography].

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.

A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies.

Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organization (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralization test (MNT). We also compared the MNT results of two laboratories. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13 to 150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA, 100% specificity and sensitivity were obtained for a shorter time window (13 to 36 and 13 to 28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralizing antibodies (NAbs). Anti-spike IgG concentrations correlated strongly (ρ = 0.77 to 0.84, P < 2.2 × 10-16) with NAb titers, and the two laboratories' NAb titers displayed a very strong correlation (ρ = 0.95, P < 2.2 × 10-16). Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against the WHO international standard did not, however, improve the comparability of FMIA and EIA results. IMPORTANCE SARS-CoV-2 serological assays with excellent clinical performance are essential for reliable estimation of the persistence of immunity after infection or vaccination. In this paper we present a thoroughly validated SARS-CoV-2 serological assay with excellent clinical performance and good comparability to neutralizing antibody titers. Neutralization tests are still considered the gold standard for SARS-CoV-2 serological assays, but our assay can identify samples with neutralizing antibodies with 100% sensitivity and 96% specificity without the need for laborious and slow biosafety level 3 (BSL-3) facility-requiring analyses.

Performance of the LumiraDx Microfluidic Immunofluorescence Point-of-Care SARS-CoV-2 Antigen Test in Asymptomatic Adults and Children.

OBJECTIVES: The LumiraDx SARS-CoV-2 Ag Test has previously been shown to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals symptomatic for coronavirus disease 2019 (COVID-19). This evaluation investigated the LumiraDx SARS-CoV-2 Ag Test as an aid in the diagnosis of SARS-CoV-2 infection in asymptomatic adults and children. METHODS: Asymptomatic individuals at high risk of COVID-19 infection were recruited in 5 point-of-care (POC) settings. Two paired anterior nasal swabs were collected from each participant, tested by using the LumiraDx SARS-CoV-2 Ag Test at the POC, and compared with results from reverse transcription-polymerase chain reaction (RT-PCR) assays (cobas 6800 [Roche Diagnostics] or TaqPath [Thermo Fisher Scientific]). We calculated positive percent agreement (PPA) and negative percent agreement (NPA), then stratified results on the basis of RT-PCR reference platform and cycle threshold. RESULTS: Of the 222 included study participants confirmed to be symptom-free for at least 2 weeks before testing, the PPA was 82.1% (95% confidence interval [CI], 64.4%-92.1%). The LumiraDx SARS-CoV-2 Ag Test correctly identified 95.8% (95% CI, 79.8%-99.3%) of the samples confirmed positive in fewer than 33 RT-PCR cycles and 100% (95% CI, 85.1%-100%) in fewer than 30 RT-PCR cycles while maintaining 100% NPA. CONCLUSIONS: This rapid, high-sensitivity test can be used to screen asymptomatic patients for acute SARS-CoV-2 infection in clinic- and community-based settings.

An Insight Into Detection Pathways/Biosensors of Highly Infectious Coronaviruses.

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.

MOV10 Helicase Interacts with Coronavirus Nucleocapsid Protein and Has Antiviral Activity.

Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.

Characterization of T lymphocytes in severe COVID-19 patients.

In this observational study, 13 patients with severe COVID-19 and 10 healthy controls were enrolled. The data concerning the analysis of circulating T cells show that, in severe COVID-19 patients, the expansion of these cell compartments is prone to induce antibody response, inflammation (CCR4+ and CCR6+ TFH) and regulation (CD8+ Treg). This pathogenic mechanism could lead us to envision a possible new form of biological target therapy.

Monocytes and Macrophages, Targets of Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 2019 Immunoparalysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during COVID-19 and if monocytes and macrophages could be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Monocytes and monocyte-derived macrophages (MDMs) from COVID-19 patients and controls were infected with SARS-CoV-2 and extensively investigated with immunofluorescence, viral RNA extraction and quantification, and total RNA extraction followed by reverse-transcription quantitative polymerase chain reaction using specific primers, supernatant cytokines (interleukins 6, 10, and 1ß; interferon-ß; transforming growth factor-ß1, and tumor necrosis factor-α), and flow cytometry. The effect of M1- vs M2-type or no polarization prior to infection was assessed. RESULTS: SARS-CoV-2 efficiently infected monocytes and MDMs, but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In COVID-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity. CONCLUSIONS: SARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of COVID-19 progression.SARS-CoV-2 infection of macrophages induces a specific M2 transcriptional program. In Covid-19 patients, monocyte subsets were decreased associated with up-expression of the immunoregulatory molecule CD163 suggesting that SARS-CoV-2 drives immune system for the benefit of Covid-19 disease progression.