Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1-100 µM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

Patients hospitalized with active tuberculosis and Covid-19 coinfection: A matched case-control from the Brazilian Covid-19 Registry.

Although control of Covid-19 has improved, the virus continues to cause infections, such as tuberculosis, that is still endemic in many countries, representing a scenario of coinfection. To compare Covid-19 clinical manifestations and outcomes between patients with active tuberculosis infection and matched controls. This is a matched case-control study based on data from the Brazilian Covid-19 Registry, in hospitalized patients aged 18 or over with laboratory confirmed Covid-19 from March 1, 2020, to March 31, 2022. Cases were patients with tuberculosis and controls were Covid-19 patients without tuberculosis. From 13,636 Covid-19, 36 also had active tuberculosis (0.0026%). Pulmonary fibrosis (5.6% vs 0.0%), illicit drug abuse (30.6% vs 3.0%), alcoholism (33.3% vs 11.9%) and smoking (50.0% vs 9.7%) were more common among patients with tuberculosis. They also had a higher frequency of nausea and vomiting (25.0% vs 10.4%). There were no significant differences in in-hospital mortality, mechanical ventilation, need for dialysis and ICU stay. Patients with TB infection presented a higher frequency of pulmonary fibrosis, abuse of illicit drugs, alcoholism, current smoking, symptoms of nausea and vomiting. The outcomes were similar between them.

Psychotropic Medication Prescribing for Children and Adolescents After the Onset of the COVID-19 Pandemic.

Importance: Numerous studies have provided evidence for the negative associations of the COVID-19 pandemic with mental health, but data on the use of psychotropic medication in children and adolescents after the onset of the COVID-19 pandemic are lacking. Objective: To assess the rates and trends of psychotropic medication prescribing before and over the 2 years after the onset of the COVID-19 pandemic in children and adolescents in France. Design, Setting, and Participants: This cross-sectional study used nationwide interrupted time-series analysis of outpatient drug dispensing data from the IQVIA X-ponent database. All 8 839 143 psychotropic medication prescriptions dispensed to children (6 to 11 years of age) and adolescents (12 to 17 years of age) between January 2016 and May 2022 in France were retrieved and analyzed. Exposure: Onset of COVID-19 pandemic. Main outcomes and Measures: Monthly rates of psychotropic medication prescriptions per 1000 children and adolescents were analyzed using a quasi-Poisson regression before and after the pandemic onset (March 2020), and percentage changes in rates and trends were assessed. After the pandemic onset, rate ratios (RRs) were calculated between estimated and expected monthly prescription rates. Analyses were stratified by psychotropic medication class (antipsychotic, anxiolytic, hypnotic and sedative, antidepressant, and psychostimulant) and age group (children, adolescents). Results: In total, 8 839 143 psychotropic medication prescriptions were analyzed, 5 884 819 [66.6%] for adolescents and 2 954 324 [33.4%] for children. In January 2016, the estimated rate of monthly psychotropic medication prescriptions was 9.9 per 1000 children and adolescents, with the prepandemic rate increasing by 0.4% per month (95% CI, 0.3%-0.4%). In March 2020, the monthly prescription rate dropped by 11.5% (95% CI, -17.7% to -4.9%). During the 2 years following the pandemic onset, the trend changed significantly, and the prescription rate increased by 1.3% per month (95% CI, 1.2%-1.5%), reaching 16.1 per 1000 children and adolescents in May 2022. Monthly rates of psychotropic medication prescriptions exceeded the expected rates by 11% (RR, 1.11 [95% CI, 1.08-1.14]). Increases in prescribing trends were observed for all psychotropic medication classes after the pandemic onset but were substantial for anxiolytics, hypnotics and sedatives, and antidepressants. Prescription rates rose above those expected for all psychotropic medication classes except psychostimulants (RR, 1.12 [95% CI, 1.09-1.15] in adolescents and 1.06 [95% CI, 1.05-1.07] in children for antipsychotics; RR, 1.30 [95% CI, 1.25-1.35] in adolescents and 1.11 [95% CI, 1.09-1.12] in children for anxiolytics; RR, 2.50 [95% CI, 2.23-2.77] in adolescents and 1.40 [95% CI, 1.30-1.50] in children for hypnotics and sedatives; RR, 1.38 [95% CI, 1.29-1.47] in adolescents and 1.23 [95% CI, 1.20-1.25] in children for antidepressants; and RR, 0.97 [95% CI, 0.95-0.98] in adolescents and 1.02 [95% CI, 1.00-1.04] in children for psychostimulants). Changes were more pronounced among adolescents than children. Conclusions and Relevance: These findings suggest that prescribing of psychotropic medications for children and adolescents in France significantly and persistently increased after the COVID-19 pandemic onset. Future research should identify underlying determinants to improve psychological trajectories in young people.

Long-Term Taste and Smell Outcomes After COVID-19.

Importance: Self-report surveys suggest that long-lasting taste deficits may occur after SARS-CoV-2 infection, influencing nutrition, safety, and quality of life. However, self-reports of taste dysfunction are inaccurate, commonly reflecting deficits due to olfactory not taste system pathology; hence, quantitative testing is needed to verify the association of post-COVID-19 condition with taste function. Objective: To use well-validated self-administered psychophysical tests to investigate the association of COVID-19 with long-term outcomes in taste and smell function. Design, Setting, and Participants: This nationwide cross-sectional study included individuals with and without a prior history of COVID-19 recruited from February 2020 to August 2023 from a social media website (Reddit) and bulletin board advertisements. In the COVID-19 cohort, there was a mean of 395 days (95% CI, 363-425 days) between diagnosis and testing. Exposure: History of COVID-19. Main Outcomes and Measures: The 53-item Waterless Empirical Taste Test (WETT) and 40-item University of Pennsylvania Smell Identification Test (UPSIT) were used to assess taste and smell function. Total WETT and UPSIT scores and WETT subtest scores of sucrose, citric acid, sodium chloride, caffeine, and monosodium glutamate were assessed for groups with and without a COVID-19 history. The association of COVID-19 with taste and smell outcomes was assessed using analysis of covariance, χ2, and Fisher exact probability tests. Results: Tests were completed by 340 individuals with prior COVID-19 (128 males [37.6%] and 212 females [62.4%]; mean [SD] age, 39.04 [14.35] years) and 434 individuals with no such history (154 males [35.5%] and 280 females [64.5%]; mean (SD) age, 39.99 [15.61] years). Taste scores did not differ between individuals with and without previous COVID-19 (total WETT age- and sex-adjusted mean score, 33.41 [95% CI, 32.37-34.45] vs 33.46 [95% CI, 32.54-34.38]; P = .94). In contrast, UPSIT scores were lower in the group with previous COVID-19 than the group without previous COVID-19 (mean score, 34.39 [95% CI, 33.86-34.92] vs 35.86 [95% CI, 35.39-36.33]; P < .001]); 103 individuals with prior COVID-19 (30.3%) and 91 individuals without prior COVID-19 (21.0%) had some degree of dysfunction (odds ratio, 1.64 [95% CI, 1.18-2.27]). The SARS-CoV-2 variant present at the time of infection was associated with smell outcomes; individuals with original untyped and Alpha variant infections exhibited more loss than those with other variant infections; for example, total to severe loss occurred in 10 of 42 individuals with Alpha variant infections (23.8%) and 7 of 52 individuals with original variant infections (13.5%) compared with 12 of 434 individuals with no COVID-19 history (2.8%) (P < .001 for all). Conclusions and Relevance: In this study, taste dysfunction as measured objectively was absent 1 year after exposure to COVID-19 while some smell loss remained in nearly one-third of individuals with this exposure, likely explaining taste complaints of many individuals with post-COVID-19 condition. Infection with earlier untyped and Alpha variants was associated with the greatest degree of smell loss.

[Identifying Novel Coronavirus Pneumonia With CT Images: A Deep Learning Approach With Detail Upsampling and Attention Guidance]. / 从CTå›¾åƒä¸­æ£€æµ‹æ–°åž‹å† çŠ¶ç— æ¯’æ„ŸæŸ“å¯¼è‡´çš„è‚ºç‚Žï¼šä¸€ç§ç»†èŠ‚ä¸Šé‡‡æ ·å’Œæ³¨æ„åŠ›å¼•å¯¼çš„æ·±åº¦å­¦ä¹ æ–¹æ³•.

Objective: To construct a deep learning-based target detection method to help radiologists perform rapid diagnosis of lesions in the CT images of patients with novel coronavirus pneumonia (NCP) by restoring detailed information and mining local information. Methods: We present a deep learning approach that integrates detail upsampling and attention guidance. A linear upsampling algorithm based on bicubic interpolation algorithm was adopted to improve the restoration of detailed information within feature maps during the upsampling phase. Additionally, a visual attention mechanism based on vertical and horizontal spatial dimensions embedded in the feature extraction module to enhance the capability of the object detection algorithm to represent key information related to NCP lesions. Results: Experimental results on the NCP dataset showed that the detection method based on the detail upsampling algorithm improved the recall rate by 1.07% compared with the baseline model, with the AP50 reaching 85.14%. After embedding the attention mechanism in the feature extraction module, 86.13% AP50, 73.92% recall, and 90.37% accuracy were achieved, which were better than those of the popular object detection models. Conclusion: The feature information mining of CT images based on deep learning can further improve the lesion detection ability. The proposed approach helps radiologists rapidly identify NCP lesions on CT images and provides an important clinical basis for early intervention and high-intensity monitoring of NCP patients.

SARS-CoV-2 infects cells lining the blood-retinal barrier and induces a hyperinflammatory immune response in the retina via systemic exposure.

SARS-CoV-2 has been shown to cause wide-ranging ocular abnormalities and vision impairment in COVID-19 patients. However, there is limited understanding of SARS-CoV-2 in ocular transmission, tropism, and associated pathologies. The presence of viral RNA in corneal/conjunctival tissue and tears, along with the evidence of viral entry receptors on the ocular surface, has led to speculation that the eye may serve as a potential route of SARS-CoV-2 transmission. Here, we investigated the interaction of SARS-CoV-2 with cells lining the blood-retinal barrier (BRB) and the role of the eye in its transmission and tropism. The results from our study suggest that SARS-CoV-2 ocular exposure does not cause lung infection and moribund illness in K18-hACE2 mice despite the extended presence of viral remnants in various ocular tissues. In contrast, intranasal exposure not only resulted in SARS-CoV-2 spike (S) protein presence in different ocular tissues but also induces a hyperinflammatory immune response in the retina. Additionally, the long-term exposure to viral S-protein caused microaneurysm, retinal pigmented epithelium (RPE) mottling, retinal atrophy, and vein occlusion in mouse eyes. Notably, cells lining the BRB, the outer barrier, RPE, and the inner barrier, retinal vascular endothelium, were highly permissive to SARS-CoV-2 replication. Unexpectedly, primary human corneal epithelial cells were comparatively resistant to SARS-CoV-2 infection. The cells lining the BRB showed induced expression of viral entry receptors and increased susceptibility towards SARS-CoV-2-induced cell death. Furthermore, hyperglycemic conditions enhanced the viral entry receptor expression, infectivity, and susceptibility of SARS-CoV-2-induced cell death in the BRB cells, confirming the reported heightened pathological manifestations in comorbid populations. Collectively, our study provides the first evidence of SARS-CoV-2 ocular tropism via cells lining the BRB and that the virus can infect the retina via systemic permeation and induce retinal inflammation.

Conserved structures and dynamics in 5'-proximal regions of Betacoronavirus RNA genomes.

Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.

Patterns of Non-influenza Respiratory Viruses Among Severe Acute Respiratory Infection Cases in Burkina Faso: A Surveillance Study.

BACKGROUND: Although influenza viruses cause only one-fifth of severe acute respiratory infections (SARI) in Burkina Faso, the other viral causes of SARI remain poorly investigated to inform clinical and preventive decision making. METHODS: Between 2016 and 2019, we prospectively enrolled inpatients meeting the World Health Organization (WHO) case definition of SARI in Burkina Faso. Results of viral etiologies among inpatients tested negative for influenza using the Fast Track Diagnostics Respiratory Kits (FTD-33) were reported. RESULTS: Of 1541 specimens tested, at least one respiratory virus was detected in 76.1% of the 1231 specimens negative for influenza virus. Human rhinoviruses (hRVs) were the most detected pathogens (476; 38.7%), followed by human adenoviruses (hAdV) (17.1%, 210/1231), human respiratory syncytial virus (hRSV) (15.4%, 189/1231), enterovirus (EnV) (11.2%, 138/1231), human bocavirus (hBoV) (7.9%, 97/1231), parainfluenza 3 (hPIV3) (6.1%, 75/1231), human metapneumovirus (hMPV) (6.0%,74/1321), parainfluenza 4 (hPIV4) (4.1%, 51/1231), human coronavirus OC43 (hCoV-OC43) (3.4%, 42/1231), human coronavirus HKU1(hCoV-HKU1) (2.7%, 33/1231), human coronavirus NL63 (hCoV-NL63) (2.5%, 31/1231), parainfluenza 1 (hPIV1) (2.0%, 25/1231), parainfluenza 2 (hPIV2) (1.8%, 22/1231), human parechovirus (PeV) (1.1%, 14/1231), and human coronavirus 229E (hCoV-229E) (0.9%, 11/1231). Among SARI cases, infants aged 1-4 years were mostly affected (50.7%; 622/1231), followed by those <1 year of age (35.7%; 438/1231). Most detected pathogens had year-long circulation patterns, with seasonal peaks mainly observed during the cold and dry seasons. CONCLUSION: Several non-influenza viruses are cause of SARI in Burkina Faso. The integration of the most common pathogens into the routine influenza surveillance system might be beneficial.

Glyceraldehyde-3-Phosphate Dehydrogenase Binds with Spike Protein and Inhibits the Entry of SARS-CoV-2 into Host Cells.

INTRODUCTION: Coronavirus disease 2019 caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long-term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of spike (S) protein S1 subunit with angiotensin converting enzyme present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. METHOD: Recombinant S protein, the RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for flow cytometry-based infectivity assays. RESULTS: Pseudovirus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD of SARS-CoV-2 with nanomolar affinity. CONCLUSIONS: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.

Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells.

Viruses evolve many strategies to ensure the efficient synthesis of their proteins. One such strategy is the inhibition of the integrated stress response-the mechanism through which infected cells arrest translation through the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α). We have recently shown that the human common cold betacoronavirus OC43 actively inhibits eIF2α phosphorylation in response to sodium arsenite, a potent inducer of oxidative stress. In this work, we examined the modulation of integrated stress responses by OC43 and demonstrated that the negative feedback regulator of eIF2α phosphorylation GADD34 is strongly induced in infected cells. However, the upregulation of GADD34 expression induced by OC43 was independent from the activation of the integrated stress response and was not required for the inhibition of eIF2α phosphorylation in virus-infected cells. Our work reveals a complex interplay between the common cold coronavirus and the integrated stress response, in which efficient viral protein synthesis is ensured by the inhibition of eIF2α phosphorylation but the GADD34 negative feedback loop is disrupted.

Coronavirus takeover of host cell translation and intracellular antiviral response: a molecular perspective.

Coronaviruses are a group of related RNA viruses that cause respiratory diseases in humans and animals. Understanding the mechanisms of translation regulation during coronaviral infections is critical for developing antiviral therapies and preventing viral spread. Translation of the viral single-stranded RNA genome in the host cell cytoplasm is an essential step in the life cycle of coronaviruses, which affects the cellular mRNA translation landscape in many ways. Here we discuss various viral strategies of translation control, including how members of the Betacoronavirus genus shut down host cell translation and suppress host innate immune functions, as well as the role of the viral non-structural protein 1 (Nsp1) in the process. We also outline the fate of viral RNA, considering stress response mechanisms triggered in infected cells, and describe how unique viral RNA features contribute to programmed ribosomal -1 frameshifting, RNA editing, and translation shutdown evasion.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Sialoglycan binding triggers spike opening in a human coronavirus.

Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion1-5. Spike opening exposes domain S1B, allowing it to bind to proteinaceous receptors6,7, and is also thought to enable protein refolding during membrane fusion4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

Detection of Alpha- and Betacoronaviruses in Small Mammals in Western Yunnan Province, China.

The genetic diversity of coronaviruses (CoVs) is high, and their infection in animals has not yet been fully revealed. By RT-PCR detection of the partial RNA-dependent RNA polymerase (RdRp) gene of CoVs, we screened a total of 502 small mammals in the Dali and Nujiang prefectures of Western Yunnan Province, China. The number of overall CoV positives was 20, including ß-CoV (n = 13) and α-CoV (n = 7), with a 3.98% prevalence in rectal tissue samples. The identity of the partial RdRp genes obtained for 13 strains of ß-CoV was 83.42-99.23% at the nucleotide level, and it is worth noting that the two strains from Kachin red-backed voles showed high identity to BOV-36/IND/2015 from Indian bovines and DcCoV-HKU23 from dromedary camels (Camelus dromedarius) in Morocco; the nucleotide identity was between 97.86 and 98.33%. Similarly, the identity of the seven strains of α-CoV among the partial RdRp sequences was 94.00-99.18% at nucleotide levels. The viral load in different tissues was measured by quantitative RT-PCR (qRT-PCR). The average CoV viral load in small mammalian rectal tissue was 1.35 × 106 copies/g; differently, the mean CoV viral load in liver, heart, lung, spleen, and kidney tissue was from 0.97 × 103 to 3.95 × 103 copies/g, which revealed that CoV has extensive tropism in rectal tissue in small mammals (p < 0.0001). These results revealed the genetic diversity, epidemiology, and infective tropism of α-CoV and ß-CoV in small mammals from Dali and Nujiang, which deepens the comprehension of the retention and infection of coronavirus in natural hosts.

Assessment and sero-diagnosis for coronaviruses with risk of human spillover.

Zoonotic coronaviruses (CoVs) caused major human outbreaks in the last two decades. One of the biggest challenges during future CoV disease is ensuring rapid detection and diagnosis at the early phase of a zoonotic event, and active surveillance to the zoonotic high-risk CoVs appears the best way at the present time to provide early warnings. However, there is neither an evaluation of spillover potential nor diagnosis tools for the majority of CoVs. Here, we analyzed the viral traits, including population, genetic diversity, receptor and host species for all 40 alpha- and beta-CoV species, where the human-infecting CoVs are from. Our analysis proposed 20 high-risk CoV species, including 6 of which jumped to human, 3 with evidence of spillover but not to human and 11 without evidence of spillover yet, which prediction were further supported by an analysis of the history of CoV zoonosis. We also found three major zoonotic sources: multiple bat-origin CoV species, the rodent-origin sub-genus Embecovirus and the CoV species AlphaCoV1. Moreover, the Rhinolophidae and Hipposideridae bats harbour a significantly higher proportion of human-threatening CoV species, whereas camel, civet, swine and pangolin could be important intermediate hosts during CoV zoonotic transmission. Finally, we established quick and sensitive serologic tools for a list of proposed high-risk CoVs and validated the methods in serum cross-reaction assays using hyper-immune rabbit sera or clinical samples. By comprehensive risk assessment of the potential human-infecting CoVs, our work provides a theoretical or practical basis for future CoV disease preparedness.

Continuous evolution and emerging lineage of seasonal human coronaviruses: A multicenter surveillance study.

The seasonal human coronaviruses (HCoVs) have zoonotic origins, repeated infections, and global transmission. The objectives of this study are to elaborate the epidemiological and evolutionary characteristics of HCoVs from patients with acute respiratory illness. We conducted a multicenter surveillance at 36 sentinel hospitals of Beijing Metropolis, China, during 2016-2019. Patients with influenza-like illness (ILI) and severe acute respiratory infection (SARI) were included, and submitted respiratory samples for screening HCoVs by multiplex real-time reverse transcription-polymerase chain reaction assays. All the positive samples were used for metatranscriptomic sequencing to get whole genomes of HCoVs for genetical and evolutionary analyses. Totally, 321 of 15 677 patients with ILI or SARI were found to be positive for HCoVs, with an infection rate of 2.0% (95% confidence interval, 1.8%-2.3%). HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 infections accounted for 18.7%, 38.3%, 40.5%, and 2.5%, respectively. In comparison to ILI cases, SARI cases were significantly older, more likely caused by HCoV-229E and HCoV-OC43, and more often co-infected with other respiratory pathogens. A total of 179 full genome sequences of HCoVs were obtained from 321 positive patients. The phylogenetical analyses revealed that HCoV-229E, HCoV-NL63 and HCoV-OC43 continuously yielded novel lineages, respectively. The nonsynonymous to synonymous ratio of all key genes in each HCoV was less than one, indicating that all four HCoVs were under negative selection pressure. Multiple substitution modes were observed in spike glycoprotein among the four HCoVs. Our findings highlight the importance of enhancing surveillance on HCoVs, and imply that more variants might occur in the future.

Searching for common ground.

The conserved coronavirus fusion peptide is a target of broadly neutralizing antibodies.

Flies (Insecta, Diptera) collected in the environment of dairy farms as carriers of Rotavirus A and betacoronavirus.

AIMS: We aimed to investigate the prevalence of rotavirus and coronavirus in dipterans that commonly inhabit the environment of dairy farms. METHODS AND RESULTS: We collected 217 insect specimens from nine dairy farms, which were examined through hemi-nested RT-PCR followed by Sanger sequencing in search of VP1 and N genes for rotavirus and bovine coronavirus-BCoV, respectively. With a predominance of Muscidae (152/217 = 70%) 11 families of Diptera were identified. Rotavirus A (RVA) and betacoronavirus (BCoV) were detected in 14.7% (32/217) and 4.6% (10/217) of the dipterans, respectively. Sequencing of the amplicons was possible for 11.5% (25/217) of RVA and 0.5% (1/217) of BCoV, confirming the presence of these pathogens. CONCLUSIONS: Our findings highlight the role of dipterans as carriers of RVA and BCoV of great relevance for public and animal health.

Identification and Genetic Characterization of MERS-Related Coronavirus Isolated from Nathusius' Pipistrelle (Pipistrellus nathusii) near Zvenigorod (Moscow Region, Russia).

Being diverse and widely distributed globally, bats are a known reservoir of a series of emerging zoonotic viruses. We studied fecal viromes of twenty-six bats captured in 2015 in the Moscow Region and found 13 of 26 (50%) samples to be coronavirus positive. Of P. nathusii (the Nathusius' pipistrelle), 3 of 6 samples were carriers of a novel MERS-related betacoronavirus. We sequenced and assembled the complete genome of this betacoronavirus and named it MOW-BatCoV strain 15-22. Whole genome phylogenetic analysis suggests that MOW-BatCoV/15-22 falls into a distinct subclade closely related to human and camel MERS-CoV. Unexpectedly, the phylogenetic analysis of the novel MOW-BatCoV/15-22 spike gene showed the closest similarity to CoVs from Erinaceus europaeus (European hedgehog). We suppose MOW-BatCoV could have arisen as a result of recombination between ancestral viruses of bats and hedgehogs. Molecular docking analysis of MOW-BatCoV/15-22 spike glycoprotein binding to DPP4 receptors of different mammals predicted the highest binding ability with DPP4 of the Myotis brandtii bat (docking score -320.15) and the E. europaeus (docking score -294.51). Hedgehogs are widely kept as pets and are commonly found in areas of human habitation. As this novel bat-CoV is likely capable of infecting hedgehogs, we suggest hedgehogs can act as intermediate hosts between bats and humans for other bat-CoVs.