Epitopes screening and vaccine molecular design of SADS-CoV based on immunoinformatics.

The regional outbreak of the Swine acute diarrhea syndrome coronavirus (SADS-CoV) has seriously threatened the swine industry. There is an urgent need to discover safe and effective vaccines to contain them quickly. The coronavirus spike protein mediates virus entry into host cells, one of the most important antigenic determinants and a potential vaccine target. Therefore, this study aims to conduct a predictive analysis of the epitope of S protein B cells and T cells (MHC class I and class II) by immunoinformatics methods by screening and identifying protective antigenic epitopes that induce major neutralized antibodies and activate immune responses to construct epitope vaccines. The study explored primary, secondary, and tertiary structures, disulfide bonds, protein docking, immune response simulation, and seamless cloning of epitope vaccines. The results show that the spike protein dominant epitope of the screening has a high conservativeness and coverage of IFN-γ, IL-4-positive Th epitope, and CTL epitope. The constructed epitope vaccine interacts stably with TLR-3 receptors, and the immune response simulation shows good immunogenicity, which could effectively activate humoral and cellular immunity. After codon optimization, it was highly likely to be efficiently and stably expressed in the Escherichia coli K12 expression system. Therefore, the constructed epitope vaccine will provide a new theoretical basis for the design of SADS-CoV antiviral drugs and related research on coronaviruses such as SARS-CoV-2.

Acute Diarrhea in Adults

Acute diarrheal disease accounts for 179 million outpatient visits annually in the United States. Diarrhea can be categorized as inflammatory or noninflammatory, and both types have infectious and noninfectious causes. Infectious noninflammatory diarrhea is often viral in etiology and is the most common presentation;however, bacterial causes are also common and may be related to travel or foodborne illness. History for patients with acute diarrhea should include onset and frequency of symptoms, stool character, a focused review of systems including fever and other symptoms, and evaluation of exposures and risk factors. The physical examination should include evaluation for signs of dehydration, sepsis, or potential surgical processes. Most episodes of acute diarrhea in countries with adequate food and water sanitation are uncomplicated and self-limited, requiring only an initial evaluation and supportive treatment. Additional diagnostic evaluation and management may be warranted when diarrhea is bloody or mucoid or when risk factors are present, including immunocompromise or recent hospitalization. Unless an outbreak is suspected, molecular studies are preferred over traditional stool cultures. In all cases, management begins with replacing water, electrolytes, and nutrients. Oral rehydration is preferred;however, signs of severe dehydration or sepsis warrant intravenous rehydration. Antidiarrheal agents can be symptomatic therapy for acute watery diarrhea and can help decrease inappropriate antibiotic use. Empiric antibiotics are rarely warranted, except in sepsis and some cases of travelers' or inflammatory diarrhea. Targeted antibiotic therapy may be appropriate following microbiologic stool assessment. Hand hygiene, personal protective equipment, and food and water safety measures are integral to preventing infectious diarrheal illnesses.Copyright © 2022 American Academy of Family Physicians.

Role of heat shock protein 90 as an antiviral target for swine enteric coronaviruses.

A variety of swine enteric coronaviruses (SECoVs) have emerged and are prevalent in pig populations, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome (SADS)-CoV, a newly identified bat-origin CoV with zoonotic potential. Unfortunately, available traditional, inactivated and attenuated SECoV vaccines are of limited efficacy against the variants currently circulating in most pig populations. In this study, we evaluated the role of host factor heat shock protein 90 (Hsp90) as an antiviral target against SECoVs, exemplified by SADS-CoV. Pharmacological inhibition of Hsp90 diminished SADS-CoV replication significantly in porcine and human cell lines, and also decreased replication of SADS-CoV in a porcine intestinal enteroid model. Further mechanistic experiments revealed that both porcine and human isoforms of Hsp90 interact with the SADS-CoV nucleocapsid (N) protein, and inhibition of Hsp90 resulted in autophagic degradation of N protein. Moreover, we linked Hsp90 to virus-induced cellular pyroptosis, as SADS-CoV was found to trigger caspase-1/gasdermin-d-mediated pyroptotic cell death, which was mitigated by inhibition of Hsp90. Finally, we demonstrated that Hsp90 also associated with N proteins and was involved in propagation of PEDV, PDCoV and TGEV. This study thus extends our understanding of immune responses to SADS-CoV infection and offers a new potential therapeutic option against four SECoVs.

The origin and evolution of emerged swine acute diarrhea syndrome coronavirus with zoonotic potential.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered alphacoronavirus with zoonotic potential that causes diarrhea and vomiting mainly in piglets. Having emerged suddenly in 2017, the prevailing opinion is that the virus originated from HKU2, an alphacoronavirus whose primary host is bats, and at some unknown point achieved interspecies transmission via some intermediate. Here, we further explore the evolutionary history and possible cross-species transmission event for SADS-CoV. Coevolutionary analysis demonstrated that HKU2 may have achieved host switch via SADS-related (SADSr)-CoV, which was isolated from the genus Rhinolophus in 2017. SADS-CoV, HKU2, and SADSr-CoV share similar codon usage patterns and showed a lower tendency to use CpG, which may reflect a method of immune escape. The analyses of virus-host coevolution and recombination support SADSr-CoV is the direct source of SADS-CoV that may have undergone recombination events during its formation. Structure-based spike glycoprotein variance analysis revealed a more nuanced evolutionary pathway to receptor recognition for host switch. We did not find a possible positive selection site, and the dN/dS of the S gene was only 0.29, which indicates that the current SADS-CoV is slowly evolving. These results provide new insights that may help predict future cross-species transmission, and possibly surveil future zoonotic outbreaks and associated public health emergencies.

Preparation of a new monoclonal antibody against nucleocapsid protein of swine acute diarrhea syndrome coronavirus and identification of its linear antigenic epitope.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), which causes severe diarrhea in newborn piglets, was first identified in Southern China in 2017. Since the Nucleocapsid (N) protein in SADS-CoV is highly conserved and plays a key role in virus replication, it is often used as a target protein in scientific research. In this study, the N protein of SADS-CoV was successfully expressed, and a new monoclonal antibody (mAb), 5G12, against the protein was generated successfully. The mAb 5G12 can be used to detect SADS-CoV strains by indirect immunofluorescence assay (IFA) and western blotting. The mAb 5G12 epitope was located to amino acids 11 EQAESRGRK 19 by evaluating the antibody for reactivity with a series of truncated N protein segments. The biological information analysis showed that the antigenic epitope had a high antigenic index and conservation. This study will help further understand the protein structure and function of SADS-CoV and in the establishment of specific SADS-CoV detection methods.

Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1 ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. KEY POINTS: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread.

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope.

The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.

Molecular investigation of Campylobacter spp. and salmonella infection in children with community acquired diarrhea

Introduction: Acute gastroenteritis is a typical disorder that accounts for 8-12% of pediatric outpatient visits. Campylobacter and Salmonella infections account for about 8.4% and 11% of global diarrhea cases. Due to the importance of these bacteria in pediatric diseases, the aim of this study was to determine the infectious rate of Salmonella and Campylobacter species and also the frequency of the gene encoding Cytholethal distending toxin in children with community-acquired diarrhea. Material(s) and Method(s): Stool samples of children under 5 years of age with diarrhea were collected. The samples were related to children referred to hospitals in Hamadan, Ardabil, Bandar Abbas and two hospitals in Tehran. DNA was extracted from the samples using a DNA extraction kit from stool. The presence of Campylobacter in the studied samples was detected by polymerase chain reaction using specific primers. A control stool sample was spiked with 10-fold dilution of C. jejuni suspension for LOD (detection limit determination) measurement. Result(s): In this study, PCR results showed a LOD of 100 CFU per gram in the spiked feces sample. Accordingly, out of 144 fecal samples of children with acute diarrhea, one case was positive for Campylobacter jejuni;this sample was also positive for the presence of cdtB gene. Presence of Salmonella was confirmed in two samples of the patients (1.4%). Conclusion(s): Low prevalence of Campylobacter and Salmonella was detected in symptomatic children under 5 years of age during the Covid-19 pandemic. Examination of these samples for viruses and other microbial agents can clarify the etiology of diarrhea in children referred to the hospitals. Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.

Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses.

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV M gene, PDCoV N gene, TGEV S gene and SADS-CoV RdRp gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 105 copies/µL, 6.48 × 105 copies/µL, 8.54 × 105 copies/µL and 7.79 × 106 copies/µL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

Post-Covid Gastrointestinal Sequelae 7 Months after Acute Sars-Cov-2 Infection

Introduction: During 3rd year of COVID-19 pandemic there is emerging published and experienced evidence that a significant number of COVID-19 survivors experience long term negative health impact. Post-COVID symptoms arise mostly from impaired function of respiratory tract although in many patients the dysfunction of gastrointestinal tract and liver (among others systems) may persist. Aims & Methods: This prospective single center study was performed from February through October 2021. Adult patients (>18 years) with both asymptomatic PCR confirmed acute SARS-CoV-2 infection and symptomatic (cough, fever or dyspnea) COVID-19 disease patients confirmed by antigen or PCR test were included in the study. Severity of the illness was evaluated according to the National Institute of Health (NIH) guidelines and these were used for distinguishing two main patient groups (with asymptomatic to mild and moderate to severe course of the disease). Patients with negative result of PCR test for SARS-CoV-2 were considered for the control group. Primary data collection was based on the short gastrointestinal symptom questionnaire developed at our department. A brief telephone survey within the patient and control group was performed five to eight months after the initial screening. Patients from the control group, who had been tested positive in the time between initial screening and telephone survey were excluded from the analysis.R ver. 4.0.5 was used for data explorations and analyses. Imbalanced RandomForest (RF) machine-learning algorithm was trained on the data to predict presence/absence of symptoms during acute phase in post-COVID phase. Result(s): 590 patients were included in the study. Of them, 337 patients responded to questions during the telephone survey after 7 months (205 SARS-CoV-2 positive patients, 132 SARS-CoV-2 negative controls). The general presence of gastrointestinal symptoms 208.2 days (153-230 days) after the initial acute SARS-CoV-2 infection was 19% for patients with moderate to serious course of the disease and 7.3% for patients with mild course compared to 3.0% in SARS-CoV-2 negative controls (p<0.001) (table). Persistent diarrhea and abdominal pain are the most prevalent post-COVID gastrointestinal symptoms. To identify possible predictor factors for post COVID gastrointestinal symptoms we performed machine-learning analysis with random forest algorithm with handpicked factors: acute symptoms (diarrhea, abdominal pain, heartburn, bloating, nausea), sex, age and antibiotics administration. Acute diarrhea and antibiotics administration seem to be the strongest predictors for gastrointestinal sequelae with Area under curve (AUC) 0.68. Conclusion(s): Presence of gastrointestinal sequelae 7 months after the initial SARS-CoV-2 infection is significantly higher in patients with moderate to severe course of the acute COVID-19 compared to asymptomatic patients or those with mild course of the disease. Most prevalent post-COVID gastrointestinal symptoms are diarrhea and abdominal pain. The strongest predictors for persistence of these symptoms are antibiotics administration and acute diarrhea during the initial infection.

Unraveling the role of calcitonin gene-related peptide b in gastrointestinal disorders: Implications for migraine

Introduction: Calcitonin gene-related peptide (CGRP) a is well-known to be implicated in migraine pathophysiology. Its b isoform, released mainly in the enteric nervous system, has not been as extensively studied. Previous research prompt CGRP has a role in gastrointestinal motility, as well as immune and intestinal blood flow regulation. It is postulated to help peptic ulcer healing, and immune cell migration and regulation in acute gastrointestinal infections. In COVID-19 between 2% and 50% of patients develop diarrhea, and its prevalence increases with the severity of the disease. The pathophysiology of the diarrhea in this infection is not completely clear but CGRP has been proposed to play a role in different aspects of the symptomatology. Inflammatory bowel disease (IBD), known to be associated with migraine, is a chronic gastrointestinal autoimmune disease. Neuropeptides like CGRP might play a role in the complex pathophysiology of the disease, but this has not been well established yet. On the other hand, the most frequent adverse event of new monoclonal antibodies against CGRP for migraine is constipation, which points out that the blockage may affect CGRPbeta release. Objective(s): To assess the role of CGRPbeta in two gastrointestinal disorders: COVID-19 with acute diarrhea;and IBD. Method(s): CGRPbeta were measured by ELISA (CUSABIO, China) in early morning serum samples in patients with IBD at diagnosis, as well as in COVID-19 inpatients experiencing diarrhea. We compared each group with a cohort of healthy controls matched by age and sex. Image: Results: Twenty-six COVID-19 inpatients with diarrhea were included (mean age=62+/-16 years, range 31-91 years;69.2% females) who were matched with 30 healthy controls (mean age=61+/-15 years, range 29-89 years, 66.6% women). Fifty-nine patients with early IBD (mean age 48.9+/-16.4 years, range 21-79 years;62,7% females were matched with 59 healthy controls (mean age 49.0+/-14.9 years, range 23-77 years;62,7% females). While CGRPbeta levels were significantly elevated in COVID-19 patients (6,3+/-2.6 pg/mL) vs controls (4.2+/-2.4 pg/mL) (+26.2%;p<0.01), CGRPbeta levels in patients with IBD were significantly decreased (3.1+/-1.8 pg/mL) as compared to controls (4.8+/-2.6 pg/mL) (-35.4%, p<0.001). Conclusion(s): CGRPbeta seems to exert different actions depending on the underlying conditions. While its increase with diarrhea in COVID-19 patients fits very well with the known acute effects of increase gastrointestinal motility in CGRP infusion in volunteers, the decrease in CGRPbeta levels in IBD confirm a protective role of this peptide in the homeostasis of the intestinal mucosa. These findings may help to explain the role of CGRPbeta in digestive manifestations of migraine and in the constipation seen in migraine patients on CGRP antibodies as well.

A Case of Eosinophilic Colitis after BNT162b2 mRNA COVID-19 Vaccination

Background: BNT162b2 is a widely used mRNA COVID-19 vaccine for which 8.2% of participants above the age of 56 years have reported diarrhea as an adverse event. This case report highlights the possibility of eosinophilic colitis in post-vaccination diarrhea. Case report: A 72-year-old male patient presented with generalized colicky abdominal pain and acute diarrhea after receiving the first dose of the BNT162b2 vaccine. Laboratory examination revealed peripheral blood eosinophilia with cecal and ascending colon mucosal eosinophilia with 100-130 cells/HPF and eosinophilic cryptitis. The patient's symptoms and eosinophilia resolved spontaneously and did not recur after the second dose of vaccination. More research is needed to confirm eosinophilic colitis as a possible vaccine adverse reaction. Copyright © 2022 Selva Yuwaraj Vadioaloo et al., published by Sciendo.

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV).

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.

Emodin from Aloe inhibits Swine acute diarrhea syndrome coronavirus in cell culture.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteropathogenic coronavirus that causes severe diarrhea in neonatal piglets, leading to serious economic losses to the pig industries. At present, there are no effective control measures for SADS, making an urgent need to exploit effective antiviral therapies. Here, we confirmed that Aloe extract (Ae) can strongly inhibit SADS-CoV in Vero and IPI-FX cells in vitro. Furthermore, we detected that Emodin from Ae had anti-SADS-CoV activity in cells but did not impair SADS-CoV infectivity directly. The time-of-addition assay showed that Emodin inhibits SADS-CoV infection at the whole stages of the viral replication cycle. Notably, we found that Emodin can significantly reduce virus particles attaching to the cell surface and induce TLR3 (p < 0.001), IFN-λ3 (p < 0.01), and ISG15 (p < 0.01) expressions in IPI-FX cells, indicating that the anti-SADS-CoV activity of Emodin might be due to blocking viral attachment and the activation of TLR3-IFN-λ3-ISG15 signaling axis. These results suggest that Emodin has the potential value for the development of anti-SADS-CoV drugs.

The burden of SARS-CoV-2 infection among children in Lagos, Nigeria

Introduction/ Background: Most reports are silent on the burden of COVID-19 in paediatric population. Is the incidence and severity of COVID-19 truly low as reported? The objective of this study was to describe the prevalence and clinical features of coronavirus disease among children at a COVID-19 testing centre in Lagos, Nigeria. Methods: This is a retrospective study describing the prevalence and pattern of symptoms of COVID-19 among children at a testing centre in Lagos, Nigeria. Data was collected from the electronic records of individuals (< 18 years) visiting the SARS-CoV-2 testing centre from March 1, 2020, to June 30, 2020. The sociodemographic data, travel history, preexisting comorbidities, clinical symptoms, and outcomes of the qualitative (SARS-CoV-2) RT-PCR results were extracted and analyzed using SPSS software, version 22.0. Results: Three hundred and seven children were screened for SARS-CoV-2 infection and fifty children were found to be positive, giving a prevalence of 16.3%. The median age (interquartile range (IQR)) was 9 (4- 14) years. Seventeen (34.0%) of the SARS-CoV-2- positive children presented with symptoms while the rest were asymptomatic. The common presenting symptoms among those positive for SARS-CoV-2 infection were fever (40.0%), cough (32.9%), sore throat (17.1%), and runny nose (15.7%). Fever and sore throat occurring together was the symptom combination most predictive of SARS-CoV- 2 infection among the population (OR 0.49,95% CI: 0.12-1.98 and OR 4.59 95% CI 0.93-22.73). Impact: This is the first study to describe the epidemiological and clinical characteristics of children screened for SARS-CoV-2 infection in sub-Saharan Africa. The pattern of symptoms seen in SARS-CoV-2 infections among children is similar to common childhood infections (malaria, acute diarrhea diseases, and respiratory tract infection). Conclusion: Prevalence of paediatric SARS-CoV-2 infection was 16.3%, but majorly of mild disease and was commonly predicated by fever and sore throat symptom combination. There is a need for a high level of suspicion in the management of common febrile diseases in paediatric settings especially in presence of sore throat.

ATYPICAL PRESENTATION OF LEGIONNAIRES DISEASE WITH DIVERTICULITIS IN IMMUNOSUPPRESSED PATIENT

CASE: A 69-year-old male smoker with stage 3b prostate cancer managed with abiraterone and prednisone, prior severe COVID-19 pneumonia requiring mechanical ventilation, and history of perforated sigmoid diverticulitis presented with 3 days of anorexia, watery diarrhea, and left lower abdominal pain. Two weeks earlier he developed a mild dry cough without fever, dyspnea, or chest pain. There were no sick contacts or recent travel. He was afebrile, and initial routine chemistries and a complete blood count were unremarkable. An abdomino-pelvic CT revealed acute diverticulitis of the distal descending and sigmoid colon. A consolidation at the right lung base was also incidentally noted. Follow up imaging confirmed a multifocal pneumonia on chest Xray. Legionella antigen was detected in the urine. Metronidazole and levofloxacin were initiated with clinical improvement and the patient was discharged home to complete a 10-day course of antibiotics IMPACT/DISCUSSION: Legionella bacteria are gram negative organisms found widespread in soil and bodies of water including lakes, streams, and artificial reservoirs. Transmission is via inhalation of aerosols and a high innoculum is typically needed to cause infection. Host risk factors for infection include older age, impaired cellular immunity, smoking, male sex, and medical co-morbidities such as diabetes mellitus, renal, lung and cardiovascular disease. The two most commonly known syndromes associated with Legionella infection are Legionnaire's disease, a pneumonia occurring typically in the late summer or early autumn months (as in our patient), and Pontiac fever, an acute self- limited febrile illness. The mortality rate for hospitalized Legionnaire's is up to 10%. Extra-pulmonary manifestations are rare and can include skin and soft tissue infections, septic arthritis, endocarditis, myocarditis, peritonitis, pyelonephritis, meningitis, brain abscesses, and surgical site infections. The diagnosis of extra-pulmonary disease requires detection of Legionella at the affected site by culture or polymerase chain reaction. In the absence of a known local Legionella outbreak, our patient's age, sex, smoking status, and underlying immune suppression most likely increased his risk for this sporadic infection. We postulate that the acute diarrhea associated with Legionnaire's disease may have triggered inflammation of his diverticula or the acute diverticulitis was an extra-pulmonary manifestation. To our knowledge, we are the first to report a case of Legionnaire's disease presenting as acute diverticulitis. CONCLUSION: Legionnaire's is a typical disease with many atypical and extra-pulmonary presentations. We present a case of Legionnaire's disease masquerading as acute diverticulitis and urge timely consideration and testing for Legionella in at-risk patients presenting with predominantly GI symptoms and subtle or no respiratory complaints, as it can be life-saving.

Furin cleavage is required for swine acute diarrhea syndrome coronavirus spike protein-mediated cell - cell fusion.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was reported in China in 2017 and is a causative agent of porcine enteric disease. Recent studies indicate that cells from various hosts are susceptible to SADS-CoV, suggesting the zoonotic potential of this virus. However, little is known about the mechanisms through which this virus enters cells. In this study, we investigated the role of furin in SADS-CoV spike (S)-mediated cell - cell fusion and entry. We found that the SADS-CoV S protein induced the fusion of various cells. Cell - cell fusion was inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, and between cells transfected with mutant S proteins resistant to furin cleavage. These findings revealed that furin-induced cleavage of the SADS-CoV S protein is required for cell - cell fusion. Using mutagenesis analysis, we demonstrated that furin cleaves the SADS-CoV S protein near the S1/S2 cleavage site, 446RYVR449 and 543AVRR546. We used pseudotyped viruses to determine whether furin-induced S cleavage is also required for viral entry. Pseudotyped viruses expressing S proteins with a mutated furin cleavage site could be transduced into target cells, indicating that furin-induced cleavage is not required for pseudotyped virus entry. Our data indicate that S cleavage is critical for SADS-CoV S-mediated cell - cell fusion and suggest that furin might be a host target for SADS-CoV antivirals.