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Introduction: Currently, isolated from SARS-CoV-2 virus exceed 600 million cases in the world. Objective(s): Isolation and characterization of the SARS-CoV-2 virus causing COVID-19 at the beginning of the pandemic in Peru. Method(s): Twenty nasal and pharyngeal swab samples were isolated from SARS-CoV-2 using two cell lines, Vero ATCC CCL-81 and Vero E-6;virus identification was performed by RT-PCR and the onset of cytopathic effect (CPE) was evaluated by indirect immunofluorescence and subsequent identification by genomic sequencing. One of the most widely circulating isolates were selected and named the prototype strain (PE/B.1.1/28549/2020). Then 10 successive passages were performed on Vero ATCC CCL-81 cells to assess mutation dynamics. Result(s): Results detected 11 virus isolates by cytopathic effect, and subsequently confirmed by RT-PCR and indirect immunofluorescence. Of these, six were sequenced and identified as the lineages B.1, B.1.1, B.1.1.1, and B.1.205 according to the Pango lineage nomenclature. The prototype strain corresponded to lineage B.1.1. The analysis of the strains from the successive passages showed mutations mainly at in the spike (S) protein of the virus without variation in the identity of the lineage. Conclusion(s): Four lineages were isolated in the Vero ATCC CCL-81 cell line. Subcultures in the same cell line showed mutations in the spike protein indicating greater adaptability to the host cell and variation in pathogenicity in vitro, a behavior that allows it to have more survival success.Copyright © 2023 Anales de la Facultad de Medicina. All rights reserved.
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The re-emergence of SARS-CoV, known as SARS-CoV-2, has proven extremely infectious that has infected a huge population worldwide. SARS-CoV-2 genome is translated into polyproteins that is processed by virus-specific protease enzymes. 3CLprotease is named as the main protease (Mpro) enzyme that cleaves nsp4 to nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. Till date, there is no effective commercially available drug against COVID-19 and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. We have used a high-throughput virtual screening approach to examine FDA approved library, natural compound library, and LOPAC 1280 (Library of Pharmacologically Active Compounds, Sigma-Aldrich, St. Louis, MO) library against Mpro. Primary screening identified potential drug molecules for the target, among which ten molecules were studied further using biophysical and biochemical techniques. SPR was used to validate the binding of inhibitors to purified Mpro and using FRET-based biochemical protease assay these inhibitors were confirmed to have Mpro inhibitory activity. Based on the kinetic studies, the antiviral efficacy of these compounds was further analysed by cell-culture based antiviral assays. Four out of ten molecules inhibited SARS-CoV-2 replication in Vero cells at a concentration range of 12.5 to 50 muM. The antiviral activity was evaluated by RT-PCR assay and TCID50 experiments. The co-crystallization of Mpro in complex with inhibitor for determining their structures is being carried out. Collectively, this study will provide valuable mechanistic and structural insights for development of effective antiviral therapeutics against SARS-CoV-2.
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This retrospective case-series analysis evaluated 403 fully vaccinated with Vero Cell or Sputnik V vaccines patients hospitalized in the 6th City Clinical Hospital of Minsk in the period between January 01 and February 28, 2022 with the main diagnosis of "coronavirus infection (COVID-19)". The diagnosis was confirmed by PCR or SARS-CoV-2 virus antigen tests, as well as chest computed tomography data. The study revealed higher prevalence of older patients (over 65 years) infected with the SARS-CoV-2 virus and hospitalized in early 2022, at the height of the wave of the pandemic due to the spread of the Omicron variant. Most patients (91.8 %) had moderate symptoms. More than half of them received oxygen support. A relatively small number of inpatient, only 8 persons (1.9 %), were hospitalized in the intensive care unit (ICU) and four of them needed mechanical ventilation. Comor-bid conditions and high incidence of mortality (63.5 %) were common in ICU patients. Hypertension and obesity prevailed in the structure of comorbid pathology of all inpatient persons (74.2 and 24.3 %, respectively). Patients of therapeutic departments had relatively short length of stay in the hospital, as well as low in-hospital mortality (0.5 %) and low incidence of complications (5.3 %).Copyright © 2023 The authors.
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Background: With the COVID-19 vaccine now available, there have been occasional reports of post-vaccination neurological complications. Case presentation: In this report, we present a case of neuromyelitis optica spectrum disorder (NMOSD) that developed one month after the patient received the second dose of BIBP COVID-19 vaccine (SARS-CoV-2-Vaccine [Vero Cell] Inactivated). The patient presented with itching, numbness in the hand and right side of the face, as well as nausea, vomiting, and hiccups. Brain MRI revelead lesions in the area postrema, medulla, and bilateral hypothalamus, which are typical of NMOSD. Serum antibodies to anti-AQP4 and anti-MOG were negative. Conclusion(s): The pathogenesis of NMOSD development after vaccination is still unknown. NMOSD is generally aggressive and disabling, it is important for the neurologist to be attentive to the highly variable clinical presentation after COVID-19 vaccination for early diagnosis and effective treatment.Copyright © 2023
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Objective: This study aims to identify the adverse events of COVID-19 vaccine in PD patients including the general and specific PD-related side effects. Background(s): SARS-CoV-2 is one of the most contagious and invasive respiratory viruses emerging in the past 3 years and causing serious, life-threatening diseases and sequelae (long COVID-19 syndrome) [1]. Before the development of COVID-19 vaccine, morbidity and mortality of COVID-19 infection were as high as 1% and increased with age and co-morbidity, especially in more advanced PD. The safety of COVID-19 vaccine was approved in the general population, but no study of side effects in PD. [1] There were only 2 case reports of worsening PD symptoms after the mRNA-1273 vaccine (Spikevax) and BNT162b2 mRNA vaccine (Comirnaty). [2],[3] Methods: The study was conducted between July 2021 - January 2022. The online survey was conducted during COVID - 19 lockdown. 230 PD patients responded to the online survey, and 151 patients were included, 11 patients were excluded due to being unvaccinated. 72 patients were excluded due to incomplete responses. 5 patients were excluded due to multiple entries. Result(s): The most common COVID -19 vaccine in our PD patients was ChAdOx1-S vaccine and inactivated COVID-19 Vaccine (Vero Cell). 21.9% (n=33) of the patients had reported adverse effects of COVID-19 vaccination. The most common side effects are fever (60.6%), and headache (48.5%). These adverse effects occurred mostly on the 1st day after vaccination and rarely persisted for more than 7 days. The most reported were worsening bradykinesia (24.2 %) and insomnia (15.2%). We found no statistically significant difference in side effect and PD-related symptoms between ChAdOx1-S vaccine and inactivated COVID-19 vaccine. Conclusion(s): According to the benefits and risks of COVID-19 vaccines do not appear to be different than in the general population, we recommend the approved COVID-19 vaccination to all PD patients. Some symptoms should be observed in the severe motor fluctuation PD, especially bradykinesia because they might experience more motor fluctuation phenomena. If the symptoms do not disturb the activity of daily living, we can wait and see because most of them can spontaneously improve. Further study with mRNA and protein subunit type of vaccines should be done. (table1)(table2)(table3).
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Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
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Background: There is increasing evidence for aerosol-based transmission of SARS-CoV-2, with particulate matter (PM) a possible vector. Air surveillance is necessary to safeguard public spaces. Aim(s): To characterise SARS-CoV-2 distribution in aerosols collected in hospital and public spaces and determine best sampling methods for surveillance. Method(s): Over 8 months in 2021, 8 samplers collected liquid bioaerosols and size-fractioned particulate matter(PM) in hospitals (ICU, Respiratory ward and communal waiting areas), a London railway and underground station, a university, and a primary school. RNA was extracted from samples and RT-qPCR targeting the N-gene of SARSCoV-2 was performed. Samples were cultured on Vero cells. Result(s): 209 air samples were obtained with 20 positive for SARS-CoV-2. 15 positive samples were from hospitals, 10 from outpatient waiting areas (ED waiting area, Chemotherapy Day Unit), 2 of which had the B.1.1.7 mutation (alphavariant) on sequencing, and 5 positive samples from rooms housing SARS-CoV-2 positive patients on ICU and respiratory wards. 5 positive samples were obtained via a portable sampler on two separate journeys in a London underground carriage. SARS-CoV-2 was detected mostly in PM samplers (n=17) compared to liquid bioaerosol samplers (positive sample pick-up 13% vs 4%respectively), in fine particles <=2.5mum(PM2.5) in diameter (n=14). No samples were cultured on Vero cells. Conclusion(s): Size-fractioned particulate matter samplers may be more efficient than liquid bioaerosol samplers in detecting and monitoring SARS-CoV-2 in the air. SARS-CoV-2 is most detected on fine particles, giving support to PM2.5 acting as a vector for aerosol-based transmission.
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Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment. The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection. Material(s) and Method(s): the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung-body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann-Whitney test. Result(s): RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells;the EC50 values of RPH-137 amounted to 4.69 mug/mL (21.3 nM) and 16.24 mug/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 mug/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection. Conclusion(s): both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.
ABSTRACT
Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment. The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection. Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung-body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test. Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells;the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection. Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.
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Objetivos: Avaliar as taxas de seroconversao apos duas doses da vacina contra SARS-CoV-2 em pacientes com Leucemia Mieloide Cronica (LMC). Material e Metodos: Foram coletadas amostras para teste sorologico de triagem para avaliacao da presenca de anticorpos IgG (CMIA, SARS-CoV-2 IgM, IgG - Alinity System, Abbott Laboratories, Ireland) no periodo de 1-3 meses apos duas doses de vacina para COVID-19. Nas amostras positivas, foi realizada analise quantitativa de IgG (anti-S1 - SARS-CoV-2 IgG II Quant, Alinity System, Abbott Laboratories, Ireland) e os Titulos de Anticorpos Neutralizantes (TAN), que detectam o efeito citopatico do virus em cultura celular induzidos pelas vacinas (Vero CCL-81 cells). Resultados: Entre agosto e novembro de 2021, foram avaliados 102 pacientes com LMC com media de idade de 56,2 anos (33-85), sendo 58,8% do sexo masculino, 98,5% em Fase Cronica (FC), 80% apresentavam ao menos Resposta Molecular Maior (RMM). 87% dos pacientes estavam em uso de ITQ e 13% estavam em descontinuacao da medicacao. 66,7% receberam a vacina ChAdOx1 nCoV-19 (AZD1222)-Covishield (Oxford/AstraZeneca/Fiocruz), 29,41% CoronaVac (Sinovac/Butatan), 1,96% BNT162b2 (Pfeizer/BioNTech/Fosun Pharma) e 1,96% Ad26.COV2.S (Janssen-Cilag). 15% dos pacientes apresentaram COVID-19 antes da vacinacao. O teste sorologico de triagem (CMIA) foi positivo em 25% dos pacientes. COVID-19 previa foi associada a presenca de anticorpos IgG (p<=0,001). Os TAN foram maiores que 1:320 em 13/26 casos, dentre os quais 5 haviam apresentado COVID-19 antes de completar o esquema vacinal. Neste grupo, 76% receberam a vacina ChAdOx1 nCoV-19, 19% Coronavac e 1% BNT162b2. Dentre os casos com infeccao previa pelo SARS-CoV-2, 7 apresentaram confirmacao laboratorial e 2 tinham quadro clinico sugestivo. Oito casos ocorreram antes da vacinacao e um paciente apresentou quadro leve, apos ter recebido a primeira dose da vacina ChAdOx1 nCoV-19. Onze pacientes estavam em tratamento com Imatinibe, 6 com Dasatinibe e um com Nilotinibe (6 com RMM), 5 em terceira ou quarta linha (sem RMM) e 3 pacientes em descontinuacao de ITQ. A proporcao de pacientes com TAN >1:320 foi superior no grupo que recebia terceira ou quarta linha (p=0,022). Entretanto, neste grupo havia mais pacientes com infeccao previa por COVID-19. Nao houve diferenca estatistica entre as taxas de seroconversao (CMIA, IgM and IgG) entre pacientes que receberam Coronavac ou ChAdOx1 nCoV-19. Nao houve diferencas nas taxas de seroconversao entre pacientes que receberam ITQ e naqueles em descontinuacao (p=0,77). Discussao: Estudos observacionais demonstraram que pacientes com LMC-FC produzem anticorpos em niveis semelhantes a populacao geral, apos receberem as vacinas ChAdOx1 nCoV-19 ou BNT162b2. No estudo conduzido por Rotterdam et al., 100/101 pacientes apresentaram seroconversao apos duas doses. Outro estudo avaliou as taxas de conversao em pacientes que receberam Coronavac, demonstrando menores taxas de conversao se comparados a vacina BNT162b2. Nosso estudo demonstrou que a resposta sorologica apos duas doses de vacina contra SARS-CoV-2 foi menor do que a descrita previamente na literatura. Conclusoes: Nao foram demonstradas diferencas entre os tipos de vacina (Coronavac vs. ChAdOx1 nCoV-19) ou em relacao a fase da doenca na taxa de seroconversao. Duas doses de vacinas para COVID-19 foram insuficientes para imunizacao adequada em pacientes com LMC. Agradecimentos: Brazilian National Council for Scientific and Technological Development (CNPq), grant ndegree 401977/2020-0. Copyright © 2022
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Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.
Subject(s)
Herpes Simplex , Herpesviridae Infections , Herpesviridae , Herpesvirus 1, Human , Humans , CRISPR-Cas Systems/genetics , Herpesvirus 1, Human/genetics , Herpes Simplex/genetics , Herpesviridae Infections/genetics , CRISPR-Associated Protein 9/geneticsABSTRACT
Purpose: To determine if IgM has a direct effect in preventing SARS-CoV-2 replication in Vero E6 cells, and delaying or preventing disease in infected K18- hACE2 mice. Method(s): 1) Vero E6 cells, grown to confluence in 12 well plates, were used to test the effect of IgM in reducing the number of plaque-forming units (PFU).There were 4 groups: a) 25PFU WA-1 SARS-CoV-2 was combined with 20, 5 or 0.8mug IgM in growth medium, and incubated for 1hr in a final volume of 500ul. 100mul was added to Vero E6 cells in replicate wells and incubated for 1hr;b) 100mul of 20, 5 or 0.8mug IgM was added to Vero E6 cells and incubated. Media was aspirated and the cells were then inoculated with 25PFU WA-1 and incubated for 1hr;c) Virus control - as above, but with no IgM;d) No virus or IgM. FBS growth medium containing Avicel was overlain in the wells and incubated for 48 hours. Virus replication was stopped by incubating with 10% buffered formalin. Following removal of formalin, plates were stained with Giemsa violet, dried, and photographed. 2) A COVID -19 Spike- ACE2 binding assay kit was used to determine if IgM (2ug, 4.5ug, 20ug, 45ug IgM) inhibits the interaction between the Spike-receptor binding domain (S-RBD) and Angiotensin I ConvertingEnzyme 2 (ACE2) receptor. 3) K18-hACE2 mice were divided into 3 groups based on treatment regimen;Group 1: with IgM, No virus;2: with Saline, with virus;3: with IgM, with virus. 35ug IgM was injected intraperitoneal in a single dose, 2 days prior to infection. Mice were innoculated intranasally with 1250 pfu of HK SARS-CoV-2. Result(s): 1) Exposure of 25PFU SARS-CoV-2 to IgM (at all concentrations) prior to incubation with Vero E6 cells, inhibited its replication in Vero E6 cells. When Vero E6 cells were incubated with IgM prior to infection, no plaques were seen in wells with 20ug and 5ug IgM but were observed in wells with 0.8ug IgM. Plaques were also observed in the Virus alone group, but none were seen in the 'No IgM-No virus' group. 2) 45ug IgM/100uls inhibited the binding of S-RBD to ACE2 by ~94-100%, 20ug IgM/100uls inhibited it by ~80%, and 2 or 4.5ug/100ul by ~70-75%. Control without IgM did not inhibit the S-RBD-ACE-2 binding. 3) Pretreatment with a single low dose IgM injection delayed weight loss and mortality. Conclusion(s): IgM inhibits the replication of SARS-CoV-2 in Vero cells in vitro. It also inhibits the interaction between S-RBD that is present on the viral surface and the ACE2 receptor, by binding to S-RBD. A single low dose of IgM given prechallenge delayed disease in infected mice. The discovery that IgM interferes with the formation of the S-RBD-ACE2 complex, and that a single low dose can delay disease, indicates its translational potential as a vaccine/therapeutic to prevent or treat COVID-19.
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Covid-19 was mainly treated by a broad-spectrum antiviral called Remdesivir. A truncated cone molecular structure of Hydroxypropyl-ß-cyclodextrin can enhance the solubility and cellular uptake of the poorly soluble drug's through biological membranes. This study aimed to synthesize, characterize, observe cellular uptake and evaluate the cytotoxicity of remdesivirhydroxypropyl-ß-cyclodextrin (RDV-HPßCD) inclusion complex. The RDV-HPßCD inclusion complex was synthesized by the solvent evaporation method. Furthermore, the inclusion complex characteristic was evaluated by ultraviolet-visible (UV-Vis) spectrophotometry;particle size analyzer (PSA);Fourier infrared spectrophotometry (FTIR);X-ray diffraction (XRD);and differential scanning calorimetry (DSC). Further, fluorescence microscopy was used to evaluate the cellular uptake and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used in the cytotoxicity study. In the UV-Vis spectrum, both the inclusion complex and pure remdesivir showed a maximum peak at 246 nm. The inclusion complex has a particle size of 1697 ± 738.02 nm with -22.4 ± 1.58 mV of zeta potential. Shifted FTIR spectrum, broad XRD peak, and broad DSC thermogram peak at 72.93 °C indicated the successful formation of the RDV-HPßCD inclusion complex. Furthermore, cellular uptake observation of RDV-HPßCD inclusion complex conjugated to FITC showed better intensity inside the Vero cell than pure remdesivir conjugated to FITC. Further, Inclusion complex showed higher cell viability than pure remdesivir at a certain concentration.
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Since 2019, the world is experiencing a COVID-19 pandemic period caused by the new betacoronavirus SARS-CoV-2. This pathogen has led more than millions of people to death and many questions about the molecular mechanisms of interactions with the host cell are still unanswered. Viral entry and egress are important steps for the virus cycle. Although SARS-CoV-2 internalization has been largely studied, the egress steps of SARS-CoV-2 are still not fully described. In this study, we address the morphological characteristics of SARS-CoV-2 morphogenesis and egress by transmission and high-resolution scanning microscopy, with the aim of adding more information about the route of nascent virions towards the extracellular medium. Our results reinforce the role of small secretory vesicles as a vehicle to the individual egress, which could be the predominant via to the SARS-CoV-2 egress in Vero cells.
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A worldwide shortage of molecular biology consumables is in surge. This includes filter tips, nucleic acid purification kits, polymerases, reverse-transcriptase, and different types of reagents which are included in viral diagnostic kits. In developing countries, the problem is even worse, since there is few capital enterprise to adopt this kind of industry. So, our aim is to develop a suitable, functional, comparable to commercial ones, and affordable in-house protocol to purify viral RNA. We sought some published and commercial RNA purification solutions to set-up an in-house protocol for viral RNA extraction. Solution was prepared accordingly. Also, LPA (linearized polyacrylamide) carrier was evaluated. The whole setting of in-house solutions with addition of LPA carrier was compared to QIAamp viral RNA minikit solutions. Our results showed that linearized polyacrylamide (LPA) carrier in homemade solutions is comparable to poly A carrier which is used in the most commercial kit. In addition, the whole setting of RNA purification solutions did achieve the purpose of viral RNA purification. Also, the result was confirmed using sputum of a Sars-Cov2 infected patient. Our experiments did end up with an affordable homemade solutions for viral RNA purification.
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Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.
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Background: The VITROS IgG Quant assay∗ is for the quantitative detection of SARS-CoV-2 IgG antibodies with calibration traceable to the first World Health Organization International Standard for Anti- SARS-CoV-2 antibody. Results are reported in both qualitative (reactive/ non-reactive) and quantitative values (Binding Antibody Units [BAU]/ml). PRNT is considered the gold standard method for determining neutralizing antibody (nAb) titers. Aims: This study was designed to assess the correlation of the VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Quantitative Assay (VITROS IgG Quant) to a plaque reduction neutralization test developed at Colorado State University (CSU PRNT). Methods: VITROS IgG Quant: The VITROS IgG Quant assay is a fully automated, high-throughput method run on the VITROS family of immunoassay analyzers. First, antibodies to SARS-CoV-2 present in the sample bind with the S1 subunit of the Spike protein coated on wells. After washing, horseradish peroxidase (HRP)- labelled murine monoclonal anti-human IgG antibodies are added. Following a final wash, bound HRP conjugates are detected using the VITROS signal reagent. The amount of conjugate directly correlates to the amount of SARS-CoV-2 IgG antibody present and is reported in BAU/ml. CSU PRNT: Samples were heat-inactivated for 30 min at 56°C, and serial two-fold dilutions were prepared in a 96-well plate. Viral stock (strain hCoV-19/USA/WA1/2020) containing ∼200 pfu per 0.1 ml was added to each well containing serum dilutions. Following incubation at 37°C in 5% CO2, 6-well plates containing recently confluent Vero cells were inoculated with the virus-serum mixtures. After a second incubation at 37°C, 2 ml of overlay was added to each well. After 24 h incubation at 37°C, a second overlay containing neutral red was dispensed into each well and the number of plaques was counted 48-72 h after initial inoculation. The highest dilution of serum that inhibited plaque formation by 50% (PRNT50) was determined based upon the titre of the samples and the number of plaques present at each dilution. Samples with PRNT50 titers less than or equal to 1:20 are considered negative for nAbs. One hundred forty-nine samples were blind-tested with both the VITROS IgG Quant assay and the CSU PRNT, 74 known positive for SARS-CoV-2 antibody and 75 negatives. The correlation of the VITROS IgG Quant values to CSU PRNT50 was determined. Results: The VITROS IgG Quant results ranged from <2 to 2009 BAU/ml. The CSU PRNT50 results ranged from <1:20 to >1:2560. Pearson correlation coefficient was calculated to be 0.867, demonstrating a good correlation between the VITROS IgG Quant results and the CSU PRNT50 titers. Summary/Conclusions: The VITROS Anti-SARS-CoV-2 IgG Quant assay demonstrates a strong correlation to PRNT50 for the measurement of SARS-CoV-2 nAb titers.
ABSTRACT
BACKGROUND: This paper presents the results of studying the characteristics of the antibody response in kidney recipients who are at high risk of severe COVID-19. METHOD: The study of features of the formation of post-infectious anti-SARS-CoV-2 IgG was carried out in the group of kidney recipients (n = 171) with PCR confirmed diagnosis of COVID-19. Studies of the characteristics of post-vaccination immunity were carried out in a group of vaccinated recipients (n = 49) with Sputnik V (Russia) or Vero Cell (China). ELISA was used to detect IgG to S and N proteins of the SARS-CoV-2. Comparative studies used a simple randomized selection of immunocompetent COVID-19 patients (n = 163). Statistical data processing was carried out using the χ2 andWald's methods. RESULTS: It was found that 30 days after the onset of clinical symptoms of COVID- 19 in kidney recipients, IgG to S and/or N proteins of SARS-CoV-2 were detected in 89.5% (83.9%-93.3%) of them. The detection rate of IgG to the S protein was higher than that to the N protein [87.7% (81.9%-91.9%) and 62.0% (54.5%-68.9%), respectively). At the same time, the seroprevalence to the pathogen varied by age: in the 18-34-year-old group it was 77.7% (59.9%-88.9%), in the 50-64-year-old group it was 96.7% (88.2%-99.8%) and in the group >64 years old it was 80.0% (66.8%-89.0%). This trend of antibody production in older recipients correlated with the highest frequency of registration in them of moderate and severe forms [66.7% (56.4%-75.6%)]. Differences due to the severity of the disease were noted both in the frequency of detectable antibodies [80.3% (69.5%-88.0%) in recipients with a mild form of COVID-19 and 96.0% in recipients with a severe form of infection (P < .001)] and in the intensity of the formed anti-SARS-CoV-2 immunity. Thus, high values of PC (positivity coefficient) (>12) to S protein were recorded in 52.7% (39.8%-65.3%) and 63.2% (53.1%-72.2%) patients with mild and severe forms of COVID-19, respectively, which indicated a direct dependence of the production of antiviral antibodies on the severity of the infection. It was found that in 62.6% (55.1%-69.5%) of recovered recipients anti-SARS-CoV-2 IgG persisted for a period of 3 months from the onset of infection. In a significant proportion of recipients [42.8% (35.5%-50.2%)], antibodies were detected for up to 15 months. In general, the post-infectious antibody response in kidney recipients and immunocompetent patients had similar patterns of development. Despite the general mechanism of antibody production, in immunocompetent patients, the frequency of detection of antiviral antibodies (to N protein: 82.9% and to S protein: 91.2%), tension indicators. (high values of PC to N protein: 51.0% and to S protein: 75.5%) and the duration of retention (in 50.3% at 15 months of monitoring) were slightly higher than the same parameters in recipients with COVID-19. In kidney recipients after immunization with Sputnik V and Vero Cell (n = 34), a rather low detection rate of antiviral IgG [52.9% (36.7%-68.6%) compared with a similar parameter (P < .001) in vaccinated immunocompetent individuals [96.8% (94.8%-98.1%)] was found. The seroprevalence in the group of recipients with hybrid immunity (after illness and vaccination, n = 15) was 86.7% (60.9%-97.5%). In a comparative analysis of the intensity of post-vaccination immunity, high values of PC to S protein (>12) were recorded in 44.0% (26.7%-62.9%) of recipients vaccinated with Sputnik V and 50.0% (31.4%-68.6%) of recipients vaccinated with Vero Cell. The inverse relationship was observed in immunocompetent individuals: 64.7% (58.1%- 70.7%) for Sputnik V and 44.2% (36.8%-51.8%] for Vero Cell. CONCLUSION: The patterns of antibody response to the causative agent in recipients with COVID-19 are comparable to those in immunocompetent patients, while for vaccinated recipients, a low frequency of detection of antiviral antibodies was shown, which indicates the need to continue research on the humoral immunity in people with vulnerable immunity in order to select the best t ctics for COVID-19 immunization.
ABSTRACT
The emergence of SARS-CoV-2 has triggered a pandemic with devastating consequences to the world. One of the proteins essential to the virus life cycle is nsp14, which is a bifunctional protein that encodes a 3'to 5' exoribonuclease activity in its N-terminus, and a methyl transferase activity in its C-terminus. Nsp14 in complex with the accessory protein nsp10 is involved in a proofreading mechanism that ensures the genetic stability of its massive viral genome, and is associated to the resistance against nucleotide analogs targeting the polymerase nsp12. Because of its key role, nsp14-nsp10 complex constitutes an attractive target for antiviral development. Here we present a fluorescence polarization (FP) assay development to measure the exoribonuclease activity and its inhibition in vitro. The FP method is sensitive, robust, amenable to miniaturization and offers confirmation by visualizing the degradation of the fluorescent RNA in acrylamide gels. We performed a screening of a focused library of 113 metal chelators at 20 and 5 μM compound concentration and IC50 measurement of 9 hits showing efficiency at micromolar level. We also tested the focused library in SARS-CoV-2 infected Vero cells and we confirmed 3 hits previously detected in the in vitro screening out of 6 promising inhibitors. In conclusion the FP method proposed is a reliable tool to discover inhibitors of the SARS-CoV-2 exoribonuclease activity and will help to find new antivirals to be used in combination with nucleoside analogs.
ABSTRACT
For many years, our laboratory has been developing cellular models for the study of human pathogenic viruses with RNA genomes, in order to study the replication of these pathogens, to propose new therapeutic pathways, to screen and test inhibitors. In response to the COVID-19 outbreak, we have set up the tools for the study of SARS-CoV-2 replication. First, clinical and reference SARS-CoV-2 strains have been successfully isolated and amplified using Vero E6 cells in the BSL3 facility of Bordeaux University (UB'L3, www.mfp.cnrs.fr/wp/larecherche/ andevir/ubl3/). We set up the monitoring of SARS-CoV-2 replication using conventional RT-qPCR quantification as well as evaluation of the cytopathic effect by microscopic observation or content analysis. Using VERO cells, we are now able to precisely titer viral supernatant (determination of the TCID50) and screen for potential antiviral molecule (determination of EC50 and CC50). We have developed a full-length Spike sequencing based on a Sanger approach1 as well as whole genome sequencing by nanopore technology, allowing the tracking of emerging variants. In parallel, we developed various other models to study SARS-CoV-2 replication including Calu-3 cells, modified human cells expressing Ace2 (e.g. 293T, U2OS) or even more complex cellular models (reconstituted human airway epithelium, vessels) according to the biological question to resolve. As an example, bronchial epithelia reconstituted from biopsies of adult or child donors were used to evaluate the inflammatory response upon SARS-CoV-2 infection in an age-dependent manner [2] (see poster G. Beucher). Similarly reconstituted blood vessels were used to study the impact of SARS-CoV- 2 infection on the vascular system and determine whether clinical observations (blood brain barrier damages, myocarditis) are due to direct infection of cells or indirect effects. Finally, we evaluate the efficacy of different chemical or physical processes for viral inactivation in air or on surfaces.