First isolation and characterization of the prototype strain of the SARSCoV-2 virus at the beginning of the COVID-19 pandemic in Peru

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.

Differential of antiviral activity between chloroquine and hydroxychloroquine sulfate against SARS-CoV-2 in vitro.

Objective To investigated the in vitro antiviral activity of chloroquine and hydroxychloroquine sulfate against different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Prototype, Beta, Delta, Omicron) by changing the sequence of drug and virus introduction. Methods Prophylactic treatment: Vero E6 cells were treated with Chloroquine or hydroxychloroquine sulfate (200.00, 150.00, 100.00, 50.00, 16.70, 5.55, 1.85, 0.62, 0.21 micromol.L-1) for 1 h, then the virus was added and incubated for another 2 h. The virus-drug mixture was repalced with fresh medium until the end of the experiment. Post-entry treatment: Vero E6 cells were incubated with virus for 2 h, then the virus was removed and the cells were cultured with drug-containing medium until the end of the experiment. Full-time treatment: Vero E6 cells were pretreated with the drug for 1 h ahead, then virus was added and incubated for another 2 h. The virus-drug mixture was discarded and the cells were cultured with drug-containing medium until the end of the experiment. After 72 h of culture, the cells were observed to see whether they became round and shed to determine the cytopathic situation, and the semi-maximum effect concentration (EC50) and drug selection index (SI) were calculated. Results Both drugs were less effective in preventing SARS-CoV-2. Chloroquine/hydroxychloroquine sulfate showed good antiviral activity under both therapeutic and full-time treatment. EC50 of hydroxychloroquine sulfate was less than chloroquine, SI was greater than chloroquine, antiviral effect of hydroxychloroquine sulfate was better than chloroquine. The antiviral effect of chloroquine (EC50 = 0.904 micromol.L-1) and hydroxychloroquine sulfate (EC50 = 0.143 micromol.L-1) was more significant against Omicron variant than other variants under therapeutic and full-time treatment conditions. Conclusion Chloroquine/hydroxychloroquine sulfate showed good antiviral activity under both therapeutic and full-time treatment, and both drugs were significantly more active against the Omicron variant than the other variants.Copyright © 2023 Authors. All rights reserved.

Relationship between COVID-19 infection and erectile dysfunction, a literature review examining the link and proposed mechanisms behind this phenomenon

It is now only in the wake of coronavirus disease 2019 (COVID-19) that we are beginning to understand many of the extra-respiratory manifestations of the condition. There is now growing evidence that erectile dysfunction (ED) is closely linked with the disease. We carry out one of the first literature reviews to consolidate the current evidence of the causal link between COVID-19 and ED and explore the proposed mechanisms that underpin this phenomenon. We carried out a literature search of the databases;PubMed (MEDLINE), Scopus, Web of Science and the Cochrane library. Search time frame was between December 2019 and March 2022. Only studies deemed of acceptable quality were included. Five studies were found highlighting the link between COVID-19 and ED. A further Nineteen studies were utilized to illustrate the proposed biological mechanisms underpinning COVID-19 related ED. Clear evidence has been documented through multiple studies internationally recognizing reduction in erectile scores and reduced sexual activity. It appears there is likely indirect and direct cytopathic effects on endothelial cells, in addition to hormonal and psychosocial factors. The associated ED is likely a result of a multitude of mechanisms including direct and indirect endothelial dysfunction, vasoactive cytokines, endocrine dysregulation, and psychosocial factors. This is the first literature review to delve into the likely underpinning mechanisms of the virus that drive ED.Copyright ©2023 The Author(s). Published by MRE Press.

SARS-CoV-2 ANTIVIRAL ACTIVITY OF ZOTATIFIN, A HOST-TARGETING eIF4A INHIBITOR

Background: Zotatifin (eFT226) is a potent and selective inhibitor of eukaryotic initiation factor 4A (eIF4A), a host RNA helicase required for SARS-CoV-2 replication. Zotatifin selectively inhibits translation of ribonucleic acids (RNAs) containing specific short polypurine motifs in their 5-prime (5') regions. Two such highly conserved motifs are found in the SARS-CoV-2 genome. Zotatifin is currently being evaluated in a Phase 1b dose escalation study in 36 patients with mild to moderate COVID disease. In this in vitro study, we evaluated the selectivity of zotatifin's inhibition of SARS-CoV-2 translation, the antiviral activity of zotatifin alone against different human coronaviruses and the antiviral activity of zotatifin in combination with other antivirals against SARSCoV-2. Method(s): The selectivity of zotatifin for viral translation was evaluated in a cell-based reporter assay wherein luciferase translation was driven by 5'-sequences from SARS-CoV-2 or tubulin, a housekeeping gene. The antiviral activity of zotatifin was evaluated against SARS-CoV-1, SARS-CoV-2 variants (Wash/1/2020 (ancestral), delta, omicron BA.2), MERS-CoV and HCoV-299E in primary or established cell lines using cytopathic effect or infectious virus as endpoints. The antiviral activity of zotatifin in combination with remdesivir, N-hydroxycytidine (NHC;active nucleoside analogue metabolite of molnupiravir), nirmatrelvir, baricitinib or sotrovimab was evaluated against SARS-CoV-2 and analyzed by the method of Pritchard and Shipman. Result(s): Zotatifin inhibited the translation of the SARS-CoV-2 luciferase reporter construct with a mean IC50 of 3 nM and was ~14-fold less potent in inhibiting the tubulin reporter construct. Zotatifin potently inhibited the replication of all human coronaviruses tested with 50% effective concentrations (EC50s) ranging from 0.016 to 37.3 nM. The 50% cytotoxic concentration (CC50) value for zotatifin was 250 to >100,000 nM, yielding selectivity indices of 7 to >6250. Zotatifin was ~20 to >100-fold more potent than remdesivir, nirmatrelvir or NHC (figure) and demonstrated additive interactions when combined with remdesivir, NHC, nirmatrelvir, baricitinib or sotrovimab in vitro. Conclusion(s): The potent broad-spectrum activity of zotatifin against a variety of human coronaviruses and additive activity when combined with different anti-SARS-CoV-2 antivirals highlight the advantages of eIF4A as a target and warrant further evaluation in human clinical trials.

COVID-19 as a probable cause of acute pancreatic injury

Acute pancreatic pseudocysts are increasingly recognized as complications in patients with coronavirus disease 2019 (COVID-19). There-fore, it is important for healthcare providers to be aware of this phenomenon to ensure proper diagnosis and treatment. Up to 17% of patients with severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2) infection have been shown to develop pancreatic lesions. These pancreatic lesions can be caused directly by the cytopathic effects of the viral infection or indirectly by systemic responses to inflammation or respiratory failure. Several studies have shown that angio-tensin-converting enzyme 2 (ACE2) is the functional receptor used by SARS-CoV-2 to gain access to target cells, while ACE2 receptors are expressed in significant amounts in the pancreas. In this article, we present 2 cases of COVID-19. patients that presented with similar pancreatic lesions. The first case was a 47-year-old lady who presented to the emergency department (ED) with flu-like symptoms for ten days. Incidental findings on computed tomography (CT) scan showed a large, multiloculated cystic mass in the pancreatic tail. The second case was an 81-year-old Caucasian lady who presented to the outpatient clinic with multiple chronic complaints after an acute COVID-19 infection four months prior. Abdominal CT scan with oral contrast revealed multiple hypodense masses on the pancreas measuring 0.3 cm in diameter. The cases we reported in this article showed the degree of COVID-19's effect on the gastrointestinal system, with pancreatic injury occurring during the early phases of the acute phase of the infection and lasting up to 4 months post-resolution of the infection.Copyright © 2023, The Indonesian Foundation of Critical Care Medicine. All rights reserved.

PHASE 1 CLINICAL TRIALS OF A Q-GRIFFITHSIN NASAL SPRAY FOR SARS-CoV-2 PROPHYLAXIS

Background: The currently approved vaccines do not induce sterilizing immunity against SAR-CoV-2 infection, and immunity wanes over time. A robust broad spectrum topical prophylaxis strategy could protect vulnerable populations in the face of continuous evolution of SARS-CoV-2. The algal antiviral lectin Griffithsin (GRFT), and an engineered oxidation-resistant variant Q-GRFT have robust entry inhibitory activity against SARS-CoV variants of concern, in addition to other respiratory viruses with pandemic potential. We designed a nasal spray to deliver Q-GRFT to the upper respiratory tract mucosa for on-demand use as a broad-spectrum prophylactic. Two clinical trials (Phase 1a and 1b) were conducted to assess safety, tolerability, and pharmacokinetics of Q-GRFT nasal spray in healthy adults. Method(s): Healthy adult volunteers were enrolled in a Phase 1a double blinded, randomized study to receive a single dose of either intranasal Q-GRFT (3.0 mg, 2 sprays per nostril) or placebo at 2:1 ratio. Following a safety review, the Phase 1b study was initiated. Eleven volunteers in Group 1 received 3.0 mg dose once daily, for 7 days. After a safety review, 11 volunteers in Group 2 received a total of 6.0 mg Q-GRFT (3.0 mg twice daily for 7 days). Topical Q-GRFT concentrations were measured by ELISA in collected nasal and nasopharyngeal fluids. Drug levels in plasma were assayed to determine systemic exposure. Viral microneutralization cytopathic effect (CPE) assays were performed against SARS-CoV-2 Omicron BA-5 and MERS-CoV. Result(s): Eighteen adults (24-54 years;Males 58.3%, Females 41.7%;12 Q-GRFT, 6 Placebo), and 22 adults (aged 23-59 years;Males 52.4%, Females 47.6%) were enrolled in Phase 1a and 1b, respectively. In Phase 1a, a single dose of Q-GRFT maintained quantifiable levels in nasal passages and nasopharynx for up to 24 hours. Similarly, Q-GRFT was quantifiable in nasal and nasopharyngeal regions in the Phase 1b study. No dose accumulation effect or systemic exposure was observed. Nasal and nasopharyngeal swab eluates inhibited SARS-CoV-2 Omicron BA.5 and MERS-CoV in CPE assays. Q-GRFT did not modify olfactory sensation. No severe adverse events were reported. Thus, the nasal spray was deemed safe. Conclusion(s): Intranasal Q-GRFT was safe and enhanced mucosal SARSCoV-2 inhibitory activity in human volunteers. The results support further development of Q-GRFT as a broad-spectrum prophylactic against coronaviruses to curb ongoing infections, and for future pandemic preparedness.

Efficacy of Favipiravir and Molnupiravir against Novel Sars-Cov-2 Variants in Vitro and in Vivo

The COVID-19 disease pandemic remains a significant global problem, resulting in hundreds of millions of cases and millions of deaths. The search for specific inhibitors of SARS-CoV-2 for the treatment of this infection remains relevant. Drugs such as Favipiravir and Molnupiravir, which exhibit specific antiviral activity against SARS-CoV-2, are already being used to treat patients. However, there is limited evidence of their effectiveness, especially against novel genetic variants of the COVID-19 pathogen. The aim of this study was to investigate the antiviral effect of these drugs using an in vitro experimental model of SARS-CoV-2 infection in Vero E6 cell culture and an animal model of infection using Syrian hamsters. It has been established that Molnupiravir has an inhibitory effect against variants of the SARS-CoV-2 with IC50 values from 16.51 to 7.88 microM in vitro, and reduces the infectious titer of the virus in the lungs of animals by ~1.5 Log10 in vivo, in while Favipiravir shows lower activity and severe toxicity. Dose selection and frequency of use remain unexplored.Copyright © 2022 Pirogov Russian National Research Medical University. All rights reserved.

Preclinical studies of antiviral activity of the RPH-137 fusion protein and molnupiravir against COVID-19.

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.

Understanding kidney injury in covid-19, a pressing priority

The 2019 novel coronavirus disease (COVID-19) is a newly defined infectious and highly contagious acute disease caused by the severe acute respiratory syndrome coronavirus 2 ( (SARS-CoV-2). COVID-19 is mainly characterized by an acute respiratory disease however it can also affect multiple other organ systems such as the kidney, gastrointestinal tract, heart, vascular system, and the central nervous system. Kidney involvement is frequent in patients with COVID-19 and this review aims to explore the available data on kidney and COVID-19. In conclusion, COVID-19 infection can affect renal function and may cause acute kidney injury (AKI), due to several mechanisms that need to be fully elucidated. As only supportive management strategies are available for treating AKI in COVID-19, it is necessary to identify and preserve renal function during SARS-CoV-2 infection.Copyright © 2021 The Author(s).

Acute Kidney Injury and Covid-19

Introduction: COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This emerging disease has become a public health emergency worldwide. Acute Kidney Injury (AKI) secondary to COVID-19 has been described in different studies, but information characterising patients with subsequent AKI is limited. The cause of kidney involvement in COVID-19 is thought to be multifactorial. Cardiovascular comorbidity and predisposing factors (e.g. sepsis and nephrotoxins) are considered as important contributors. The tubular damage has been linked to the cytopathic effects of kidney-resident cells and cytokine storm syndrome. To gain better understanding of the effect of COVID-19 on renal function, large clinical and register based studies have been requested. The objective of this study was to quantify the risk of acute kidney injury during and after covid-19. Method(s): This was a Swedish prospective cohort study where Generalised Estimating Equation methods (GEE) was used to map the kinetics of kidney injury markers such as serum-creatinine (s-creatinine), cystatin and eGFR for the hospitalised patients in the cohort, comparing patients with moderate and severe COVID-19 during and after the acute infection. Furthermore, we will investigate if patients with kidney dysfunction during COVID-19 have more severe disease outcome compared with the whole cohort, adjusting for age, sex, and comorbidities. We will also compare start values of kidney injury markers with the latest values and count the percentage worsening among all disease severity groups. Cohort: Approximately 550 COVID-19 patients were recruited to the study following informed and signed consent at 2 Swedish University Hospitals. A case report form was filled in at pre-specified time points, and samples collected consecutively. A database was then created containing dates and information regarding symptoms, laboratory samples, complications, and disease severity (e.g., need of oxygen, intensive care, mechanical ventilation, death). Result(s): There was a significant increase in s-creatinine among hospitalised and intensive care unit patients (n=126) during the acute phase of COVID-19 (day 0-6 post disease onset) when compared to the follow up samples after 90 days from disease onset. There was also a decrease in s-creatinine in day 11-21 and 31-70 among hospitalised and intensive care unit COVID-19 patients when compared to the same follow up samples. This analysis was adjusted for age and sex. See figure 1. [Formula presented] Conclusion(s): Our preliminary results show that s-creatinine was increased during the first days of COVID-19 followed by decreased levels compared to baseline. The higher levels of s-creatinine day 0-6 of COVID-19 could be an effect of the acute infection, but it could also be caused by other factors such as dehydration or medication. The lower levels of s-creatinine might be caused by dietary changes or loss of muscle mass due to immobilisation during hospitalisation. Knowledge about fluctuations in s-creatinine in COVID-19 patients may be of use for treating physicians. Conflict of interest Potential conflict of interest: Funding from: Swedish Kidney Foundation Central and local ALF funding Vasterbotten County Council, Sweden Arnerska Research Fund, Umea University, SwedenCopyright © 2023

Preclinical studies of antiviral activity of the RPH-137 fusion protein and molnupiravir against COVID-19

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.

Nitric-oxide enriched plasma-activated water inactivates 229E coronavirus and alters antiviral response genes in human lung host cells

The ongoing pandemic caused by the novel coronavirus, SARS-CoV-2, is influencing global health. Moreover, there is a major threat of future coronaviruses affecting the entire world in a similar, or even more dreadful, manner. Therefore, effective and biocompatible therapeutic options against coronaviruses are urgently needed. To address this challenge, medical specialists require a well-informed and safe approach to treating human coronaviruses (HCoVs). Herein, an environmental friendly approach for viral inactivation, based on plasma technology, was considered. A microwave plasma system was employed for the generation of the high amount of gaseous nitric oxide to prepare nitric oxide enriched plasma-activated water (NO-PAW), the effects of which on coronaviruses, have not been reported to date. To determine these effects, alpha-HCoV-229E was used in an experimental model. We found that NO-PAW treatment effectively inhibited coronavirus infection in host lung cells, visualized by evaluating the cytopathic effect and expression level of spike proteins. Interestingly, NO-PAW showed minimal toxicity towards lung host cells, suggesting its potential for therapeutic application. Moreover, this new approach resulted in viral inactivation and greatly improved the gene levels involved in host antiviral responses. Together, our findings provide evidence of an initiation point for further progress toward the clinical development of antiviral treatments, including such coronaviruses. © 2022 The Authors

In vitro Antiviral Activity of Remdesivir Against SARS-CoV-2 and its Variants

The outbreak of COVID-19 has become a public health emergency of international concern;thus, it is important to not only develop drugs for treating COVID-19 but also develop a method for evaluating the therapeutic effect based on the characteristics of SARS-CoV-2 and its variants. To test the antiviral activity of a drug against COVID-19, in this study, we established and compared experimental conditions, such as the treatment time and mode of action (dose) of the therapeutic substance, and a test method to evaluate its effectiveness. We optimized an assay for testing antiviral activity by plaque reduction, tissue culture infectious dose 50, and quantitative RT-PCR. These methods were applied to test the antiviral efficacy of the therapeutic against SARS-CoV-2. Antiviral activity testing using in vitro assays against SARS-CoV-2 and its variants was assessed by measuring plaque-reducing or cytopathic effects in Vero-E6 cells. The in vitro assay was validated by evaluating the antiviral activity of remdesivir. Remdesivir reduced SARS-CoV-2 titer without detectable cytotoxicity and successfully inhibited viral replication in a dose-dependent manner. Therefore, we suggest this in vitro assay as an effective method for testing the antiviral activity for a potential repurposed drug against COVID-19 or rapid screening of therapeutic candidates. Copyright © 2022 Journal of Bacteriology and Virology.

Detection of Infectious SARS-CoV-2 in Specimens with High CT Values Is More Common for Omicron than for Delta Variants

Background. Although not validated, cycle threshold (Ct) values from real-time (r)RT-PCR are sometimes used as a proxy for infectiousness to inform public health decision-making. A better understanding of variant-specific viral dynamics, including RNA and infectious virus relationships, is needed to clarify implications for diagnostics and transmission. Methods. Non-hospitalized SARS-CoV-2-infected individuals were recruited <= 5 days post-onset and self-collected nasal swabs daily for two weeks. Sequencing was used to determine variant, an in-house quantitative rRT-PCR targeting N gene was used to produce Ct values and determine RNA load, and cytopathic effect was used to assess the presence or absence of infectious virus (binary outcome). We used a Ct threshold of 30 to define high-Ct (Ct > 30) or low-Ct (Ct <= 30) specimens and assessed the percentage of RNA-positive specimens that had infectious virus;variantspecific percentages were compared by chi2 test. Results. We included 113 and 200 RNA-positive specimens from 18 and 28 Omicron- and Delta-infected participants, respectively;timing of RNA-positive specimen collection was similar in both groups (median = 8d post-onset). Maximum observed RNA levels occurred at median of 5 days post-onset for both variants but were lower for participants with Omicron vs Delta [mean RNA copies/mL = 105.2 vs 107.9]. Despite lower RNA levels, infectious virus was frequently detected for both variants [Omicron: median duration = 4.5d;Delta: median = 6d;p = 0.13]. Omicron specimens with infectious virus had higher Cts vs Delta specimens [mean Ct = 29.9 vs 23.2, p < 0.001]. In high-Ct specimens (Ct > 30;Table), the percentage of specimens with infectious virus was typically higher for Omicron vs Delta, and was significantly higher in adults [27.3% vs 9.5%]. In low-Ct specimens (Ct <= 30), the percentage with infectious virus was similar or higher for Omicron vs Delta, and was significantly higher in children [87.5% vs 53.8%] and in those unvaccinated [94.1% vs 47.4%]. Conclusion. CDC does not recommend the use of Ct values as a proxy for infectiousness. These data further highlight that Ct values may not provide a reliable or consistent proxy for infectiousness across variants.

Investigation of Treatment-Emergent Amino Acid Substitutions in Nonstructural Protein 5 from Ph2a Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor

Background. Ensitrelvir is a novel oral SARS-CoV-2 3C-like protease inhibitor, and under late clinical development stage for COVID-19 disease. In Ph2a and Ph2b studies, ensitrelvir demonstrated rapid decline of viral titer and viral RNA compared with placebo, and tolerability. To investigate treatment-emergent amino acid substitutions (TEAASs), we analyzed viral RNA sequences of nonstructural protein 5 (NSP5), target of ensitrelvir from Ph2a study in Japan. Methods. In Ph2a study, 69 patients with confirmed SARS-CoV-2 infection were randomized 1:1:1 to ensitrelvir with the loading dose on Day 1/maintenance dose on Day 2-5 (375/125 mg or 750/250 mg), or placebo. Intent-to-treat (ITT) population was defined as participants confirmed with positivity by qualitative RT-PCR at baseline. TEAASs were defined as novel amino acid substitutions identified after treatment with ensitrelvir. NSP5 sequence analysis was performed with sanger sequencing using nasopharyngeal swab samples at Day 1, 6, 9, 14, and 21 with allowances from ITT population with ensitrelvir. Infectious viral titer was measured by virus-induced cytopathic effects in VeroE6/TMPRSS2 cells. Viral RNA was quantified by RT-PCR. Results. By NSP5 sequence analysis, TEAASs were observed in 1 patient from 375/125 mg group and 2 patients from 750/250 mg group. H246Y in 375/125 mg was detected from specimen on Day 8, and A234S and T198I in 750/250 mg were detected from specimens on Day 8 and Day 14, respectively. Structural analysis revealed that these mutations are located outside of the active center of 3C-like protease which is the binding site of ensitrelvir. Viral titer and viral RNA in each specimen, in which TEAASs were observed, were below lower limit of detection and lower limit of quantification, respectively. Furthermore, H246Y, A234S and T198I are rare substitution (< 0.05%) among SARS-CoV-2 variants according to Global Initiative on Sharing Avian Influenza Data. Conclusion. In Ph2a study, H246Y, A234S, and T198I in NSP5 were detected as TEAASs. However, results of viral titer and viral RNA, and structural information suggest that these mutations do not have the impact on antiviral efficacy of ensitrelvir.

Molnupiravir Exhibits a High Barrier to the Development of SARS-CoV-2 Resistance in vitro

Background. Molnupiravir is an orally available prodrug of the antiviral nucleoside analog N-Hydroxycytidine (NHC). In preclinical studies NHC has shown broad-spectrum antiviral activity against multiple RNA viruses including SARS-CoV-2. Incorporation of NHC by viral polymerases impairs replication by introducing errors into the viral genome. NHC has been shown to have a high barrier to the development of resistance in vitro with RSV, Influenza and Venezualen Equine Encephalitis viruses. In these studies, we have explored the potential for SARS-CoV-2 to develop resistance to NHC in cell culture. Methods. Vero E6 cells were infected with SARS-CoV-2 (WA-1) in triplicate in the presence of NHC or a C3L-protease inhibitor (MRK-A). Culture supernatants from wells with the highest drug concentration exhibiting a cytopathic effect (CPE) score of>=2+ were repassaged and at each passage, IC50 values were estimated based on CPE scoring. At each passage, full genome next generation sequencing (NGS) was performed on the viral RNA Results. No change in susceptibility to NHC (EC50 fold change <= 1.1) was noted in 2 of 3 cultures and a 2-fold change was observed in one culture after 30 passages. In contrast, a 3- to 4-fold decreases in susceptibility to the 3CL protease inhibitor were seen by passage by 12, with increasing resistance of 4.6- to 15.7-fold observed by passage 30. NHC passaged viruses exhibited 53 to 99 amino acid changes, including substitutions and deletions (both in-frame and frameshift), across 25 different viral proteins as compared with 10 to 13 changes in 13 proteins in the MRK-A cultures. With NHC, 3 to 4 changes were observed in the viral polymerase;however, these were randomly distributed, and none were observed more than once. In contrast, the 3CL protease passaged virus had a nsp5 T21I substitution detected in all 3 cultures. Conclusion. No evidence of SARS-CoV-2 phenotypic or genotypic resistance was observed following 30 passages with NHC. A random pattern of amino acid changes were observed across multiple proteins consistent with the mechanism of action of NHC. In the same study, resistance was readily selected to a control 3CL protease inhibitor. Together these data support previous reports demonstrating the high barrier to resistance of NHC.

Two Cases of New-Onset Lichen Planus Following BNT162b2 mRNA COVID-19 Vaccine

Various cutaneous side effects have been reported to be observed following coronavirus disease-2019 (COVID-19) vaccination. Urticaria, maculopapular eruption, vasculitis, pityriasis rosea, psoriasis and papulovesicular exanthem are some dermatological diseases which are observed after COVID-19 vaccination. Hyperinflammatory environment resulting from the cytokine storm in the setting of COVID-19, immune complex deposition and direct cytopathic effects of the virus are implicated in the etiopathogenesis of the dermatological manifestations. Infectious agents, immunologic factors, vaccinations and drugs have all been blamed in the emergence of lichen planus. With the cases presented in this case report, we would like underline that new-onset cutaneous lichen planus may be seen after mRNA COVID-19 vaccination. Copyright © 2022 by Turkiye Klinikleri.

Plaque reduction neutralization test as a method for detecting functional neutralizing antibodies against live SARS-CoV-2 virus

Introduction and objective: Since 2020, little is known about neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus emerging and causing COVID-19 in humans. Here, we established a plaque reduction neutralization test (PRNT) to use as a method for detecting functional NAbs to SARS-CoV-2. Material(s) and Method(s): Vero cells were used as target cells for infection with the new coronavirus and cytopathic effects were obviously exhibited. Result(s): The development of plaque reduction after neutralization of virus with diluted specific antiserum was assessed according to a dose-response effect and consistency of test results with pooled antibody serum, which illustrated the robustness and dynamic reduction of plaque. In addition, the PRNT was used for evaluating functional NAb responses in Thai patients after symptom onset. Conclusion(s): The PRNT can be a method for measuring antibodies against live SARS-CoV-2 and future studies will be planned to investigate functional NAb responses elicited by any COVID-19 vaccine and to evaluate long-term protection of Thai COVID-19 vaccination. Copyright © 2022, Faculty of Pharmaceutical Sciences, Chulalongkorn University. All rights reserved.