Uncovering the implications of IFN-I in severe COVID-19 neutrophil inflammasome (preprint)

Excessive inflammation defines COVID-19 pathophysiology. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation. We conducted transcriptomic profiling of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, which revealed a pronounced type I interferon (IFN-I) gene signature in severe COVID-19, compared to mild COVID-19 and healthy controls. Notably, low-density granulocytes (LDGs) from severe COVID-19 did not exhibit this signature and displayed a distinct immature neutrophil phenotype. PMNs from severe COVID-19 patients showed heightened nigericin-induced caspase1 activation but reduced responsiveness to exogenous inflammasome priming. Interestingly, while mature neutrophils efficiently released IL-1? upon inflammasome activation, they were poor producers of IL-18. Furthermore, IFN-I emerged as a priming stimulus for neutrophil inflammasomes, which was confirmed in a COVID-19 mouse model. Overall, these findings underscore the crucial role of neutrophil inflammasomes in driving inflammation during severe COVID-19 and opens promising avenues for targeted therapeutic interventions to mitigate the pathological processes associated with the disease.

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors (preprint)

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.

Low dose (ICMR) hydroxychloroquine prophylaxis for COVID-19 fails - a prospective study

Since the COVID-19 pandemic, the world began a frantic search for possible prophylactic options. We conducted a study to assess the role of hydroxychloroquine for COVID-19 prophylaxis in health-care workers. The study was a prospective cohort with four arms (high, medium, low dose, and control) of HCQ prophylaxis. Participants were grouped as per their opting for any one arm on a voluntary basis as per institute policy. The outcomes studied were COVID-19 positivity by RT-PCR and its severity assessed by WHO COVID-19 severity scale. Total 486 participants were enrolled, of which 29 (6%) opted for low dose, 2 (<1%) medium dose, and none for high dose HCQ while 455 (93.6%) were in the control arm. Of the 164 participants who underwent RT-PCR, 96 (58.2%) tested positive. Out of these 96 positive cases, 79 [82.3%] were ambulatory and were managed conservatively at home. Only 17.7% participants, all from the control group, required hospitalization with the mild-moderate disease. None of the participants had severe disease, COVID-related complications, ICU stay, or death. The difference in the outcome was statistically insignificant (p value >0.05). This single-centre study demonstrated that HCQ prophylaxis in healthcare workers does not cause a significant reduction in COVID-19 as well as mitigating its severity in those infected. At present, most of the trials have not shown any benefit. Though COVID-19 vaccines have reduced the need for prophylaxis, the search for a safe and reasonable chemoprophylaxis should continue until a large population of individuals gets vaccinated, especially in underdeveloped countries.

The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition (preprint)

Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Despite vaccinations, the development and use of neutralising antibodies against the viral surface spike proteins, and small molecule inhibitors targeting the viral replication machinery, COVID-19 remains a global public health crisis. Emerging mutations in the viral genome have the potential to reduce prophylactic and therapeutic efficacy of virus-directed treatments. Targeting host cell factors required for infection could, therefore, be a potential strategy to overcome this problem since mutations in the viral genome are unlikely to bypass the requirement for the targeted host factor or function. The enzymatic activity of N-myristoyltransferases (NMTs) are essential to mediate stable membrane binding and function of a diverse class of cellular proteins, many of which regulate intracellular membrane trafficking. Here we report that nanomolar concentrations of the NMT inhibitor IMP-1088 inhibited SARS-CoV-2 spreading in human cells by compromising the infectivity of released viral particles, which was reduced by up to 90%. IMP-1088 also inhibited human Respiratory syncytial virus, the main cause of viral death in infants world-wide, but not the mosquito-delivered alphavirus Semliki Forest virus and the vesiculovirus Vesicular stomatitis virus. The antiviral effect of IMP-1088 against SARS-CoV-2 displayed remarkably slow reversibility, was well tolerated by cells, and is, therefore, a promising candidate for COVID-19 prophylaxis and therapy.

Acute bilateral central serous chorioretinopathy in a young male after the first dose of Oxford/AstraZeneca AZD1222 (Covishield) vaccine.

We case of acute bilateral central serous chorioretinopathy (CSCR) after receiving the first dose of Covishield vaccine in a young, otherwise healthy male with no associated risk factors.

Molecular dynamics simulation and docking analysis of NF-κB protein binding with sulindac acid.

It is of interest to document the Molecular Dynamics Simulation and docking analysis of NF-κB target with sulindac sodium in combating COVID-19 for further consideration. Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) of the arylalkanoic acid class that is marketed by Merck under the brand name Clinoril. We show the binding features of sulindac sodium with NF-κB that can be useful in drug repurposing in COVID-19 therapy.

Wild red deer (Cervus elaphus) do not play a role as vectors or reservoirs of SARS-CoV-2 in northeastern Poland (preprint)

Several studies reported a high prevalence of SARS-CoV-2 among white-tailed deer in North America. Monitoring cervids in all regions to better understand SARS-CoV-2 infection and circulation in other deer populations has been urged. To evaluate deer exposure to SARS-CoV-2 in Poland, we sampled 90 reed deer individuals shot by hunters in five hunting districts in northeastern Poland. Serum and nasopharyngeal swabs were collected, and then the Immunofluorescent Assay (IFA) to detect anti-SARS-CoV-2 antibodies was performed as well as real-time PCR with reverse transcription for direct virus detection. No positive samples were detected. There is no evidence of spillover of SARS-CoV-2 from the human to deer population in Poland.

Nanobody engineering for SARS-CoV-2 neutralization and detection (preprint)

In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, 'nanobodies'. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel diagnostic assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.

Improving performance of classifiers for diagnosis of critical diseases to prevent COVID risk.

The risk of developing COVID-19 and its variants may be higher in those with pre-existing health conditions such as thyroid disease, Hepatitis C Virus (HCV), breast tissue disease, chronic dermatitis, and other severe infections. Early and precise identification of these disorders is critical. A huge number of patients in nations like India require early and rapid testing as a preventative measure. The problem of imbalance arises from the skewed nature of data in which the instances from majority class are classified correct, while the minority class is unfortunately misclassified by many classifiers. When it comes to human life, this kind of misclassification is unacceptable. To solve the misclassification issue and improve accuracy in such datasets, we applied a variety of data balancing techniques to several machine learning algorithms. The outcomes are encouraging, with a considerable increase in accuracy. As an outcome of these proper diagnoses, we can make plans and take the required actions to stop patients from acquiring serious health issues or viral infections.

Serological surveillance for wild rodent infection with SARS-CoV-2 in Europe (preprint)

We report serological surveillance for exposure to SARS-CoV-2 in 1,237 wild rodents and other small mammals across Europe. All samples were negative with the exception of one. Given the ongoing circulation of this virus in humans and potential host jumps, we suggest such surveillance be continued.

Understanding the Effect of COVID-19 Pandemic on Emergency Surgical Care Delivery in India: A Multicenter Cross-sectional Study (preprint)

BackgroundThe COVID-19 pandemic and subsequent lockdowns adversely affected global health care services to varying extent. Emergency Services were affected along-with elective surgeries, to accommodate the added burden of COVID19 affected patients. We aimed to reflect, quantify and analyse the trends of essential surgeries and bellwether procedures during the waxing and waning of the pandemic, across various hospitals in India.MethodologyA research consortium led by WHO Collaboration Centre (WHOCC) for Research in Surgical Care Delivery in Low-and Middle-Income countries, India, conducted this study with 5 centres. All surgeries performed during the months of April 2020 (wave 1), November 2020 (recovery 1) and April 2021 (wave 2) were compared with those performed in April 2019 (pre-pandemic period). ResultsThe total number of surgeries reduced by 77% during wave 1, which improved to 52% reduction in recovery 1, as compared to pre-pandemic period. However, surgeries reduced again during wave 2 to 68%, but reduction was less as compared to wave 1. Emergency and essential surgeries were affected along-with the elective ones, but to a lesser extent.ConclusionOur study quantified the effects of the pandemic on surgical-care delivery across a timeline and documented reduction in overall surgical volumes during the peaks of the pandemic (wave 1 and 2) with minimal improvement as the surge of COVID19 cases declined (recovery 1). The second wave showed improved surgical volumes as compared to the first one which may be attributable to improved preparedness. Caesarean sections were affected the least. 

Introduction and rapid spread of SARS-CoV-2 Omicron variant and the dynamics of its sub-lineages BA.1 and BA.1.1, December 2021, Finland (preprint)

Multiple introductions of SARS-COV-2 Omicron variant BA.1. and BA.1.1. lineages to Finland were detected early December 2021, and comprised the majority over Delta variant in 3 weeks in the capital region. Our sequence analysis demonstrates emergence of a large cluster of BA.1.1 in community transmission.  

Experimental infection of mink with SARS-COV-2 Omicron (BA.1) variant leads to symptomatic disease with lung pathology and transmission (preprint)

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that mink remain virus RNA positive for days, develop clinical sings and histopathological changes, and transmit the virus to uninfected recipients warranting further studies and preparedness.

Intranasal inhibitor blocks omicron and other variants of SARS-CoV-2 (preprint)

The emergence of the SARS-CoV-2 Omicron variant capable of escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a need for SARS-CoV-2 blocking agents that are more economical to produce in large scale, while less vulnerable to mutational variation in the neutralization epitopes of the viral Spike glycoprotein. Here we describe TriSb92, a highly manufacturable trimeric human nephrocystin SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of the Spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Delta and Omicron. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before the challenge with SARS-CoV-2 B.1.351 efficiently protected mice from infection. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the Spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. Our results highlight the potential of intranasal inhibitors in protecting susceptible individuals from SARS-CoV-2 infection, and describe a novel type of inhibitor that could be of use in addressing the challenge posed by the Omicron variant.

Intranasal inhibitor blocks Omicron and other variants of SARS-CoV-2 (preprint)

The emergence of the SARS-CoV-2 Omicron variant capable of escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models. However, there remains a need for SARS-CoV-2 blocking agents that are more economical to produce in large scale, while less vulnerable to mutational variation in the neutralization epitopes of the viral Spike glycoprotein. Here we describe TriSb92, a highly manufacturable trimeric human nephrocystin SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of the Spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Delta and Omicron. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before the challenge with SARS-CoV-2 B.1.351 efficiently protected mice from infection. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the Spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. Our results highlight the potential of intranasal inhibitors in protecting susceptible individuals from SARS-CoV-2 infection, and describe a novel type of inhibitor that could be of use in addressing the challenge posed by the Omicron variant.

ClusTRace, a bioinformatic pipeline for analyzing clusters in virus phylogenies (preprint)

Summary: SARS-CoV-2 is the highly transmissible etiologic agent of coronavirus disease 2019 (COVID-19) and has become a global scientific and public health challenge since December 2019. Several new variants of SARS-CoV-2 have emerged globally raising concern about prevention and treatment of COVID-19. Early detection and in depth analysis of the emerging variants allowing pre-emptive alert and mitigation efforts are thus of paramount importance. Here we present ClusTRace, a novel bioinformatic pipeline for a fast and scalable analysis of sequence clusters or clades in large viral phylogenies. ClusTRace offers several high level functionalities including outlier filtering, aligning, phylogenetic tree reconstruction, cluster or clade extraction, variant calling, visualization and reporting. ClusTRace was developed as an aid for COVID-19 transmission chain tracing in Finland and the main emphasis has been on fast and unsupervised screening of phylogenies for markers of super-spreading events and other features of concern, such as high rates of cluster growth and/or accumulation of novel mutations. Availability: All code is freely available from https://bitbucket.org/plyusnin/clustrace/

Common laboratory mice are susceptible to infection with SARS-CoV2 beta variant (preprint)

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes consistent with COVID-19.

The phylodynamics of SARS-CoV-2 during 2020 in Finland — Disappearance and re-emergence of introduced strains. (preprint)

Finland has had a low incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infections as compared to most European countries. Here we report the origins and turnover of SARS-CoV-2 lineages circulating in Finland in 2020. SARS-CoV-2 introduced to Finland in January 2020 and spread rapidly across southern Finland during spring. We observed rapid turnover among Finnish lineages during this period. Clade 20C became the most prevalent among sequenced cases and was replaced by other strains in fall 2020. Bayesian phylogeographic reconstructions suggested 42 independent introductions into Finland during spring 2020, mainly from Italy, Austria, and Spain, which might have been the source for a third of cases. The investigations of the original introductions of SARS-CoV-2 to Finland during the early stages of the pandemic and of the subsequent lineage dynamics could be utilized to assess the role of transboundary movements and effects of early intervention and public health measures.

The rise and fall of Alpha and Beta variants of SARS-CoV2 in Finland in spring of 2021 (preprint)

Two SARS-CoV-2 Variants of Concern, Alpha (~ 80%) and Beta (~ 23%) rapidly became dominant in Finland in the spring of 2021 but diminished near summer. To assess their temporal epidemiological dynamics among Finnish cases, we began large-scale sequencing efforts to identify spreading events and sources via phylogenetic clustering analyses. The results show the majority belonged to clusters spreading in the community while few sequenced samples were singletons. The results highlight the importance of surveillance and preventative policies in controlling the epidemic.

Molecular dynamics simulation and docking studies reveal NF-κB as a promising therapeutic drug target for COVID-19 (preprint)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is rampant worldwide and is a deadly disease for humans. Our current work emphasizes on molecular dynamics simulation (MDS) targeting nuclear factor-kappa B (NF-κB), the well-known human transcription factor controlling innate and adaptive immunity, to understand its mechanism of action during COVID-19 in humans. NF-κB was interacted with the SARS-CoV-2 spike protein in an in silico MDS experiment, revealing the NF-κB site at which the SARS-CoV-2 spike protein interacts. We screened some known drugs via docking studies on NF-κB used as a receptor. The MDS software Schrodinger generated more than 2000 complexes from these compounds and using the SMILES format of these complexes, 243 structures were extracted and 411 conformers were generated. The drug used as a ligand that docked with NF-κB with the best docking score and binding affinity was Sulindac sodium as its trade name. Furthermore, RMSF data of sulindac sodium and NF-κB displayed minimal fluctuations in the protein structures, and the protein-ligand complex had reduced flexibility. Sulindac sodium is hence suggested as a suitable drug candidate for repurposing in clinical trials for SARS-CoV-2 infections. This drug potently blocked the spike protein’s interaction with NF-κB by inducing a conformational change in the latter. Arguably, NF-κB inaction is desired to have normal immunity and can possibly be retained using proposed drug. This work provides a significant lead for drug repurposing to combat SARS-CoV-2 and its various mutant forms and reveals new approach for controlling SARS-CoV-2-induced disease.