A novel coronavirus (SARS-CoV-2) that is initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has killed 2,718 people in China by February 26, 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 38 countries around the world. However, the drug has not yet been officially licensed or approved to treat SARS-Cov-2 infection. NSP12-NSP7-NSP8 complex of SARS-CoV-2, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12-NSP7-NSP8 complex (PDB ID: 6NUR) structure of SARS, two homologous models were established for virtual screening in the present study, namely NSP12-NSP7 interface model and NSP12-NSP8 interface model. Seven compounds (Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All seven compounds can combine well with NSP12-NSP7-NSP8 in the homologous model, providing drug candidates for the treatment and prevention of SARS-CoV-2.

SARS-CoV-2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that Vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a major outbreak of coronavirus disease 2019 (COVID-19) and instigated a widespread fear and has threatened global health security. Although phenomenal efforts are in progress to effectively combat this COVID-19 outbreak. Still, no licensed antiviral drugs or vaccines are available, and treatment is limited to supportive care and few repurposed drugs. In this urgency situation, computational drug discovery methods provide both an alternative and a supplement to tiresome high-throughput screening, particularly in the hit-to-lead-optimization stage. Identification of small molecules that specifically target viral replication apparatus has shown the most successful strategy in antiviral drug discovery. The present study deals with the identification of potential compounds that specifically interact with SARS-CoV-2 vital proteins, including main protease (Mpro), Nsp12 RNA-dependent-RNA-polymerase (RdRp) and Nsp13 helicase. A constructive and integrated virtual screening efforts together with molecular dynamics simulations identified potential binding modes and favourable molecular interaction profile of corresponding compounds. Moreover, structurally important binding site residues in conserved motifs located inside the active site are elucidated, which displayed relative importance in ligand binding based on residual energy decomposition analysis. Although the current study lacks experimental validation, the structural information obtained from this computational study paved the way to identify and design specific targeted inhibitors to combat COVID-19 outbreak.

Mitochondria are classically termed as powerhouse of a mammalian cell. Most of the cellular chemical energy in the form of adenosine tri phosphate (ATP) is generated by mitochondria and dysregulation of mitochondrial functions thus can be potentially fatal of cellular homeostasis and health. Acute respiratory distress has been earlier linked to mitochondrial dysfunction. SARS-CoV-2 infection severity leads to acute respiratory distress syndrome (ARDS) and can be fatal. We tried to investigate possible connection between SARS-CoV-2, ARDS and mitochondria. Here, we report identification of SARS-CoV-2 non-structural proteins (particularly Nsp12 and 13) that have recognition sequence with respect to mitochondrial entry. We also report that these proteins can potentially shuttle between cytoplasm and mitochondria based on the localization signals and help in downstream maintenance of the virus. Their properties to use ATP for enzymatic activities may cause ATP scavenging allowing viral RNA functions in lieu of host cell health.

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKUI1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to human, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.

Starting from December 2019, coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures and supporting apparatuses. Remdesivir was reported by World Health Organization (WHO) as the most promising drug currently available for the treatment of COVID-19. Here, we use molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). In the absence of a crystal structure of the SARS-CoV-2 RdRp, we first construct the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identify of 95.8%). We then build the putative binding mode by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp. The putative binding structure is further optimized with molecular dynamics simulations and demonstrated to be stable, indicating a reasonable binding mode for remdesivir. The relative binding free energy of remdesivir is calculated to be -8.28 ± 0.65 kcal/mol, much stronger than the natural substrate ATP (-4.14 ± 0.89 kcal/mol) which is needed for the polymerization. The ~800-fold improvement in the K_d from remdesivir over ATP indicates an effective replacement of APT in blocking of the RdRp binding pocket. Key residues D618, S549 and R555 are found to be the contributors to the binding affinity of remdesivir. These findings demonstrate that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA reproduction, with key residues also identified for future lead optimization and/or drug resistance studies.

Recent emergence of novel coronavirus (SARS-CoV-2) all over the world has resulted more than 33,106 global deaths. To date well-established therapeutics modules for infected patients are unknown. In this present initiative, molecular interactions between FDA-approved antiviral drugs against the Hepatitis-C virus (HCV) have been investigated theoretically against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. HCV and SARS-CoV-2 are both +ssRNA viruses. At 25^o C beclabuvir, a non-nucleoside inhibitor of the RdRp_HCV can efficiently bind to RdRp _SARS-CoV-2 (ΔG_AutoDock = -9.95 kcal mol^-1) with an inhibition constant of 51.03 nM. Both the ΔG_London and ΔG_{GBVI / WSA} values were - 9.06 and - 6.67 kcal mol^-1, respectively for binding of beclabuvir to RdRp_SARS-CoV-2. In addition, beclabuvir has also shown better binding free energy with RdRp_SARS-CoV-2 (ΔG_vina = -8.0 kcal mol^-1) than that observed with the Thumb 1 domain of RdRp_HCV (ΔG_vina = -7.1 kcal mol^-1). InterProScan has suggested the RNA-directed 5'-3' polymerase activity exists within 549^th to 776^th amino acid residues of RdRp_SARS-CoV, where the major amino acid residues interacting being I591, Y621, C624, D625, A690, N693, L760, D762, D763, and E813-N817. Molecular interaction suggests occupancy of beclabuvir inside the active site environment of the RdRp_SARS-CoV-2, the enzyme essential for viral RNA synthesis. In conclusion, results suggest beclabuvir may serve as an anti-SARS-CoV-2 drug.

The severity of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), greatly varies from patient to patient. In the present study, we explored and compared mutation profiles of SARS-CoV-2 isolated from mildly affected and severely affected COVID-19 patients in order to explore any relationship between mutation profile and disease severity. Genomic sequences of SARS-CoV-2 were downloaded from GISAID database. With the help of Genome Detective Coronavirus Typing Tool, genomic sequences were aligned with the Wuhan seafood market pneumonia virus reference sequence and all the mutations were identified. Distribution of mutant variants was then compared between mildly and severely affected groups. Among the numerous mutations detected, 14,408C>T and 23,403A>G mutations resulting in RNA-dependent RNA polymerase (RdRp) P323L and spike protein D614G mutations, respectively, were found predominantly in severely affected group (>82%) compared with mildly affected group (<46%, p<0.001). The 241C>T mutation in the non-coding region of the genome was also found predominantly in severely affected group. The 3,037C>T, a silent mutation, also appeared in relatively high frequency in severely affected group. We concluded that RdRp P323L and spike protein D614G mutations predominate in severely affected COVID-19 patients. Further studies will be required to explore whether these mutations have any impact on the severity of COVID-19.

Lumpy skin disease (LSD), caused by a virus within the Poxviridae family and Capripoxvirus genus, induces nodular skin lesions in cattle, spreading through direct contact and insect vectors, significantly affecting global cattle farming. Despite available vaccines, their effectiveness is limited by poor bioavailability and adverse effects. Our study aimed to identify potential inhibitors targeting LSD-associated DNA polymerase protein, selecting LSDV039 for further investigation through comprehensive analysis and computational methods. Virtual screening revealed rhein and taxifolin as potent binders among 380 phytocompounds, with respective affinities of -8.965 and -7.195 kcal/mol. Canagliflozin and tepotinib exhibited strong affinities (-9.858 and -8.856 kcal/mol) among 718 FDA-approved antiviral drugs. Molecular dynamics simulations of canagliflozin, tepotinib, rhein, and taxifolin highlighted taxifolin's superior stability and binding energy. Rhein displayed compactness in RMSD and RMSF but fluctuations in Rg and SASA, while canagliflozin demonstrated stability compared to tepotinib. The study highlights the promising potential of repurposed drugs and phytocompounds as LSD therapeutics. However, extensive validation through in-vitro, in-vivo, and clinical trials is crucial for their practical application.

Since mid-December of 2019, coronavirus disease 2019 (COVID-19) has been spreading from Wuhan, China. As of February 21, total 75,773 confirmed cases worldwide have spread to more than two dozen countries. Transmission of COVID-19 can occur early in the course of infection since SARS-CoV-2 viral loads in asymptomatic patients are similar to that in the symptomatic patients. Therefore, more sensitive diagnostic methods are needed to detect early phase of the infection to prevent secondary or tertiary spreads. Here, we compare the RT-PCR confirmatory test results using two different SARS-CoV-2 viral RNAs from two Korean COVID-19 confirmed cases.RT-PCR method targeting the RdRP gene, which was recommended by WHO guideline, was less sensitive than targeting N genes (as per CDC guideline). Because many countries follow the WHO guideline, our findings may contribute to the early diagnosis of COVID-19.

Establishment of diagnostic methods with low detection limits plays a critical role in the maintenance of early diagnosis, prevention of serious neurological complications, and control of the spread of ZIKA. In this study, we established the micro-droplet digital polymerase chain reaction (ddPCR) and real-time fluorescent quantification PCR (qPCR) protocols for the detection of Zika virus based on the NS5 gene. For the Zika standard plasmid, the standard curve of R² was 0.999, and the amplification efficiency was 92.203%, as determined by qPCR. Both ddPCR and qPCR were positive for cell culture of Zika nucleic acid.The minimum detection limit of ddPCR is 1–2 times lower than qPCR. Moreover, all tests of Dengue virus (1–4 serotypes) were negative in cell culture. Overall, these results suggested than ddPCR may have a lower limit of detection than qPCR.

Background: Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2. In Colombia, many commercial methods are now available to perform the RT-qPCR assays, and the laboratories must evaluate its diagnostic accuracy to ensure reliable results to suspected COVID-19 patients. The purpose of the study was to compare four commercial RT-qPCR assays for detection of SARS-CoV2 virus, from nasopharyngeal swab samples referred to Laboratorio Carvajal IPS, SAS of Tunja, Boyacá - Colombia. Methods: This prospective study was conducted on 152 samples of respiratory tract samples (Nasopharyngeal Swab) from patients with suspected SARS-CoV-2 infection. Diagnostic accuracy of GeneFinderTM COVID-19 Plus RealAmp (In Vitro diagnostic), One-Step Real-Time RT-PCR (Vitro Master diagnostica), Berlin modified protocol and gold standard Berlin protocol (Berlin Charite Probe One-Step RT-qPCR Kit, New England Biolabs) as reference was assessed. Operational characteristics were estimated in terms of sensitivity, specificity, agreement, and predictive values. Results: Using Berlin Charite Probe One-Step RT-qPCR Kit as reference, the sensitivity/specificity for the diagnostic tests were found to be GeneFinderTM COVID-19 Plus RealAmp Kit 100%/92.7%, One-Step Real-Time RT-PCR, One-Step Real-Time RT-PCR 92.75%/67.47%, and Berlin modified protocol 100%/96.39%. The results of four commercially available methods were found to be consistent with those obtained from Berlin Modified protocol analysis for % of the samples and showed good agreement (κ= 0.96). Concordant SARS-CoV2 negative and positive RT-qPCR results were reported for xxx and xxx samples, respectively. Summarize something about the Ct. Conclusion: Our data demonstrate that all commercially available methods are rapid and reliable for the identification of SARS-CoV-2 virus associated with COVID-19. One-Step RT-qPCR Kit and GeneFinderTM COVID-19 Plus RealAmp assay show optimal sensitivity compared with Belin modified protocol. In addition, there is no significant correlation between xxxxx

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, PDIP46 and PDIP38, both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the splicesosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, Mdm2 alternative splicing and the regulation of the Nox4 NADPH oxidase.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3’-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and found to occur with the homologous domains of influenza B and C FluPols. Residues [1-72] of RPB4 were found critical in this interaction. Numerous punctual mutants generated at conserved positions between influenza A, B and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defect. These results suggest that FluPol interacts with several domains/subunits of Pol II, the CTD to bind Pol II initiating host transcription and a second on RPB4 to locate FluPol at the proximity of the 5’-end of nascent host mRNA.

The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values &lt; 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzo[d]isothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure-activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzo[d]isothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values ~ 1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection.

Polymerase I (Pol I) is at the epicenter of ribosomal RNA (rRNA) synthesis. Pol I is a target for treatment of cancer. Given the many cellular commonalities between cancer and neurodegeneration (i.e., different faces of the same coin) it seems rational to consider targeting of Pol I or, more generally, rRNA synthesis for treatment of disorders associated with the death of terminally differentiated neurons. Principally, ribosomes synthesize proteins and, accordingly, Pol I can be considered the starting point for protein synthesis. Given that cellular accumulation of abnormal proteins such as α-synuclein and tau is an essential feature of neurodegenerative disorders such as Parkinson disease and fronto-temporal dementia, reduction of protein production is now considered as a viable target for treatment of these and closely related neurodegenerative disorders. Abnormalities in polymerase I activity and rRNA production may also be associated with nuclear and nucleolar stress, DNA damage, and childhood onset neuronal death as is the case for the UBTF E210K neuroregression syndrome. Moreover, restraining the activity of Pol I may be a viable strategy to slow aging. Before starting down the road of Pol I inhibition for treating non-cancerous disorders of the nervous system, many questions must be answered. First, how much Pol I inhibition can neurons tolerate and for how long? Should inhibition of Pol I be continuous or pulsed? Will cells compensate for Pol I inhibition by upregulating the number of active rDNAs? At present, we have no effective and safe disease modulatory treatments for Alzheimer disease, α-synucleinopathies, or tauopathies, and novel therapeutic targets and approaches must be explored.

DNA polymerases replicate cell genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with E. coli DNA polymerase I (pol I), have been grouped in Family A. Pol I participates in Okazaki fragment maturation and in bacterial genome repair. Since its discovery in 1956, pol I has been extensively studied, primarily to get deeper insights into the mechanism of DNA replication. As research on DNA polymerases advances, many novel functions of this group of polymerases are uncovered. For example, human DNA polymerase θ (a Family A DNA pol) has been shown to synthesize DNA using RNA as a template, a function typically attributed to retroviral reverse transcriptase. A heightened interest in drug discovery against pol θ has emerged due to its roles in cancer. Likewise, Pol I family enzymes also appear attractive drug-development targets against microbial infections. Development of antimalarial compounds targeting apicoplast apPOL, an ortholog of Pol I, further extends the druggability of this family of enzymes. Here, we summarize reported drug-development efforts against Family A polymerases, and future perspective regarding these enzymes as antibiotic targets. Current techniques such artificial intelligence can be used to facilitate the goal of new drugs.

Transcriptional regulations have been widely studied as one of the main bridges between biology and other basic sciences as well as medicine. The traffic across it has been mostly unidirectional: chemistry and physics provided a lot of tools for biology, although the supply is now saturating. The traffic in opposite direction, the supply of subjects to develop chemistry and physics, has been only a little. However, if there are any, the supply will be at least from transcription, because the notion of chemical reaction is the strongest. This topic is aimed to increase the opposite traffic by introducing the forefront of physicochemical studies of transcription.

Previously, we described that Adenine, Thymine, Cytosine, and Guanine nucleobases were superconductors in a quantum superposition of phases on each side of the central Hydrogen bond acting as a Josephson Junction. Genomic DNA has two strands wrapped helically around one another, but during transcription, they are separated by the RNA polymerase II to form a molecular condensate called the transcription bubble. Successive steps involve the bubble translocation along the gene body. This work discusses how quantum information transfer and entanglement can be achieved by modeling DNA as a combination of ? nonperturbative circuits quantum electrodynamics with nine Radio-Frequency- Superconducting Quantum Interference Devices (SQUIDs) inside. A bus can be coupled capacitively to a single-mode microwave resonator. The cavity mode and the bus can mediate long-range, fast interaction between neighboring and distant DNA SQUID qubits. RNA polymerase II produces decoherence during transcription. This enzyme is a multifunctional biomolecular machine working like an artificially engineered device. Phosphorylation reactions catalyzed by protein kinases constitute the driving force. The coupling between n phosphorylation pulses and any particular SQUID qubit can be obtained selectively via frequency matching.

Immunosuppressed individuals, such as people living with HIV (PLWH), remain vulnerable to severe COVID-19. We analyzed the persistence of specific SARS-CoV-2 humoral and cellular immune responses in a retrospective, cross-sectional study in PLWH on antiretroviral therapy. Among 104 participants, 70.2% had anti-S IgG antibodies and 55.8% had significant neutralizing activity against the Omicron variant in a surrogate virus neutralization test. Only 38.5% were vaccinated (8.76 ± 4.1 months prior), all displaying anti-S IgG, 75% with neutralizing antibodies and anti-S IgA. 29.8% had no SARS-CoV-2 serologic markers and displayed significantly lower CD4 counts and HIV viral load. Severe immunosuppression (present in 12.5% of participants) was linked to lower levels of detectable anti-S IgG (0.0003), anti-S IgA (p &lt; 0.0001) and lack of neutralizing activity against Omicron variant (p &lt; 0.0001). T-cell responses were present in 86.7% of tested participants, even in those lacking serological markers. In PLWH without severe immunosuppression, neutralizing antibodies and T-cell responses persisted for up to 9 months post-infection or vaccination. Advanced immunosuppression led to diminished humoral immunity, but retained cellular immune.

During the last few years, the world witnessed one of the worst calamities to humankind- the COVID-19 pandemic. We also witnessed the development of vaccines at an unprecedented pace against the SARS-CoV-2- the causative agent. India was among the hard-hit countries by COVID-19, developed its indigenous vaccine and implemented vaccines developed in other countries as well. Initially, the antibody level and the neutralizing antibody titer against the pathogen were considered as the correlates of immune protection from the disease. However, the long-term protection from the pathogen was provided by the immune memory cells viz. T and B memory cells and their ability to efficiently make recall responses. In this regard, globally the research studies focused mainly on the mRNA-based vaccines. The studies on immune memory response, particularly- the B cell memory response bestowed by the vaccines given to Indians is relatively obscure. We studied the level of RBD specific memory B cells in the peripheral circulation of the study participants and the ability of B cells to secrete antigen-specific antibodies among Indians vaccinated with Covaxin (BBV152), Covishield (AZD1222), Corbevax (BECOV2D) and Sputnik Light (Gam-COVID-Vac) as well as unvaccinated individuals. Our study revealed that Corbevax and Sputnik Light conferred better antibody-secreting cell (ASC) responses.

(1) Background: Several chronic respiratory diseases could be a risk factor for acquiring SARS-CoV-2 infection: among them, Primary Ciliary Dyskinesia (PCD) is a rare (about 1:10.000) inherited ciliopathy (MIM 242650) characterized by recurrent upper and lower respiratory tract infections due to a dysfunctioning of the respiratory cilia; We aimed to investigate if some polymorphisms of the TAS2R38 bitter taste receptor correlate with an increased prevalence of SARS-CoV-2 infection and severity of symptoms in a cohort of PCD subjects; (2) Methods: Patients answered several questions about possible SARS-CoV-2 infection, experienced symptoms and vaccinations; in case of infection, they also filled out a SNOT-22 and ARTIQ questionnaires; (3) Results: Forty PCD adult patients (mean age 36.6 ± 16.7 years, 23 females) participated to the study, out of which 30 % had tested positive for COVID-19 during the last four years; most of them reported a mildly symptomatic disease. We found no differences in age or sex, but a statistic significant difference (p=0.03) was observed in body mass index (BMI), that was higher in the COVID-acquired group (23.2 ± 3.3 vs 20.1 ± 4.1 kg/m2). Genotyping for TAS2R38 polymorphisms showed a prevalence of 28.6% PAV/PAV, 48.6% PAV/AVI and 22.8% AVI/AVI individuals in our cohort. In contrast to our hypothesis, we did not observe a protective role of PAV allele towards SARS-CoV-2 infection; (4) Conclusions: Our findings suggest that subjects with PCD may not be at increased risk of severe outcomes from COVID-19 and the TAS2R38 bitter taste receptor genotype type does not affect SARS-CoV-2 infection.

Introduction: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection may cause acute respiratory failure but also remains responsible for many other pathologies, including electrolyte disorders. SARS-CoV-2 infection can cause dysfunction of the renin-angiotensin-aldosterone system and disrupt water hemostasis with thirst and appetite abnormalities. Dysnatremia affects prognosis and may be associated with mortality in patients admitted to SARS-CoV2 intensive care. Patients and Methods: The study included 209 patients admitted to the Intensive Care Unit between April 12, 2021, and March 1, 2022 who were over 18 years old and diagnosed with SARS-CoV-2 infection by clinical and thoracic tomography findings or with a positive reverse transcription polymerase chain reaction (RT-PCR) test result. The laboratory markers, treatment modalities, nutritional, and respiratory support also for outcome evaluation, length of stay in the ICU, total hospitalization duration, and mortality in ICU were recorded.The laboratory marker comparison was made admission with the final assessment performed before the time of mortality in ICU or discharge. Results: Inotropic requirements among patients were high which was reflected in the first-month mortality. Hypernatremia presence was associated with an increase in enteral support and inotropic support requirement and mortality. Hypernatremia was correlated with diabetes mellitus, chronic renal failure, and a longer duration under mechanical ventilation. Discussion: Hypernatremia was an important risk factor in ICU patients hospitalized for SARS-CoV-2 infection, which was also affected by the treatment regimens given itself. This complex relationship underlies the importance of proper electrolyte management, especially in patients who were under severe stress and organ failure.

The continues evolution of coronaviruses has gained considerable attention prior to the outbreak of COVID 19. Although, COVID-19 is currently not recognized as a global pandemic, the virus's ability to evolve and cause disease outbreaks in both humans and animals necessitates ongoing research into its genetic and structural characteristics. Therefore, understanding the molecular origin, rapid evolution, as were as the immunogenicity of coronaviruses is a critical step towards preparing for and preventing future outbreaks. This study aimed to uncover the genetic diversity and evolutionary relationships among the main viral proteins of SARS-CoV-2 and other related coronaviruses. Amino acid sequences of structural viral proteins spike glycoprotein, nucleocapsid protein, membrane protein, and envelope protein were retrieved from the NCBI database for SARS-CoV-2 and 13 other coronaviruses. Here, we identified that SARS-CoV-2 is closely related to BatCoV RaTG13 and Pangolin-CoV, suggesting their potential role in the emergence of the virus. Additionally, we predicted novel epitopes, glycosylation sites, and cleavage sites, identifying three novel glycosylation sites (NATR, NXSN, and NGTK) and six new CTL epitopes (XSNQVAVLY, NSFTRGVYY, NATRFASVY, STQDLFLPF, NSASFSTFK, and ASFSTFKCY). Our comparative analysis revealed that BatCoV RaTG13 has the closest cleavage site to SARS-CoV-2, with significant similarities also observed with Pangolin-CoV. The study highlighted the unique features of SARS-CoV-2, including its furin cleavage site, which may account for its high infectivity during the pandemic. These findings contribute to the understanding of SARS-CoV-2 evolution and its relationship with other coronaviruses, providing critical insights for the development of broad-spectrum vaccines and antiviral therapies.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Typically, mouse-adaptation studies are conducted in inbred laboratory strains BALB/c and C57BL/6, which have defects in the antiviral Mx1 gene that results in increased susceptibility to infection and disease severity. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering these three adaptive PB1 and PA mutations into the parental CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions, and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

Neurological involvement following COronaVIrus Disease 19 (COVID-19) is thought to have a neuroinflammatory etiology. Co-ultraPEALut (an antiinflammatory molecule) and luteolin (an antioxidant) yielded promising results as neuroinflammation antagonists. This study aimed at describing cognitive impairment in post-COVID patient treated with co-ultraPEALut. Montreal Cognitive Assessment (MoCA), Prospective-Retrospective Memory Questionnaire (PRMQ), Fatigue Severity Scale (FSS) and a subjective evaluation were administered at baseline and after 10 months. Co-ultraPEALut-treated patients were retrospectively compared with controls. 26 co-ultraPEALut-treated patients showed significant improvement in PRMQ (T0: 51.94±10.55, T1: 39.67±13.02, p

Early identification of fetal abnormalities is a huge challenge for modern obstetrics. Quantitative fluorescent polymerase chain reaction (QF-PCR) has quickly become an effective means of chromosome anomaly detection due to its advantages in terms of timing, manpower and accuracy. The QF-PCR results also make a significant change in clinicians’ attitude to some extent, assisting them providing parents with professional and valuable advice on pregnancy management. In this review, the advantages and drawbacks of QF-PCR will be explored. By reviewing studies published in Vietnamese Medical Journals, we conclude QF-PCR can become a potential screening test in the field of prenatal diagnosis.

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. In this respect the identification of single agents with multitarget potential might represent the ideal solution. Accordingly, a small library of biphenylhydrazo 4-arylthiazoles derivatives has been synthesised and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity.

The outbreak of novel coronavirus (COVID-19) infections in 2019 is in dire need of finding potential therapeutic agents. In this study, we used molecular docking to repurpose HIV protease inhibitors and nucleoside analogues for COVID-19, with evaluations based on docking scores calculated by AutoDock Vina and RosettaCommons. Our results suggest that Indinavir and Remdesivir possess the best docking scores, and comparison of the docking sites of the two drugs reveal a near perfect dock in the overlapping region of the protein pockets. After further investigation of the functional regions inferred from the proteins of SARS coronavirus, we discovered that Indinavir does not dock on any active sites of the protease, which may give rise to concern in regards to the efficacy of Indinavir. On the other hand, the docking site of Remdesivir is not compatible with any known functional regions, including template binding motifs, polymerization motifs and nucleoside triphosphate (NTP) binding motifs. However, when we tested the active form (CHEMBL2016761) of Remdesivir, the docking site revealed a perfect dock in the overlapping region of the NTP binding motif. This result suggests that Remdesivir could be a potential therapeutic agent. Clinical trials still must be done in order to confirm the curative effect of these drugs.

In this study, we investigated the features of co-infection with SARS-CoV-2 and nonpathogenic strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-СoV-2 and LEV-8 or EV-A71 in the cell model was shown that a competitive inhibitory effect for these viruses were especially significant against SARS-CoV-2. Pre-infection with enteroviruses in animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tracts and more rapid clearance of the lower respiratory tract from infectious SARS-CoV-2. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection for animals. Additionally, the histological data illustrated that co-infection with nonpathogenic coxsackievirus decreased the level of pathological changes by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of antiviral immune response, which can be associated with significant inhibition of SARS-CoV-2 infection. These results demonstrate that there exists a strong viral interference between the studied nonpathogenic strain of coxsackievirus A7 or enteroviruses A71 and SARS-CoV-2 pathogenic for humans in vitro and in vivo.

Background: The rise of new SARS-CoV-2 mutations brought challenges and progress in the global fight against COVID-19. Mutations in spike and accessory genes affect transmission, vaccine efficacy, treatments, testing, and public health strategies. Monitoring emerging variants is crucial to halt virus spread. Methods: 44 nasopharyngeal/oropharyngeal swabs from Kenyan patients were sequenced with the Illumina platform. Galaxy's bioinformatic tools were used for genomic analysis. SARS-CoV-2 genome classification was done using PANGOLIN and mutation annotation with the COVID-19 Annotator tool. Results: The study showed 5 clades to be circulating in the region. 38(86%) were BA.1.1; 2(5%) were BA.1.1.1; 1(2%) was BA.1; 1(2%) was BA.1.14 and 2(5%) were AY.46. These clades had a cumulative of 173 mutations among them with 50 novel mutations. Forty-eight of these novel mutations occurred in a low frequency of 2.3% of the sequences tested while the other two, S:R214R, and NSP2:A555A, were for 43.2% and 18.2% of the cases respectively. Conclusions: The high-frequency novel mutations were synonymous mutations, a phenomenon that was previously viewed as phenotypically silent but recent studies indicate they can affect viral fitness with potential functional associations. These findings add to the understanding of the SARS-CoV-2 virus future evolutionary and immunological dynamics in the region

Objective: The aim of our study was to perform a retrospective analysis of the volume of cervical screening tests, the number of patients treated with an excision method, and the incidence of invasive and non-invasive cervical during a pandemic and pre-pandemic period of 24 months. Results: There was a statistically significant age difference between the two study periods, both by the average age of patients and by age group. The mean difference was 32 years before the pan-demic and 35 years during the pandemic (p-value >0.05). The majority of patients presenting for investigations before and during the pandemic were in the 30-39-year-old age group (31.95%, respectively; 34.2% (p-value = 0.003). The biggest patient loss ratio identified by age group was in the 50–59 years: 14,53% in the pre-pandemic period and 9,1% in the pandemic period. In the pan-demic period, patients from rural areas presented in the clinical trial with a lower rate of 39.52% (83 patients) vs. 60.47% (127 patients) in urban areas. A higher percentage of patients experiencing cervicorrhagia as a clinical manifestation in the pandemic period vs. the prepandemic period, with an increase in more severe lesions in the pandemic period, has a statistical significance of 8% more newly diagnosed compared to the pre-pandemic period. Conclusion: The addressability of the patients during the COVID period was not affected in a drastic way in our study. We encountered a decrease in appointments in the age group 50–59 years and a decrease in patients with rural residence. In our study, we found an increase in cervicorrhagia as a reason for consultation in the pandemic period with a higher lesion degree, both on a pap smear and on a cervical biopsy.

Based on screening in computational biology and biological in vitro assays, five natural products isolated from extracts of the herbal medicine toad skin, such as cinobufagin (CBFi), bufalin (BFi), arenobufagin (ABFi), telocinobufagin (TBFi), bufotalin (BFTi), were subjected to molecular docking calculations with the use of SARS-CoV-2 main protease (PDB 6LU7 and 7BTF) and top-scoring ligand-receptor complexes were obtained. The results showed that the binding energy of ABFi to the 3CL protein was -17.044kcal/mol, which was higher than CBFi and TBFi. However, the binding energy of ABFi to the RdRp protease was -23.250 kcal/mol, which was much lower than that of CBFi and TBFi, EVEN lower than that of ABFi to the 3CL protein. ABFi also has polar interactions with amino acids such as Glu811, Ser814, Ser681 and Thr680 of RdRp enzyme. The results revealed that ABFi had a moderate inhibitory effect on the cell proliferation of SARS-CoV-2 in vitro, with an inhibition rate of 61.12%, even weaker than Remdesivir. This new discovery provides us with new ideas for in-depth studies on the development of natural products with this class of structural generalizations as inhibitors of SARS-CoV-2, and provides an experimental basis for the next step of mechanistic studies.

The detection, characterization, and monitoring of SARS-CoV-2 recombinant variants is a challenge to public health authorities worldwide. Recombinant variants, composed of two or more SARS-CoV-2 lineages, often have unknown impacts on transmission, immune escape, and virulence in the early stages of emergence. We examined 4,213 SARS-CoV-2 recombinant SARS-CoV-2 genomes collected between 2020 and 2022 from California to describe regional and statewide trends in prevalence. Many of these recombinant genomes, such as those belonging to the XZ lineage, or novel recombinant lineages, likely originated within the state of California. We discuss the challenges and limitations surrounding Pango lineage assignments, the use of publicly available sequence data, and adequate sample sizes for epidemiologic analyses. Although these challenges will continue as SARS-CoV-2 sequencing volumes decrease globally, this study enhances our understanding of SARS-CoV-2 recombinant genomes to date while providing a foundation for future insights into emerging recombinant lineages.

The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase–DNA–dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease of PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) continues to wreak havoc threatening the public health services and imposing economic collapse worldwide. Tailoring public health responses to the SARS-CoV-2 pandemic depends on understanding the mechanism of viral replication, disease pathogenesis, and accurately identifying acute infections and mapping the spreading risk of hotspots across the globe. However, effective identification and isolation of persons with asymptomatic and mild SARS-CoV-2 infections remain the major obstacles to efforts in controlling the SARS-CoV-2 spread and hence the pandemic. Understanding the mechanism of persistent viral shedding, reinfection, and the post-acute sequalae of SARS-CoV-2 infection (PASC) is crucial in our efforts to combat the pandemic and provide better care and rehabilitation to survivors. Here we present a living literature review on SARS-CoV-2 viral persistence, reinfection and PASC. We also highlight potential areas of research to uncover putative links between viral persistence, intra-host evolution, host immune status, and protective immunity to guide and direct future basic science and clinical research priorities.

Abstract: The emergence of the COVID-19 pandemic has led to the rapid and worldwide devel-opment and investigation of multiple vaccines. While most side effects of these vaccines are mild and transient, potentially severe adverse events may occur and involve the endocrine system. This narrative review aims to explore the current knowledge on potential endocrine adverse effects following COVID-19 vaccination, with thyroid disorders being the most common. Data about pi-tuitary, adrenal, diabetes, and gonadal events will also be reviewed. This review also provides a comprehensive understanding of the pathogenesis of endocrine disorders associated with SARS-CoV-2 vaccines. A PubMed/MEDLINE, Embase database (Elsevier), and Google Scholar research were performed. Case reports case series, original studies, and reviews written in English and published online up to 31 August 2023 were selected and reviewed. Data on endocrine adverse events of SARS-CoV-2 vaccines is accumulating. However, their causal relationship with COVID-19 vaccines is not strong enough to make a definite conclusion, and further studies are needed to clarify the pathogenesis mechanisms of endocrine disorders linked to COVID-19 vac-cines.

The study investigated the application of Wastewater-Based Epidemiology (WBE) as a tool for monitoring SARS-CoV-2 prevalence in a city in Northern Italy from October 2021 to May 2023. Starting from a widely applied deterministic model, this study proposed a variation to take into account population characteristics and virus biodegradation in the sewer network. The model calculated virus loads and corresponding COVID-19 cases over time in different areas of the city and was validated with healthcare data while considering viral mutations, vaccinations, and testing variability. The correlation between the predicted and reported cases was high across the three waves that occurred in the considered period, demonstrating the model’s capacity to predict relevant fluctuations in the number of cases. Population characteristics did not substantially influence the predicted and reported infection rates. Conversely, biodegradation significantly reduced the virus load reaching the wastewater treatment plant, resulting in a 30% reduction in the total virus load produced in the study area. This approach can be applied for comparing virus load values across cities with varying population demographics and sewer network structures, enhancing the comparability of WBE data for effective surveillance and intervention strategies.

Nuclear DNA mutations, mitochondrial DNA mutations, combined nuclear and mitochondrial DNA defects, and random occurrences are the main causes of mitochondrial disease. Black pigmentation loss is a major sign of Parkinsonism. Because of the loss of dopaminergic neurons in the substantia nigra, it drops to less than 80%. The degree of motor impairment was correlated with the amount of black pigmentation loss. After RNA extraction, reverse transcription is carried out because reverse transcriptase was used to create cDNA. One microliter of total RNA supplemented with IORT Buffer, dNTPs, random hexamers, Multi-Scribe Reverse Transcriptase, and nuclease-free was used to create cDNA. The following was how the reaction was carried out in a thermal cycler: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C, and lastly 4°C. The data was expressed as a v-value, which shows how much the target gene is enriched in comparison to the reference gene. Earlier research using mice injected with functional HtrA2/Omi protease activity revealed similar outcomes, including induced up-regulation of CHOP, decreased mitochondrial function, and early brain cell aging because of an increase in mtDNA deletions. The cell viability of the genotypes exhibits a dmg concentration-dependent effect in these results, suggesting that high content ratios of drug treatment with 6-OHDA may be the cause of cellular content depletion. After 6-OHDA neurotoxin treatment, CHOP is up-regulated. Prior in vitro research has shown that treatment with a model based on 6-OHDA neurotoxin causes the activation of mitochondrial stress pathways, which in turn promotes the death of neurons. 6-OHDA reversibly inhibited complexes I and IV's activities in rat brain cells, which led to dopaminergic neurodegeneration, a defining feature of PD pathogens. As a result, the number of living cells will directly correlate with the dmg concentration, supporting the results of the current study. Real-time PCR can generally demonstrate that the removal of HtrA 2 results in a significant up-regulation of Hsp60 expression when compared to WT cells. This could be linked to a decreased threshold for mitochondrial stress in HtrA2 KO cells, which would explain why, following ADEP4 and ACP5 treatment, Hsp60 is significantly up-regulated in HtrA2 KO cells compared to WT cells.

From gene expression studies to identifying microbes quantitative polymerase chain reaction (qPCR) is widely used in research and medical diagnostics. In transmittable diseases like the Ebola outbreak in West Africa (2014-2016), or the present SARS-CoV2 pandemic qPCR plays a key role in the detection of infected patients. Although the technique itself is decades old with reliable approaches (eg. TaqMan essay) in the diagnosis of pathogens many people showed distrust in it during the SARS-CoV2 outbreak. This came mainly from not understanding or misunderstanding the principles of qPCR. This situation motivated us to design a simple laboratory practical class, in which students have opportunities to understand the underlying principles of qPCR and its advantages in microbiological diagnosis. Moreover, during the exercise, students can develop skills such as handling experimental assays, and the ability to solve problems, discuss their observations. Finally, this activity brings them closer to the clinical practice and they can see the impact of the science on real life. The class is addressed to undergraduate students of biological sciences.

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery has fundamental functional consequences for gene expression, its detailed studies have been for long time abrogated by the limits of classical light microscopy. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

Global public health systems have continuously faced challenges from the advent of novel infectious illnesses, necessitating quick and efficient actions to stop their spread.First discovered in Saudi Arabia in 2012, Middle East respiratory syndrome (MERS) is a viral respiratory disorder caused by the Middle East respiratory syndrome coronavirus (MERS-CoV). Since then, the nation has reported most cases and fatalities related to this sickness, with occasional outbreaks happening in other nations and regions. Severe sickness and high mortality are caused by MERS-CoV, especially in older persons and those with underlying chronic conditions. Through intimate contact, the virus can spread from people to dromedary camels and vice versa. It has sparked multiple epidemics both domestically and abroad in the Middle East, raising the possibility of fatalities and widespread transmission, especially in susceptible groups. The Riyadh region was the main target of the most current MERS-CoV outbreak in Saudi Arabia, which began in 2023 and ended on September 1, 2023, with 312 confirmed cases and ninety-seven fatalities. With the use of published studies and their contributions to the field, we hope to present a thorough overview of the MERS-CoV outbreak in Saudi Arabia in this review of the literature, including its epidemiological investigation, public health response, impact, and lessons learned. In addition to highlighting the importance and continued relevance of this problem, our work suggests ways to lower the risk of MERS-CoV transmission and avert further outbreaks.

Currently, SARS-CoV-2 has evolved into various variants, including the numerous highly mutated Omicron sub-lineages, significantly increasing immune evasion ability. The development raises concerns about possibly diminished effectiveness of available vaccines and antibody-based therapeutics. Here, we describe those representative categories of broadly neutralizing antibodies (bnAbs) that retain prominent effectiveness against emerging variants including Omicron sub-lineages. The molecular characteristics, epitope conservation, and resistance mechanisms of these antibodies are further detailed, aiming to offer suggestion or direction for the development of therapeutic antibodies, and facilitate the vaccine design with broad-spectrum potential.

Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of metal ions removed and overall binding capacity. Microorganisms are a key component in wastewater treatment plants and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose-sialic acid (Gal—NeuNAc) heteropolysaccharide binds lead effectively, thereby demonstrating the potential for using this capsular polysaccharide in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis N-acetylgalactosamine-sialic acid (GalNAc—NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics simulations (MD) with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a duplex RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). TPHD-RPA assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 minutes at 42 oC. The duplex RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Compared with commercial multiplex real-time PCR, the TPHD-RPA assay demonstrated 94-95% sensitivity for T. pallidum and H. ducreyi confirmed samples, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication programs by ensuring effective diagnosis as well as enable monitoring of eradication efforts success or failure and planning of follow-up interventions at the community level.

COVID-19 pandemic in first seven months has led to more than 15 million confirmed infected cases and 600,000 deaths. SARS-CoV-2, the causative agent for COVID-19 has proved a great challenge for its ability to spread in asymptomatic stages and a diverse disease spectrum it has generated. This has created a challenge of unimaginable magnitude not only affecting human health and life but also potentially generating a long-lasting socioeconomic impact. Both medical sciences and biomedical research have also been challenged consequently leading to a large number of clinical trials and vaccine initiatives. While known proteins of pathobiological importance are targets for these therapeutic approaches, it is imperative to explore other factors of viral significance. Accessory proteins are one such trait that have diverse roles in coronavirus pathobiology. Here we analyze certain genomic characteristics of SARS-CoV-2 accessory protein ORF8, predict upon its protein features and review current available literature regarding its function. We have also undertaken review of ORF8 homolog ORF8ab from SARS-CoV with a purpose of developing holistic understanding of these proteins for reason that coronaviruses have been infecting humans repeatedly and might continue to do so. Despite low nucleotide and protein identity and differentiating genome level characteristics, there appears to be significant structural integrity and functional proximity between these proteins pointing towards their high significance. There is further need for comprehensive genomics and structural-functional studies to lead towards definitive conclusions regarding their criticality and that can eventually define their relevance to therapeutics development.

The novel coronavirus disease (COVID-19) pandemic has resulted in over 720 million confirmed cases and 7 million deaths worldwide, with insufficient treatment options. Innumerable efforts are being made around the world for faster identification of therapeutic agents to treat the deadly disease. Postacute sequelae of SARS-CoV-2 infection or COVID-19 (PASC), also called Long COVID, is still being understood and lacks treatment options as well. A growing list of drugs are being suggested by various in silico, in vitro and ex vivo models, however currently only two treatment options are widely used: the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir, and the main protease inhibitor nirmatrelvir in combination with ritonavir. Computational drug development tools and in silico studies involving molecular docking, molecular dynamics, entropy calculations and pharmacokinetics can be useful to identify new targets to treat COVID-19 and PASC, as shown in this work and our recent paper that identified alendronate as a promising candidate. We have now investigated all bisphosphonates which can bind competitively to nidovirus RdRp-associated nucleotidyl (NiRAN) transferase domain, and systematically down selected seven candidates (CHEMBL608526, CHEMBL196676, CHEMBL164344, CHEMBL4291724, CHEMBL4569308, CHEMBL387132, CHEMBL98211), two of which closely resemble the approved drugs minodronate and zoledronate. This work and our recent paper together provide an in silico mechanistic explanation for alendronate and zoledronate users having dramatically reduced odds of SARS-CoV-2 testing, COVID-19 diagnosis, and COVID-19-related hospitalizations, and indicate that similar observational studies with minodronate could be valuable.

We discuss the role of water bridging the DNA-enzyme interaction by resorting to recent 1 results showing that London dispersion forces between delocalized electrons of base pairs of DNA 2 are responsible for the formation of dipole modes that can be recognized by Taq polymerase. 3 We describe the dynamical origin of the high efficiency and precise targeting of Taq activity in 4 PCR. The spatiotemporal distribution of interaction couplings, frequencies, amplitudes, and phase 5 modulations comprise a pattern of fields instantiating the electromagnetic image of DNA in its 6 water environment, which is what the polymerase enzyme actually recognizes at long range. The 7 experimental realization of PCR amplification, achieved through replacement of the DNA template 8 by the treatment of pure water with electromagnetic signals recorded from viral and bacterial DNA 9 solutions, is found consistent with the gauge theory paradigm of quantum fields.

We construct a Bayesian estimator for the dilution of a wastewater sample. When wastewater is diluted by rainwater an estimate of the level of dilution is required if we are to normalise sample measurements of disease markers such as SARS-CoV-2 RNA. We consider the situation where flow measurements are available, but will regularly be unreliable because of the action of combined sewer outflows (CSOs) and wastewater storage tanks, which divert wastewater away from treatment plants when the flow is excessive. However, we also have proxies for the flow from various chemical markers (e.g.\ phosphate, ammonium, electrical conductivity), whose level per unit of population is fixed. The new flow estimator has multiple advantages compared to existing procedures: credible intervals for the estimates; optimal weighting of the chemical markers; systematic handling of missing and censored values; and model based smoothing without lags.

The quality of cellular products used in biological research can directly impact the ability to ob-tain accurate results. Epstein-Barr virus (EBV) is a latent virus that spreads extensively world-wide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. We developed three EBV detection systems: (1) a polymerase chain reac-tion (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based de-tection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The mini-mum EBV detection limits were 1×103 copy numbers for the RPA-based and RPA-LFA systems and 1×104 copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those obtained by the EBV assay methods specified in the pharma-ceutical industry standards of the People's Republic of China. 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid results visualization. This system can be implemented in laborato-ries and cell banks as part of a daily quality control strategy to ensure cell quality and experi-mental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

Tuberculosis (TB) is a contagious disease, caused by Mycobacterium tuberculosis (MTB) that has infected and killed a lot of people in the past. At present treatments against TB are available at a very low cost. Since these chemical drugs have many adverse effects on health, more attention is now given on the plant-derived phytochemicals as potential agents to fight against TB. In this study, 5 phytochemicals, 4-hydroxybenzaldehyde, benzoic acid, bergapten, psoralen, and p-hydroxybenzoic acid, are selected to test their potentiality, safety, and efficacy against two potential targets, the MTB RNA polymerase and enoyl-acyl carrier protein (ACP) reductase, the InhA protein, using various tools of in silico biology. The molecular docking experiment, drug-likeness property test, ADME/T-test, P450 SOM prediction, pharmacophore mapping, and modeling, solubility testing, DFT calculations, and PASS prediction study had confirmed that all the molecules had the good potentiality to inhibit the two targets. However, two agents, 4-hydroxybenzaldehyde and bergapten were considered as the best agents among the five selected agents and they also showed far better results than the two currently used drugs, that function in these pathways, rifampicin (MTB RNA polymerase) and isoniazid (InhA protein). These two agents can be used effectively to treat tuberculosis.

The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have abnormally hard outer shell (low M disorder; i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far was found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2 especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 amd later in Omicron, is associated with attenuation according to the Shell-Disorder-Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis.

Background: The effectiveness of the anti-COVID-19 vaccine remains a matter to be clarified. Methods: The humoral and cellular immunity after the administration of three doses of the Pfizer–BioNTech and Oxford AstraZeneca vaccines against SARS-CoV-2 over one year and the appearance of post-vaccination COVID-19 have been studied. Results: Anti-SARS-CoV-2 IgG and IgA antibodies showed a progressive increase throughout the vaccination period. This increase was greatest after the third dose. The highest levels were observed in subjects who had anti-SARS-CoV-2 antibodies prior to vaccination. There was an increase in CD4+ αβ, CD8+ γδ and TEM CD8+ γδ T cells, and a decrease in apoptosis in CD4+CD8+ and CD56+ αβ and γδ T cells. Post-vaccination SARS-CoV-2 infection was greater than 60%. The symptoms of COVID-19 were very mild and were related to γδ T cell deficit, specifically CD8+ TEMRA and CD56+ γδ TEM, as well as lower pre-vaccine apoptosis levels. Conclusions: Those results demonstrate the important role that γδ T cells play in SARS-CoV-2 vaccine immunization.

Papaya is a globally important crop, with production primarily based on hermaphrodite plants. Papaya has three sex types: male, female, and hermaphrodite, determined by flower morphology until the flowering stage. In this study, a recombinase polymerase amplification (RPA) assay was developed and optimized to identify the three sexes of papaya. Recombinant uvsX, uvsY, gp32, and Bsu DNA polymerase were used to study the effects of temperature, reaction time, and sensitivity conditions for RPA reaction efficiency. The optimal conditions were found to be 41°C and a 30-minute reaction time, allowing the detection of the target sex from specific DNA markers, even when using crude extract. The study shows that RPA could be used for sex determination in papaya, and the findings could contribute to developing a point-of-need strategy due to their sensitivity and specificity.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) shows that inflammation starts early and progresses with age. B cells play a central role in this process, contributing to cytokine production, defective regulatory functions, and abnormal immunoglobulin production, even in the central nervous system. Anti-CD20 (aCD20) therapies, which deplete CD20+ B cells, are effective for both relapsing-remitting (RR) and progressive-relapsing (PR) MS. While effective against MS symptoms and lesions detectable by magnetic resonance imaging, aCD20 therapies can reduce the immune response to COVID-19 vaccination. By using high-parameter flow cytometry, we examined the antigen-specific (Ag+) immune response six months post-third COVID-19 mRNA-vaccination in MS patients with RR and PR forms on aCD20 therapy. Despite lower Ag+ B cell responses and lower levels of anti-SARS-CoV2, both total and neutralizing antibodies, RR and PR patients developed strong Ag+ T cell responses. We observed similar percentages and numbers of Ag+ CD4+ T cells and high proportion of Ag+ CD8+ T cells, with slight differences in T cell phenotype and functionality, that however suggested the presence of differences in immune responses driven by age and disease severity.

SARS-CoV2 RNA depended RNA polymerase is an essential enzyme for the survival of the virus in hosts as it helps in the replication of viral RNA. There are no human polymerases that share either sequence or structural homology with viral RNA depended RNA polymerase. These make it a good target for inhibitor discovery, as a specific inhibitor cannot cross-react with the human polymerases. We have used virtual screening, docking, binding energy calculation and simulation to show that valproic acid Co-A, a metabolite from prodrug valproic acid, forms stable interaction with nsP12 of CoV. Our results suggest valproic acid Co-A could be a potential inhibitor of nsP12 of SARS-CoV2.

The ongoing SARS-CoV-2 pandemic has raised concerns surrounding immunological protection against the virus, particularly for people with inborn errors of immunity (IEI). While COVID-19 vaccination induces robust antibody, memory B-cell, and T-cell responses in healthy individuals, how well vaccination protects people with IEI against infection and severe disease remains unclear – especially in the context of new viral variants and vaccine formulations – leading to anxiety, ongoing self-isolation, and repeated vaccination with limited evidence of increased efficacy. Whilst most people with IEI generate some level of cellular and/or humoral immunity to COVID-19 vaccination, the level of protection and durability of the response are unclear. Alongside vaccination, antibody-based therapeutics are aimed at limiting infection and severe disease in this cohort. Immunoglobulin replacement therapy (IgRT) provides passive immunity in antibody-deficient individuals. However, to successfully prevent SARS-CoV-2 infection, a sufficient number of neutralizing antibodies must be present in product. While antibodies against circulating variants are eventually present in IgRT products (both from natural infection and vaccination of the donors), evidence of their capacity to recognize new variants is limited. Furthermore, while antibody-based pre- and post-exposure prophylaxis can be effective, they are susceptible to decreased efficacy in the context of variant evolution. This review provides an in-depth overview of current knowledge about COVID-19 vaccine efficacy in IEIs, the efficacy of SARS-CoV-2-specific antibody products, and knowledge and technological advances required for continued protection of people with IEI.

The SARS-CoV-2 polybasic furin cleavage site is still a missing link. Remarkably, the two arginine residues of this protease recognition site are encoded by the CGG codon, which is rare in Betacoronavirus. However, the arginine pair is common at viral furin cleavage sites, but are not CGG-CGG encoded. The question is: Is this genetic footprint unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers of the Betacoronavirus proteins. The main result reveals that a group of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) also have the CGG-CGG arginine pair in the spike protein polybasic furin cleavage region. In addition, CGG-CGG encoded arginine pairs were found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the probability of finding the arginine CGG-CGG codon pair in Betacoronavirus, the likelihood ratio (LR) and a Markov model were defined. In conclusion, it is highly unlikely to find this genetic marker in betacoronaviruses wildlife, but they are there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine pair in the S1/S2 cleavage site.

The high transmissibility, rapid evolution, and immune escape of SARS-CoV-2 variants can influence the course of infection and, in turn, morbidity and mortality in COVID-19, posing a challenge in controlling transmission rates and contributing to the emergence and spread of new variants. The factors that shape viral genetic variation are consequently essential for understanding the evolution and transmission of SARS-CoV-2, especially in vaccinated individuals where immune response plays a role in the progression and spread of this disease. This study assessed. In this context, we evaluated the impact of immunity induced by the CoronaVac vaccine (Butantan/Sinovac) on intra-host genetic diversity, analyzing 118 whole-genome sequences of SARS-CoV-2 from unvaccinated and vaccinated patients infected with the Gamma variant. Vaccination with CoronaVac favors negative selection at the intra-host level in different genomic regions and prevent greater genetic diversity of SARS-CoV-2, which may help reduce the emergence of new mutations, reinforcing the importance of vaccination in reducing virus transmission.

The COVID-19 pandemic over the past years has shown a great need for rapid, low-cost and on-site detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, an aptamer-based colloidal gold nanoparticle lateral flow test strip was well developed to realize the visual detection of wild type SARS-CoV-2 spike protein (SP) and multiple variants. Under the optimal reaction conditions, a low detection limit of SARS-CoV-2 S protein of 0.68 nM was acquired, and the actual detection recovery was 83.3% to 108.8% for real world samples. This suggests a potential tool for the prompt detection of SARS-CoV-2 with good sensitivity and accuracy, and a new method for the development of alternative antibody test strips for the detection of other viral targets.

Influenza B virus (IBV) is a major respiratory pathogen of humans, particularly in the elderly and children and vaccines are the most effective way to control it. In previous work, incorporation of two mutations (E580G, S660A) along with the addition of a HA epitope tag in the PB1 segment of B/Brisbane/60/2008 (B/Bris) resulted in an attenuated strain that was safe and effective as a live attenuated vaccine. A third attempted mutation (K391E) in PB1 was not always stable. Interestingly, viruses that maintained the K391E mutation were associated with the mutation E48K. To explore the contribution of the E48K mutation for stability of the K391E mutation, a vaccine candidate was generated by inserting both mutations along with attenuating mutations E580G and S660A in PB1 of B/Bris (B/Bris PB1att 4M). Serial passage of the B/Bris PB1att 4M vaccine candidate in eggs and MDCK indicated high stability. In silico structural analysis revealed a potential interaction between amino acids at positions 48 and 391. In mice, B/Bris PB1att 4M was safe and provided complete protection against homologous challenge. These results confirm the compensatory effect of mutation E48K to stabilize the K391E mutation, resulting in a safer, yet still protective, IBV LAIV vaccine.

Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy, verified by bio-statistical analysis (Pearson correlation coefficient, R=0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Zika virus (ZIKV) has been characterized as one of the potential pathogens and placed under future epidemic outbreaks by the WHO. However, lack of potential therapeutics can result in uncontrolled pandemic like other human pandemic viruses; therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted the strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the ZIKVpro and ZIKVRdRp proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of top four common bioflavonoids, viz. rutin, nicotiflorin, isoquercitrin, and hyperoside. These compounds showed substantial docking energy (-7.9 to -11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (His51, Asp74, and Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals the stability of respective docked complexes contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp for further experimental assessment.

A number of studies suggested that influenza vaccination can provide protection against COVID-19 but the underlying mechanisms that could explain this association is still unclear. In this study, the effect of the 2021/2022 seasonal influenza vaccination on the immune response to the booster dose of anti-SARS-CoV-2 vaccination was evaluated in a cohort of healthy individuals. One hundred and thirteen participants were enrolled, and 74 of them with no prior COVID-19 diagnosis or significant co-morbidities were considered for the analysis. Participants received the anti-influenza tetravalent vaccine and the booster dose of the anti-SARS-CoV-2 vaccine or the anti-SARS-CoV-2 vaccine alone. Blood was collected before and 4 weeks after each vaccination and 12 weeks after SARSCoV2 vaccination and analyzed for anti-flu and anti-spike-specific antibody titers and for in vitro influenza and SARS-CoV-2 neutralization capacity. Results indicated an increased reactivity in subjects who received both influenza and SARSCoV2 vaccinations, as compared to those who received only the SARS-CoV-2 vaccine, with sustained anti-spike antibody titers up to 12 weeks post-vaccination. Immune response to influenza vaccine was evaluated and individuals were stratified as high or low responders. High responders showed increased antibody titers against SARSCoV2 vaccine both after 4- and 12-weeks post-vaccination. On the contrary, the individuals classified as low responders were less responsive to the SARS-CoV-2 vaccine. These data indicate that both external stimuli, such as influenza vaccination, and the host's intrinsic ability to respond to stimuli play a role in the response to vaccine.

Rapid SARS-CoV-2 self-tests have the potential to expand access to COVID-19 testing and improve community-level case detection, particularly in resource-constrained countries such as Kenya. However, prior to their introduction, their acceptability must be assessed. This qualitative study explored key decision-takers’ values towards SARS-CoV-2 self-testing in Kenya. Healthcare workers, representatives of civil society, and potential implementors from Mombasa and Taita-Taveta were selected as decision-takers. Semi-structured interviews and focus group discussions were used to collect data on their values towards self-testing. A thematic analysis approach was applied. Most informants considered that the Kenyan public is equipped to accept and use self-testing safely as an approach to help to reduce workload at public healthcare facilities, and know one’s COVID-19 status in a private manner. The informants emphasized the need to provide counselling to end-users, to support those needing to self-isolate, and to engage different civil society stakeholders in information provision on self-testing. Fear of stigma and of forced isolation were noted as potential deterrents to self-testing uptake for some individuals. In conclusion, there is high acceptability of self-testing in Kenya among decision-takers. However, enhanced education, counselling, and addressing deterrents to testing would be helpful to ensure effective use of SARS-CoV-2 self-testing in Kenya.

Introduction: Coronaviruses are zoonotic viruses that include human epidemic pathogens such as the Middle East Respiratory Syndrome virus (MERS-CoV), and the Severe Acute Respiratory Syndrome virus (SARS-CoV), among others (e.g., COVID-19, the recently emerging coronavirus disease). The role of animals as potential reservoirs for such pathogens remains an unanswered question. No systematic reviews have been published on this topic to date. Methods: We performed a systematic literature review with meta-analysis, using three databases to assess MERS-CoV and SARS-CoV infection in animals and its diagnosis by serological and molecular tests. We performed a random-effects model meta-analysis to calculate the pooled prevalence and 95% confidence interval (95%CI). Results: 6,493articles were retrieved (1960-2019). After screening by abstract/title, 50 articles were selected for full-text assessment. Of them, 42 were finally included for qualitative and quantitative analyses. From a total of 34 studies (n=20,896 animals), the pool prevalence by RT-PCR for MERS-CoV was 7.2% (95%CI 5.6-8.7%), with 97.3% occurring in camels, in which pool prevalence was 10.3% (95%CI 8.3-12.3). Qatar was the country with the highest MERS-CoV RT-PCR pool prevalence, 32.6% (95%CI 4.8-60.4%). From 5 studies and 2,618 animals, for SARS-CoV, the RT-PCR pool prevalence was 2.3% (95%CI 1.3-3.3). Of those, 38.35% were reported on bats, in which the pool prevalence was 14.1% (95%CI0.0-44.6%). Discussion: A considerable proportion of infected animals tested positive, particularly by nucleic acid amplification tests (NAAT). This essential condition highlights the relevance of individual animals as reservoirs of MERS-CoV and SARS-CoV. In this meta-analysis, camels and bats were found to be positive by RT-PCR in over 10% of the cases for both; thus, suggesting their relevance in the maintenance of wild zoonotic transmission.

Seroprevalence of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies in the postvaccination period in Kenya remains to be elucidated. This study aimed to determine the seroprevalence of anti-SARS-CoV-2 IgM and IgG and evaluate vaccination uptake in Nairobi, Kenya. This was a cross-sectional study conducted in a university setting. Serum anti-SARS-CoV-2 IgM and IgG levels were assayed using enzyme-linked immunosorbent assays. Mann–Whitney U test was used for binary comparisons and Kruskal–Wallis Test for multigroup comparisons. Statistical significance was set at p &lt; 0.05. A total of 189 participants were enrolled (median age, 21 years; female, 50.8%). The seroprevalence of anti-SARS-CoV-2 was 12.7% for IgM and 87.8% for IgG. Anti-SARS-CoV-2 IgG titers were higher among the vaccinated vs. non-vaccinated individuals (p &lt; 0.001, U = 2817.5), females vs. males (p = 0.024, U = 3616), and those vaccinated &lt; 6 months before the study vs. those vaccinated &gt;1 year earlier (p = 0.002, H = 12.359). The vaccination hesitancy rate was 43.4% and the underlying reasons included mistrust (22.4%), health concerns (19.7%), and lack of information (18.4%). Despite the high seroprevalence of anti-SARS-CoV-2 IgG, the high vaccine hesitancy rate necessitates community engagement and education prior to vaccines roll out.

The main proteinase (Mpro), or 3CLpro, is a critical enzyme in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle and is responsible for breaking down and releasing vital functional viral proteins crucial for virus development and transmission. As a catalytically active dimer, its dimerization interface has become an attractive target for antiviral drug development. Recent research has extensively investigated the enzymatic activity of Mpro, focusing on its role in regulating the coronavirus replication complex and its significance in virus maturation and infectivity. Computational investigations have identified four druggable pockets, suggesting potential allosteric sites beyond the substrate-binding region. Empirical validation through site-directed alanine mutagenesis targeted residues in both the active and allosteric regions corroborated these predictions. Structural studies of drug target proteins can inform therapeutic approaches, with metadynamics simulations shedding light on the role of H163 in regulating Mpro function and providing insights into its dynamic equilibrium to the wild-type enzyme. Despite the efficacy of vaccines and drugs in mitigating SARS-CoV-2 spread, ongoing viral evolution, selective pressures, and continued transmission pose challenges, potentially leading to resistance mutations. Phylogenetic analyses indicated the existence of several resistance variations predating drug introduction to the human population, emphasizing the likelihood of drug spread. Hydrogen/deuterium-exchange mass spectrometry reveals the structural influence of the mutation, while clinical trials on 3CLPro inhibitors underscore the clinical significance of reduced enzymatic activity and offer avenues for future therapeutic exploration. Understanding the implications of 3CLPro mutations holds promise for shaping forthcoming therapeutic strategies against COVID-19. This review delves into factors influencing mutation rates and identifies areas warranting further investigation, providing a comprehensive overview of Mpro mutations, their categorization, and terminology. Moreover, we examined their associations with clinical outcomes, illness severity, unresolved issues, and future research prospects, including their impact on vaccine efficacy and potential therapeutic targeting.

Wastewater-based surveillance has emerged as an important method for monitoring SARS-CoV-2. This study investigated the presence of SARS-CoV-2 in wastewater in Zambia. We conducted a longitudinal study in the Copperbelt and Eastern provinces of Zambia from October 2023 to December 2023 during which 155 wastewater samples were collected. The samples were subjected to three different concentration methods, namely bag-mediated filtration, skimmed milk flocculation, and polythene glycol-based concentration assays. Molecular detection of SARS-CoV-2 nucleic acid was conducted using real-time PCR. Whole genome sequencing was conducted using Illumina COVIDSEQ assay. Of the 155 wastewater samples, 62 (40%) tested positive for SARS-CoV-2. Of these, 13 sequences of sufficient length to determine SARS-CoV-2 lineages were obtained and two sequences were phylogenetically analyzed. Various Omicron subvariants were detected in wastewater including BA.5, XBB.1.45, BA.2.86, and JN.1. Some of these subvariants have been detected in clinical cases in Zambia. Interestingly, phylogenetic analysis positioned a sequence from the Copperbelt Province in the B.1.1.529 clade, suggesting that earlier Omicron variants detected in late 2021 could still be circulating and may not have been wholly replaced by newer subvariants. This study stresses the need for integrating wastewater surveillance of SARS-CoV-2 into mainstream strategies for monitoring SARS-CoV-2 circulation in Zambia.

To date, viruses closely related to SARS-CoV-2 have been reported in four bat species: Rhinolophus acuminatus, Rhinolophus affinis, Rhinolophus malayanus, and Rhinolophus shameli. Here, we analysed 343 sequences of the mitochondrial cytochrome c oxidase subunit 1 gene (CO1) from georeferenced bats of the four Rhinolophus species identified as reservoirs of SARS-CoV-2-like viruses. Haplotype networks were constructed in order to investigate patterns of genetic diversity among bat populations of Southeast Asia. No strong geographic structure was found for the four Rhinolophus species, suggesting high dispersal capacity. The ecological niche of SARS-CoV-2 like viruses was predicted using the four localities of bat SARS-CoV-2-like viruses and the localities where bats showed identical or very similar CO1 haplotypes than virus-positive bats. The ecological niche of SARS-CoV-like viruses was deduced from the localities where bat SARS-CoV-like viruses were previously detected. The results show that the ecological niche of SARS-CoV2-like viruses includes several regions of mainland Southeast Asia whereas that of SARS-CoV-like viruses is mainly restricted to China. In agreement with these results, human populations in Laos, Vietnam, Cambodia, and Thailand appear to be much less affected by the Covid-19 pandemic than other countries of Southeast Asia. In the climatic transitional zone between the two ecological niches (southern Yunnan, northern Laos, northern Vietnam, and possibly Hainan and Taiwan), genomic recombination between highly divergent viruses is more likely to occur. Since recombinant viruses can threaten the benefit of vaccination campaigns, these regions should be under surveillance.

In this study, the analysis of the changes of SARS-CoV-2 Orf3a protein during pandemic is reported. Orf3a, a conserved protein in the Coronaviruses, is involved in virus replication and release. A software workflow able to carry out a quick, systematic and repeatable screening of the SARS-CoV-2 genome isolates to detect protein mutations, was utilized to scan 70,752 high-quality SARS-CoV-2 genomes available in GISAID databank at the end of August 2020. All ORF3a mutations in the virus genomes were grouped according to the collection date interval and over the entire data set. The considered intervals were start of collection-February, March, April, May, June, July and August 2020. The top five most frequent variants were examined within each collection interval. Overall, seventeen variants have been isolated. Ten of the seventeen mutant sites occur within the transmembrane (TM) domain of ORF3a and are in contact with the central pore or side tunnels. The other variant sites are in different places of the Orf3a structure. Within the entire sample, the five most frequent mutations are V13L, Q57H, Q57H+A99V, G196V and G252V. The same analysis identified 28 sites identically conserved in all the genome isolates. These sites are possibly involved in stabilization of monomer, dimer, tetramerization and interaction with other cellular components. The results here reported can be helpful to understand virus biology and to design new therapeutic strategies.

The coronavirus disease 2019 (COVID-19) is now a global pandemic caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unlike other known coronaviruses, such as the Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 reveals new clinical, immunological, and pathologic features. The lymphocyte depletion, macrophage and neutrophil hyperactivation, cytokine dysregulation, thrombophilia, delayed antiviral response, and immune exhaustion are key immunological findings linked to the clinical progression of this disease. Understanding and identifying the underlying immunological basis of COVID-19 is crucial to designing effective therapies. Here, we provide an overview of immunopathogenesis driven by SARS-CoV-2 after its interactions with the immune system.

Wastewater-based epidemiology (WBE) has emerged as a key method for continuous monitoring of COVID-19 prevalence including circulating SARS-CoV-2 lineages. WBE addresses limitations of traditional clinical COVID-19 surveillance such as clinical test availability, fluctuating testing rates and increased reliance on rapid antigen tests. Our study in Perth, Western Australia, found a significant positive correlation between SARS-CoV-2 concentrations in wastewater and clinical PCR positivity rates (rs = 0.772; p < 0.001) over an 18-month period that included four successive COVID-19 waves. A strong positive correlation was apparent between the proportions of SARS-CoV-2 lineages in wastewater and clinical cases within the same region (rs = 0.728; p < 0.001), including earlier detection of Omicron and recombinant lineages in wastewater before clinical case confirmation. The successful integration of WBE with healthcare data underscores its critical role in enhancing public health decision-making and pandemic management. This approach not only demonstrates the value of WBE in current global health surveillance efforts but also highlights the potential of WBE to address future public health challenges, as a comprehensive disease monitoring and response approach.

Background: Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Methods: Peripheral lymphocyte lysates were analysed for Poly(ADP-Ribose) Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibody to measure automodified anti-Poly(ADP-Ribose) Polymerase levels by densitometry. Results: Poly(ADP-Ribose)Polymerase activity levels were consistent with those of enzyme auto-modification. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. Conclusions: PARP activity and auto-modification in peripheral blood lymphocytes are possible,non-invasive, and routinar tools to monitor the healthy conditions of floricoltorists.

Abstract5′,8-Cyclo-2′-deoxyadenosine (cdA), in the 5′R and 5′Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the diastereomeric cdA lesions could induce specific PARP1 binding. Here we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions 5′S-cdA and 5′R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism,fluorescence spectroscopy, immunoblotting analysis and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1were characterized. Remarkably, PARP1 efficiently binds to both cdA lesions in the double stranded (ds)-oligonucleotides. In particular, PARP1 proved to bind 5′S-cdAwith a higher affinity constant for the 5'S lesion in a model of ds DNA than 5′R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.

According to our previous investigation the total methanol extract from Graptopetalum paraguayense E. Walther demonstrates a significant inhibitory effect on HSV-1. To clarify what causes this inhibitory activity on HSV-1, a metabolic profile of the plant was performed. Three main fractions: non-polar substances, polar metabolites and phenolic compounds were obtained and GC-MS analysis was carried out. Since it is well known that phenolic compounds show a significant anti-herpes effect and that viral DNA polymerase (DNApol) appears to play a key role in HSV virus replication, we present a docking and quantum-chemical analysis of the binding of these compounds to viral DNApol amino acids. Fourteen different phenolic acids found by GS/MS analyses, were used in molecular docking simulations. According to the interaction energies of all fourteen ligands in the DNApol pockets based on docking results, DFT calculations were performed on the five optimally interacting with the receptor acids. It was found that hydroxybenzoic acids from phenolic fraction of Graptopetalum paraguayense E. Walther show a good binding affinity to the amino acids from the active site of the HSV DNApol, but significantly lower than that of acyclovir. The mode of action on virus replication of acyclovir (by DNApol) is different from that of the plant phenolic acids one, probably.

To understand the evolution of the human norovirus GII.P6-GII.6 and GII.P7-GII.6 strains, we analyzed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

SARS-CoV-2 can induce vascular dysfunction and thrombotic events in patients with severe COVID-19; however, the cellular and molecular mechanisms behind these effects remain largely unknown. In this study, we used a combination of experimental and in silico approaches to explore the mechanisms behind the vascular and thrombotic events in COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the publicly available Gene Expression Omnibus (GEO) repository. In addition, HUVECs were treated with inactivated protein C before exposure to SARS-CoV-2 infection or a severe COVID-19 serum. An RT-qPCR array containing 84 related genes was used, and the candidate genes obtained were evaluated. Activated protein C levels were measured using an ELISA kit. We identified at the single-cell level the expression of several pro-inflammatory and pro-coagulation genes in endothelial cells from the patients with COVID-19. Furthermore, we demonstrated that exposure to SARS-CoV-2 promoted transcriptional changes in HUVECs that were partly reversed by the activated protein C pretreatment. We also observed that the serum of severe COVID-19 had a significant amount of activated protein C that could protect endothelial cells from serum-induced activation. In conclusion, activated protein C protects endothelial cells from pro-inflammatory and pro-coagulant effects during exposure to the SARS-CoV-2 virus.

Summary CoV-GLUE is an online web application for the interpretation and analysis of SARS-CoV-2 virus genome sequences, with a focus on amino acid sequence variation. It is based on the GLUE data-centric bioinformatics environment and provides a browsable database of amino acid replacements and coding region indels that have been observed in sequences from the pandemic. Users may also analyse their own SARS-CoV-2 sequences by submitting them to the web application to receive an interactive report containing visualisations of phylogenetic classification and highlighting genomic variation of potentially high impact, for example linked to primer mismatches.Availability and implementation Available at http://cov-glue.cvr.gla.ac.uk. Implemented using GLUE, an open source framework for the development of virus sequence data resources. Contact [email protected]

The current globally dominant SARS-CoV-2 variants are showing immune escape and reduced susceptibility to antiviral drugs. Therefore, agencies responsible for drug evaluation and regulation such as the FDA and EMA are revising their emergency authorization use of several COVID-19 neutralizing antibodies. These MAbs proved to be unlikely effective against new variants especially Omicron descendants and several pharmaceutical companies are pursuing the development of more potent neutralizing antibodies. To address this issue, we used In Silico method we previously developed to assess 10 anti-SARS-CoV-2 antibodies propensity to neutralize the new Omicron’s subvariants EG.5, BA.2.86 and JN.1, based on comparative binding affinity of 3D generated models and previous experimental and clinical observations. Nine of these MAbs were once granted emergency use authorization, and one is currently under clinical investigation. The results showed that one antibody showed a marked increase of the binding energy for EG.5 compared to two antibodies that showed a significant increase with Pirola (BA.2.86) and JN.1. This data indicates that the new SARS-CoV-2 variant escapes neutralization of most of the available therapeutic NAbs. Furthermore, the data showed new potential therapeutic MAbs combination that could be effective for the treatment countermeasure of the new Omicron’s descendants or potential novel variants.

The polymerase chain reaction (PCR) is a widely used technique in the biosciences and has become increasingly popular in recent years. One of the key elements of this technique is the use of a DNA polymerase that is heat-stable and retains fidelity during the process. To this end, archaeal Fam-ily-B DNA polymerases are preferred due to their high thermostability and fidelity. In particular, the DNA polymerase from Thermus aquaticus (Taq DNApol) is widely utilized in PCR procedures. In this work, a novel in-silico structure-based methodology was employed to examine the most heat-tolerant DNA polymerase available. In spite of this, Thermococcus kodakarensis and Geobacillus stearothermophilus DNApol are more stable and heat-tolerant DNApols due to their high number of intra-protein interactions. Variations in the content of polar amino acids also played a significant role in the increase in heat stability. A further factor contributing to the stability of proteins is the stabilization of helix in secondary structure through the use of charged amino acids. DNApol from these organisms has been shown to be suitable for use in PCR, as well as in other biological processes able to withstand high temperatures. In this study, it has been demonstrated that im-provements in PCR performance can be easily obtained by blending elements from closely related archaeal polymerases, a strategy that may, in the future, be extended to other archaeal polymer-ases. This approach allowed for a comprehensive analysis of the enzyme's thermal stability and fidelity, leading to an improved understanding of the polymerase's properties and potential ap-plications

Background/Objectives: Survival prospects following SARS-CoV-2 infection may extend beyond the acute phase, influenced by various factors including age, health conditions, and infection se-verity. However, this topic has not been studied in detail. We therefore investigated mortality risk post-acute COVID-19 in the CRIT-COV-U cohort. Methods: We could retrieve survival data from 651 patients and also assess the association between urinary peptides and future death. Data spanning until December 2023 were collected from six countries, comparing mortality trends with age- and sex-matched non-infected controls. A death prediction classifier was developed and validated using pre-existing urinary peptidomics datasets. Results: Notably, 13.98% of post-COVID-19 patients succumbed during the follow-up, with mortality rates significantly higher than non-infected controls, particularly evident in younger individuals (&lt;65 years). These data for the first time demonstrate that SARS-CoV-2 infection highly significantly increases the risk of mortality not only during the acute phase of the disease, but also beyond, for a period of about one year. In our study we were further able to identify 201 Urinary peptides linked to mortality, and integrated these into a predictive classifier (DP201). Higher DP201 scores, alongside age and BMI, significantly predicted death. Conclusions: Our findings underscore the utility of urinary peptides in prognosticating post-acute COVID-19 mortality, offering insights for targeted interventions. Our data also suggest that mortality should be considered as one possible symptom or conse-quence of post-acute sequelae of SARS-CoV-2 infection, a fact that is currently overlooked.

SARS-CoV-2 causes severe pneumonia epidemics and probably originated in horseshoe bats, but the intermediate host is unknown. The interaction of SARS-CoV-2 spike protein and its acceptor protein ACE2 is an important issue in determining viral host range and cross-species infection, while the binding capacity of Spike protein to ACE2 of different species is unknown. Here, we used the atomic structure model of SARS-CoV-2 and human ACE2 to assess the receptor utilization capacity of ACE2s from different species including cats, chimpanzees, dogs, cattles. Our results show, domestic cats (Felis catusc) and dogs (Canis lupus familiaris) are more susceptible to infection by SARS-CoV-2 and that they can efficiently transmit the virus to previously uninfected animals that are housed with them. Especially, cats could be a choice of animal model for screening antiviral drugs or vaccine candidates against SARS-CoV-2.

2019-n-CoV is the seventh coronavirus known to infect humans and cause serious illness, and due to its lethality there was a worldwide mobilization for the development of new drugs and new vaccines for Covid-19. Therefore, the present work aims to carry out a virtual screening of terpenes with potential anti SARS-CoV-2 activity. To perform the ligand-based virtual, a model was constructed regarding the compounds which already showed inhibitory activity of the SARS-CoV-2. The test series corresponded to terpenes isolated in the Fabaceae Family. The compounds that were most likely to activity were subjected to Molecular Docking and the best poses obtained were subjected to Molecular Dynamics (MD) simulations carried. The molecules under study were submitted to in vitro evaluation to assess inhibitory activity of compounds against SARS-CoV-2 in a phenotypic screening model of the virus. The compounds selected by in silico screening corresponded to triterpenoids from the lupane series. In the in vitro test, both demonstrated activity against the SARS-CoV-2 virus. The virtual screening performed managed to identify the compounds with the highest probability of activity, as well as the most stable compounds, and the method used was validated by the in vitro study carried out.

Background: to analyze the impact of COVID-19 pandemic on characteristics and management of uveal melanoma (UM) in National Referral Center in Poland. Materials and Methods: the retrospective analysis of 1336 patients who were newly diagnosed with UM at Department of Ophthalmology and Ophthalmic Oncology, Jagiellonian University Collegium Medicum Krakow, Poland between January 1, 2018 and December 31, 2021. The demographic and clinical data were compiled including localization, size and treatment methods of tumors. Results: In total, 728 patients with UM were included before COVID-19 pandemic in years 2018-2019 and 608 were included during COVID-19 pandemic in years 2020-2021. Fixed-base dynamics indicators for the incidence of uveal melanoma (base year 2018) in the national referral center in Poland were 80.22% and 86.81% in year 2020 and 2021, respectively. UMs were statistically significantly larger and more frequently localized anterior to the equator of the eye globe in year 2021 than in year 2018 (Chi^2 Pearson test p= 0.0001 and p= 0.0077, respectively). The rate of patients treated with enucleation increased from 15.94% in year 2018 to 26.90% in year 2021 (Chi^2 Pearson test p=0.0005). Conclusions: statistically significant differences were found in the management of uveal melanoma in National Referral Center in Poland during COVID-19 pandemic with tumors being larger, more frequently localized anterior to the equator of the eye globe and more often enucleated.

Phylogenetic trees of coronaviruses are difficult to interpret because they undergo frequent ge-nomic recombination. Here, we propose a new method, named coloured genomic bootstrap (CGB) barcodes, to highlight the polyphyletic origins of human sarbecoviruses and understand their host and geographic ori-gins. The results indicate that SARS-CoV and SARS-CoV-2 contain genomic regions of mixed an-cestry originating from horseshoe bat (Rhinolophus) viruses. First, different regions of SARS-CoV share exclusive ancestry with five Rhinolophus viruses from Southwest China (RfYNLF/31C: 17.9%; RpF46: 3.3%; RspSC2018: 2.0%; Rpe3: 1.3%; RaLYRa11: 1.0%) and 97% of its genome can be related to bat viruses from Yunnan (China), supporting its emergence in Rhinolophus species of this province. Second, different regions of SARS-Cov-2 share exclusive ancestry with eight Rhi-nolophus viruses from Yunnan (RpYN06: 5.8%; RaTG13: 4.8%; RmYN02: 3.8%), Laos (RpBA-NAL103: 3.3%; RmarBANAL236: 1.7%; RmBANAL52: 1.0%; RmBANAL247: 0.7%), and Cam-bodia (RshSTT200: 2.3%), and 98% of its genome can be related to bat viruses from northern Laos and Yunnan, supporting its emergence in Rhinolophus species of this region. Although CGB barcodes are very useful to retrace the origins of human sarbecoviruses, further investigations are needed to better apprehend the diversity of coronaviruses in bats from Cambo-dia, Laos, Myanmar, Thailand and Vietnam.

Various studies indicate that vaccination, especially with pneumococcal vaccines, protects against symptomatic cases of SARS-CoV-2 infection and death. This paper explores the possibility that pneumococcal vaccines in particular, but perhaps other vaccines as well, contain antigens that might be cross-reactive with SARS-CoV-2 antigens. Comparison of the glycosylation structures of SARS-CoV-2 with the polysaccharide structures of pneumococcal vaccines yielded no obvious similarities. However, while pneumococcal vaccines are primarily composed of capsular polysaccharides, some are conjugated to CRM197, a modified diphtheria toxin, and all contain about three percent protein contaminants, including the pneumococcal surface proteins PsaA, PspA and probably PspC. All of these proteins have very high degrees of similarity, using very stringent criteria, with several SARS-CoV-2 proteins including the spike protein, membrane protein and replicase 1a. CRM197 is also present in Hib and meningitis vaccines. Equivalent similarities were found at lower rates, or were completely absent, among the proteins in diphtheria, tetanus, pertussis, measles, mumps, rubella, and poliovirus vaccines. Notably, PspA and PspC are highly antigenic and new pneumococcal vaccines based on them are currently in human clinical trials so that their effectiveness against SARS-CoV-2 disease is easily testable.

Objectives: This study aimed to compare the similarities and differences between the Kawasaki disease (KD) during the SARS-CoV-2 pandemic and multisystem inflammatory syndrome in children (MIS-C). Methods: The medical records of patients diagnosed with KD and MIS-C at a single center between July 2020 and November 2021 were retrospectively reviewed. Results: The study included 39 MIS-C patients (84.6% male) with a median age of 138 months and 17 KD patients (58.8% male) with a median age of 36 months. Compared with the KD patients, MIS-C patients were older (p<0.001), had longer hospital stays (p=0.023), elevated neutrophil count (p<0.001), C-reactive protein (p<0.001), procalcitonin (p<0.001), interleukin-6 (p<0.014), ferritin (p<0.001), fibrinogen (p<0.001), troponin I (p=0.001), NT-proBNP (p<0.001), and, D-dimer levels (p<0.001). They had more hypotension (p=0.024), decreased left ventricular function (p=0.023), and a greater need for corticosteroids (p<0.001), enoxaparin (p=0.045), and therapeutic plasma exchange (p<0.001). Patients with KD displayed a higher frequency of rash (p<0.001), oral mucosal changes (p<0.001), conjunctival injection (p<0.001), extremity changes (p<0.001), and cervical lymphadenopathy (p<0.001). They had a longer duration of fever (p<0.001), elevated white blood cell count (p<0.001), platelet count (p<0.001), and alanine aminotransferase level (p<0.001). The two groups were similar in terms of hemoglobin levels, erythrocyte sedimentation rate, albumin levels, or the frequency of coronary dilation/aneurysm, myocarditis, pericarditis, invasive mechanical ventilatory support, and intravenous immunoglobulin treatment. Conclusions: Older patient age, a greater presence of gastrointestinal and cardiac findings associated with hypotension, increased NT-proBNP levels, decreased left ventricular function, the use of various treatment modalities, and longer hospital stays suggest MIS-C, whereas a longer duration of fever and classical clinical features of KD favor KD.

Emerging viruses description have grown at an unprecedented rate since the beginning of the 21^st century. The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its related illness, Coronavirus Disease 2019 (COVID-19) has been reported as the third highly pathogenic coronavirus introducing itself into human population in the current era after the SARS-CoV and Middle East Respiratory Syndrome (MERS-CoV). Molecular and cellular studies considering the pathogenesis of this novel coronavirus are still in the early stages of research, however, regarding the similarity of SARS-CoV-2 and other coronaviruses, it could be hypothesized that the NF-κB, Cytokine regulation, ERK, and TNF-α signaling pathways are the more likely causes of inflammation upon onset of COVID-19. There are several drugs prescribed and used to alleviate the activity of these inflammatory cellular signaling pathways which might be beneficial for developing novel therapeutic modalities against COVID-19. In this review, we briefly summarized the alteration of cellular signaling pathways affected by coronavirus infection, particularly SARS-CoV and MERS-CoV and tabulated the current therapeutic agents approved for previous human diseases.

The Xpert® Xpress SARS-CoV-2 and Xpert® Xpress SARS-CoV-2/Flu/RSV tests were rapidly developed and widely used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In response to emerging genetic variability, a new SARS-CoV-2 target (RNA-dependent RNA-polymerase) has been added to both tests: Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test. A rapid evaluation of both tests was performed in South Africa, using residual respiratory specimens. Residual respiratory specimens (n=125) were used to evaluate the Xpert® Xpress CoV-2 plus test and included 50 genotyped specimens. The Xpert® Xpress CoV-2/Flu/RSV plus test was assessed using 45 genotyped SARS-CoV-2 specimens, ten influenza A, ten Influenza B and twenty respiratory syncytial virus specimens. Results were com-pared to in-country standard of care tests. Genotyped specimens tested the performance of the test under pressure from circulating SARS-CoV-2 variants of concern. Reference material was included to assess the test limits and linearity. The Xpert® Xpress CoV-2 plus test performance compared to reference results across residual respiratory specimens was good (positive per-centage agreement (PPA)=95.2%, negative percentage agreement (NPA)=95.0%) The Xpert® Xpress CoV-2/Flu/RSV plus test showed good performance across all residual respiratory specimens (PPA=100%, NPA=98.3%). All genotyped variants of concern were detected by both tests. The Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests can be used to diagnose SARS-CoV-2, and to diagnose and differentiate SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus respectively. The NPA was lower than the recommended 99%, but was influenced by the low number of negative specimens tested. The variants of concern assessed did not affect test performance. It is recommended that sites perform their own assessments compared to in-country standard of care tests.

Coronavirus Disease-19 (COVID-19) is presently wreaking havoc on public health and socio-economic development. Besides the upper and lower respiratory tract involvement, gastrointestinal symptoms are also reported in COVID-19 patients through gut-lung axis. Finding its way through the feces of infected individuals and other sources, the genetic material of SARS-CoV-2 (ssRNA) is reported widely in wastewater and is being used as a fingerprint for its detection. With millions of cases arriving every day, there is a need to level up the testing speed efficiency. Due to the restricted sampling potential of testing laboratories, clinical testing is unable to track all the symptomatic and asymptomatic cases. Wastewater-based epidemiology (WBE) bestows an auxiliary monitoring tool that will contribute in community level screening. Sample collection, concentration, RNA extraction, quantification and data analysis are the main steps involved in implementation of WBE that can be relied upon as an alarm call for an upcoming wave, emergence of a new variant or any future pandemic. WBE can be a cheaper and more practical alternative to high end and sophisticated clinical testing for community transmission detection. Worldwide, there are more than 300 reports entailing the occurrence of SARS-CoV-2 in wastewater exhibiting unique temporal trends with five of them in India. This review aims to address the present knowledge on surveillance of SARS-CoV-2 in wastewater and its implications.

Angiotensin-converting enzyme 2 (ACE2) is an important player of the renin-angiotensin-aldosterone system (RAAS) in regulating the conversion of angiotensin II into angiotensin (1-7). While expressed on the surface of human cells, such as lung, heart, kidney, neurons, and endothelial cells (EC), ACE2 is the entry receptor for SARS-CoV-2. Here, we would like to highlight that ACE2 is predominant on the EC membrane. Many of coronavirus disease 2019 (COVID-19) symptoms have been associated with the large recruitment of immune cells, directly affecting EC. Additionally, cytokines, hypoxia, and complement activation can trigger the activation of EC leading to the coagulation cascade. The EC dysfunction plus the inflammation due to SARS-CoV-2 infection may lead to abnormal coagulation, actively participating in thrombo-inflammatory processes resulting in vasculopathy and indicating poor prognosis in patients with COVID-19. Considering the intrinsic relationship between EC and the pathophysiology of SARS-CoV-2, EC-associated therapies such as anticoagulants, fibrinolytic drugs, immunomodulators, and molecular therapies have been proposed. In this review, we will discuss the role of EC in the lung inflammation and edema, in the disseminate coagulation process, ACE2 positive cancer patients, and current and future EC-associated therapies to treat COVID-19.

The novel respiratory disease COVID-19 has reached the status of worldwide pandemic and large efforts are currently being undertaken in molecularly characterizing the virus causing it, SARS-CoV-2. The genomic variability of SARS-CoV-2 specimens scattered across the globe can underly geographically specific etiological effects. In the present study, we gather the 48,635 SARS-CoV-2 complete genomes currently available thanks to the collection endeavor of the GISAID consortium and thousands of contributing laboratories. We analyze and annotate all SARS-CoV-2 mutations compared with the reference Wuhan genome NC_045512.2, observing an average of 7.23 mutations per sample. Our analysis shows the prevalence of single nucleotide transitions as the major mutational type across the world. There exist at least three clades characterized by geographic and genomic specificity. In particular, the clade G, prevalent in Europe, carries a D614G mutation in the Spike protein, which is responsible for the initial interaction of the virus with the host human cell. Our analysis may drive local modulation of antiviral strategies based on the molecular specificities of this novel virus.

A novel coronavirus has been reported as the causative pathogen of the Coronavirus disease 2019 (COVID-19) outbreak Wuhan city, China in December 2019. Due to the rapid spreading of COVID-19 worldwide, it has been announced as a pandemic by the World Health Organization (WHO). Hospitalized patients in Wuhan are associated with the Huanan seafood wholesale market where live animals, such as poultry, bats, snakes, frogs, rabbits, marmots, and hedgehogs are sold in that market which suggests a possible zoonotic infection. Therefore, it is essential to identify the potential animal reservoir, and the possibility of infection for other animal species. This short review aims to provide an overview on the relation between severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection and animals.

The emergence of SARS-CoV-2 is a challenge in the actual medical scenario. Besides the classical lung and respiratory disease, patients infected with the virus can present with cardiac injury, and pathogenic mechanisms point to a direct infection of the heart.

Since there is not a clear consensus about the possibility for COVID-19 to be an airborne disease, exists a controversy regarding the need to use surgical masks to prevent its spread. Here, using the Kepler conjecture for ideal packaging, the number of virions of different sizes that can be accommodated inside droplets was calculated and are proportional to the 3^rd potency of the droplet/virion diameter. The differences between particles of 5 um and 100 μm are around four orders of magnitude, explaining why the airborne spread is much more difficult but still possible. There is no solid evidence yet that the airborne coronaviruses may reach enough concentration to infect, but this may be the case under certain circumstances. The WHO partially recognizes now this fact in a warning to health workers (from my point of view too late, as it was the declaration of a pandemic). Another issue is whether the virus stays infective in aerosols generated from patients. This has not been directly proved yet except with artificial aerosols, but there are no reasons why the virus cannot remain in the air and be infective if the viral charge and time of exposure are enough. We must also consider whether the virus can infect the intestine; there are some signs in this sense. Finally, and most importantly, we need to reduce interactions by using surgical masks to flatten the curve, leave the quarantine and avoid a rebound. For cultural reasons, a social distance of 2 meters (2M) is extremely hard to manage. Surgical masks do reduce the interactions in conditions of proximity and, therefore, help to “flatten the curve”. The WHO and CDC “laissez-faire” on this matter do not help and we are running out of time. Anticipated actions, such as the use of surgical masks for the general population, are critical.

This study evaluated the epidemiological and genetic susceptibility profile caused by coronavirus 2 (SARS-CoV-2) in children ≤3 years of age with acute gastroenteritis (AGE) or acute respiratory infection (ARI), living in the northwestern region of the Amazon (NWAR). Fecal and saliva sam-ples collected from 202 children (n=404) were subjected to detection of SARS-CoV-2 by RT-qPCR. Polymorphisms in the angiotensin-converting enzyme (ACE rs4646994) and ACE2 G8780A; rs2285666) genes were detected by SYBR GREEN real-time PCR and PCR/Alul digestion, respec-tively. Secretory and Lewis status of histo-blood group antigen (HBGA) was performed using phenotyping/ELISA. Frequencies for the AGE group: SARS-CoV-2; 8.9%; ARI group: SARS-CoV-2; 2.9%. The ACE I/I homozygous polymorphism gene was majority. Males with the ACE2 G8780A SNP were more susceptible to ARI symptoms than AGE (OR = 3.85; 95% CI-1.42-10.38; P 0.007). Most children were secretory FUT2 phenotype (92.6%). Surveillance of diarrhea and respiratory infections in children, along with understanding their causes, are crucial for identifying preven-tion and control strategies and for prioritizing and developing new vaccines. Clinical manifesta-tions of COVID-19 in children ≤3 years of age include AGE.

Base excision repair (BER) is the predominant pathway to remove most forms of hydrolytic, oxidative and alkylative DNA damage. The precise functioning of BER is achieving by the regulation of each step by regulatory/accessory proteins, with the most important of which being poly(ADP-ribose) polymerase 1 (PARP1). The PARP1 regulatory functions extend to many cellular processes including regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of the BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study the CRISPR/Cas9 based technique has been used to knocked out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with putative PARP1 deletion were characterized by several approaches including: PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, qPCR analysis of PARP1 mRNA, western-blot analysis of whole-cell-extract proteins with anti-PARP1 antibodies, and the PAR synthesis in the whole-cell-extracts. qPCR analysis of mRNAs coding the BER related proteins: PARP2, uracil DNA glycosylase 2, AP endonuclease 1, DNA polymerase β, DNA ligase III and XRCC1 did not reveal crucial influence of PARP1 knockout. The corresponding catalytic activities in the whole-cell extracts (WCEs) evaluated in parallel did not differ significantly between mutant and parental cell lines. No considerable influence of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed.

The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist, of which Middle East Respiratory Syndrome (MERS) and SARS-CoV (SARS) showed higher mortality rates without causing a pandemic. As of December 2022, SARS-CoV-2 has resulted in over 6.6 million deaths worldwide through an array of acute to chronic pathologies. Historical pandemics include smallpox and influenza with efficacious therapeutics utilized to reduce overall disease burden. Therefore, immune system process analysis is required to compare innate and adaptive immune system interactions. Lymphatic system organs include bone marrow and thymus using a network of nodes throughout which white blood cells traverse glycolipid membranes utilizing cytokines and chemokine gradients that affect cell development, differentiation, proliferation, and migration processes as well as genetic factors affecting cell receptor expression. Innate processes involve antigen-presenting cells and B lymphocyte cellular responses to pathogens relevant to other viral and bacterial infections but also in oncogenic diseases. Such processes utilize cluster of differentiation (CD) marker expression, major histocompatibility complexes (MHC), pleiotropic interleukins (IL) and chemokines. The adaptive immune system consists of Natural Killer (NK) and T cells. Other viruses are also contributory to cancer including human papillomavirus (cervical carcinoma ), Epstein-Barr virus (EBV) ( lymphoma), hepatitis B and C (hepatocellular carcinoma) and human T cell leukemia virus-1 (adult T-cell leukemia). Bacterial infections also increase the risk of developing cancer( e.g. H. pylori). Therefore, as the above factors can cause both morbidity and mortality along-side being transmitted within clinical and community settings, it is appropriate to now examine advances in single cell sequencing, FACS analysis and many other laboratory techniques that allow insights into discoveries of newer cell types. These developments offer improved clarity and understanding that over-lap with known autoimmune conditions that could be affected by innate B cell or T cell responses to SARS-CoV-2 infection. Thus, this review quantifies and outlines the nature of specific receptors and proteins relevant to clinical laboratories and medical research by documenting both innate and adaptive immune system cells within current coronavirus immunology case study data and other pathologies to date.

Spread of severe acute respiratory syndrome coronavirus (SARS-CoV-2) made a historic transition between December 2019 to March 2020. In the present scenario SARS-CoV-2 as becomes a major burden on public health and economic stability of societies around the globe. From the substantial evidences gained from the pandemic of SARS-CoV-2 and MERS-CoV (Middle East respiratory syndrome coronavirus), scientists and clinicians strongly believes that these pathogenic viruses share common homology of some biologically active enzymes which includes RNA-dependent RNA polymerase (RdRP), 3-chymotrypsin-like protease (3CL^pro), papain-like protease (PL^pro) etc. RdRP relatively grabs higher level of clinical importance in comparison with other enzyme target. Indian system of traditional medicine pioneering the therapy towards infectious disease since several centuries. In view of this potential therapeutic leads from some of the Indian medicines along with standard drug favipiravir subjected to docking investigation targeting SARS-CoV-2- RNA dependent RNA polymerase (RdRp). Residual proximity analysis reveals 18 out of 28 compounds reveals potential binding affinity of about 100% with the target amino acid residue (618 ASP, 760 ASP,761 ASP), 7 out of 28 reveals 75% binding efficacy and 3 out of 28 reveals 25% binding efficacy with that of the target residue. Hence further clinical validation may be warranted with proper in-vitro and in-vivo studies prior to the clinical recommendation in treating COVID-19 patient’s.