Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

SARS-CoV-2 variant detection from wastewater: rapid spread of B.1.1.7 lineage in Hungary.

Wastewater-based epidemiology (WBE) is a recognised tool for tracking community transmission of COVID-19. From the second half of 2020, the emergence of new, highly infective, more pathogenic or vaccine-escape SARS-CoV-2 variants is the major public health concern. Variant analysis in sewage might assist the early detection of new mutations. Weekly raw sewage samples from 22 wastewater treatment plants (WWTPs) in Hungary (representing 40% of the population) were analysed between December 2020 and March 2021 for signature mutations N501Y and del H69/V70 of B.1.1.7 lineage by melting point genotyping and RT-digital droplet PCR (RT-ddPCR). The latter method proved to be more efficient in parallel detection of different variants and also provides quantitative information. Wastewater surveillance indicated that the B.1.1.7 variant first emerged in Budapest in early January 2021 and rapidly became dominant in the entire country. Results are in close agreement with the available clinical data (Pearson's correlation coefficient, R = 0.9153). RT-ddPCR was confirmed to be a reliable tool for tracking emerging variant ratios in wastewaters. It is a rapid and cost-effective method compared to whole-genome sequencing, but only applicable for the detection of known mutations. Efficient variant surveillance might require the combination of multiple methods.

In Vitro Determination of Inhibitory Effects of Humic Substances Complexing Zn and Se on SARS-CoV-2 Virus Replication.

(1) Background: Humic substances are well-known human nutritional supplement materials and they play an important performance-enhancing role as animal feed additives. For decades, ingredients of humic substances have been proven to carry potent antiviral effects against different viruses. (2) Methods: Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern ("Alpha Variant") in a VeroE6 cell line. (3) Results: This combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. (4) Conclusions: Even picomolar concentration ranges of humic substances, Vitamin C and Zn/Se ions in the given composition, were enough to achieve 50% viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.

Evaluating the field performance of multiple SARS-Cov-2 antigen rapid tests using nasopharyngeal swab samples.

The SARS-CoV-2 pandemic, which started in December 2019, has been posing significant challenges to the health care system worldwide. As the pandemic spreads with rapidly increasing number of positive cases, early diagnosis of infected patients is crucial to successfully limit the spread of the virus. Although the real-time reverse-transcription polymerase chain reaction (RT-qPCR) is the recommended laboratory method to diagnose COVID-19 infection, many factors such as availability of laboratory equipment, reagents and trained personnel affect the use of time-consuming molecular techniques. To facilitate on-the-spot diagnosis of COVID-19, SARS-CoV-2 rapid antigen tests were developed by several different manufacturers. The evaluation of such rapid tests is particularly important due to the recent unanimous agreement by the European Commission Member States on a recommendation setting out a framework for the use of antigen rapid tests that contains a list of the mutually recognized assays and the basis of independent validation protocols. To evaluate the on-field performance of ten commercially available SARS-CoV-2 antigen rapid tests (CLINITEST Rapid COVID-19 Antigen Test, GenBody COVID-19 Antigen Test, GENEDIA W COVID-19 Ag Test, Healgen Coronavirus Antigen Rapid Test, Humasis COVID-19 Ag Test, VivaDiag SARS-CoV-2 Ag Rapid Test, Helix i-SARS-CoV-2 Ag Rapid Test, Roche SARS-CoV-2 Rapid Antigen Test, Abbot COVID-19 Ag Rapid Test and Vazyme SARS-CoV-2 Antigen Detection Kit) and compare with RT-qPCR as a reference method, the Hungarian National Public Health Center provided 1,597 antigen rapid tests to the National Ambulance Service, COVID-testing trucks and two hospitals treating COVID-19 patients. Sensitivity, specificity and accuracy were determined by performing the rapid test directly from nasopharyngeal swab samples of symptomatic individuals. For strongly positive samples (Ct < 25) sensitivities ranged between 66.7% and 100%, while for positive samples (Ct < 30) they gave a maximum sensitivity of 87.5%. The specificity of the tests was ranging between 79% to 100%. The results presented here are of high importance to the European Commission and also help governmental decision-making regarding the application of the proper rapid tests for screening different at-risk populations. Nonetheless, SARS-Cov-2 rapid tests play an important role in early and on-the-spot diagnosis of potentially infected individuals.

Nationwide effectiveness of five SARS-CoV-2 vaccines in Hungary-the HUN-VE study.

OBJECTIVES: The Hungarian vaccination campaign was conducted with five different vaccines during the third wave of the coronavirus disease 2019 (COVID-19) pandemic in 2021. This observational study (HUN-VE: Hungarian Vaccine Effectiveness) estimated vaccine effectiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and COVID-19-related mortality in 3.7 million vaccinated individuals. METHODS: Incidence rates of SARS-CoV-2 infection and COVID-19-related mortality were calculated using data from the National Public Health Centre surveillance database. Estimated vaccine effectiveness was calculated as 1 - incidence rate ratio ≥7 days after the second dose for each available vaccine versus an unvaccinated control group using mixed-effect negative binomial regression controlling for age, sex and calendar day. RESULTS: Between 22 January 2021 and 10 June 2021, 3 740 066 Hungarian individuals received two doses of the BNT162b2 (Pfizer-BioNTech), HB02 (Sinopharm), Gam-COVID-Vac (Sputnik-V), AZD1222 (AstraZeneca), or mRNA-1273 (Moderna) vaccines. Incidence rates of SARS-CoV-2 infection and COVID-19-related death were 1.73-9.3/100 000 person-days and 0.04-0.65/100 000 person-days in the fully vaccinated population, respectively. Estimated adjusted effectiveness varied between 68.7% (95% CI 67.2%-70.1%) and 88.7% (95% CI 86.6%-90.4%) against SARS-CoV-2 infection, and between 87.8% (95% CI 86.1%-89.4%) and 97.5% (95% CI 95.6%-98.6%) against COVID-19-related death, with 100% effectiveness in individuals aged 16-44 years for all vaccines. CONCLUSIONS: Our observational study demonstrated the high or very high effectiveness of five different vaccines in the prevention SARS-CoV-2 infection and COVID-19-related death.

Tissue-Specific Accumulation and Isomerization of Valuable Phenylethanoid Glycosides from Plantago and Forsythia Plants.

A comparative phytochemical study on the phenylethanoid glycoside (PhEG) composition of the underground organs of three Plantago species (P. lanceolata, P. major, and P. media) and that of the fruit wall and seed parts of Forsythia suspensa and F. europaea fruits was performed. The leaves of these Forsythia species and six cultivars of the hybrid F. × intermedia were also analyzed, demonstrating the tissue-specific accumulation and decomposition of PhEGs. Our analyses confirmed the significance of selected tissues as new and abundant sources of these valuable natural compounds. The optimized heat treatment of tissues containing high amounts of the PhEG plantamajoside (PM) or forsythoside A (FA), which was performed in distilled water, resulted in their characteristic isomerizations. In addition to PM and FA, high amounts of the isomerization products could also be isolated after heat treatment. The isomerization mechanisms were elucidated by molecular modeling, and the structures of PhEGs were identified by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) techniques, also confirming the possibility of discriminating regioisomeric PhEGs by tandem MS. The PhEGs showed no cytostatic activity in non-human primate Vero E6 cells, supporting their safe use as natural medicines and allowing their antiviral potency to be tested.

Ahead of the second wave: Early warning for COVID-19 by wastewater surveillance in Hungary.

Wastewater based epidemiology is a potential early warning tool for the detection of COVID-19 outbreak. Sewage surveillance for SARS-CoV-2 RNA was introduced in Hungary after the successful containment of the first wave of the pandemic to forecast the resurge of infections. Three wastewater treatment plants servicing the entire population (1.8 million) of the capital, Budapest were sampled weekly. 24 h composite (n = 44) and grab samples (n = 21) were concentrated by an in-house flat sheet membrane ultrafiltration method. The efficiency and reproducibility of the method was comparable to those previously published. SARS-CoV-2 RNA was quantified using RT-qPCR of the N gene. The first positive signal in sewage was detected 2 weeks before the rise in case numbers. Viral concentration and volume-adjusted viral load correlated to the weekly new cases from the same week and the rolling 7-day average of active cases in the subsequent week. The correlation was more pronounced in the ascending phase of the outbreak, data was divergent once case numbers plateaued. Wastewater surveillance was found to be effective in predicting the second wave of the outbreak in Hungary. Data indicated that even relatively low frequency (weekly) sampling is useful and at the same time, cost effective tool in outbreak detection.

Topography, Spike Dynamics, and Nanomechanics of Individual Native SARS-CoV-2 Virions.

SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, displays a corona-shaped layer of spikes which play a fundamental role in the infection process. Recent structural data suggest that the spikes possess orientational freedom and the ribonucleoproteins segregate into basketlike structures. How these structural features regulate the dynamic and mechanical behavior of the native virion are yet unknown. By imaging and mechanically manipulating individual, native SARS-CoV-2 virions with atomic force microscopy, here, we show that their surface displays a dynamic brush owing to the flexibility and rapid motion of the spikes. The virions are highly compliant and able to recover from drastic mechanical perturbations. Their global structure is remarkably temperature resistant, but the virion surface becomes progressively denuded of spikes upon thermal exposure. The dynamics and the mechanics of SARS-CoV-2 are likely to affect its stability and interactions.