A Comparative Analysis of Molecular Biological Methods for the Detection of SARS-CoV-2 and Testing the In Vitro Infectivity of the Virus.

The virus discovered in 2019 in the city of Wuhan, China, which was later identified as SARS-CoV-2 and which spread to the level of a pandemic, put diagnostic methods to the test. Early in the pandemic, we developed a nested PCR assay for the detection of SARS-CoV-2, which we validated and applied to detect the virus in feline samples. The present study describes the application of the nested PCR test in parallel with LAMP for the detection of the virus in 427 nasopharyngeal and oropharyngeal human samples taken between October 2020 and January 2022. Of the swabs tested, there were 43 positives, accounting for 10.1% of all samples tested, with the negatives numbering 382, i.e., 89.5%, and there were 2 (0.4%) invalid ones. The nPCR results confirmed those obtained by using LAMP, with results concordant in both methods. Nasal swabs tested using nPCR confirmed the results of oropharyngeal and nasopharyngeal swab samples tested using LAMP and nPCR. The focus of the discussion is on the two techniques: the actual practical application of the laboratory-developed assays and the diagnostic value of nasal samples. The nPCR used is a reliable and sensitive technique for the detection of SARS-CoV-2 in nasopharyngeal, oropharyngeal, and nasal swab samples. However, it has some disadvantages related to the duration of the entire process, as well as a risk of contamination. Experiments were performed to demonstrate the infectivity of the virus from the positive isolates in vitro. A discrepancy was reported between direct and indirect methods of testing the virus and accounting for its ability to cause infection in vitro.

Effect of a Total Extract and Saponins from Astragalus glycyphyllos L. on Human Coronavirus Replication In Vitro.

Members of the family Coronaviridae cause diseases in mammals, birds, and wildlife (bats), some of which may be transmissible to humans or specific to humans. In the human population, they can cause a wide range of diseases, mainly affecting the respiratory and digestive systems. In the scientific databases, there are huge numbers of research articles about the antiviral, antifungal, antibacterial, antiviral, and anthelmintic activities of medicinal herbs and crops with different ethnobotanical backgrounds. The subject of our research is the antiviral effect of isolated saponins, a purified saponin mixture, and a methanol extract of Astragalus glycyphyllos L. In the studies conducted for the cytotoxic effect of the substances, CC50 (cytotoxic concentration 50) and MTC (maximum tolerable concentration) were determined by the colorimetric method (MTT assay). The virus was cultured in the MDBK cell line. As a result of the experiments carried out on the influence of substances on viral replication (using MTT-based colorimetric assay for detection of human Coronavirus replication inhibition), it was found that the extract and the purified saponin mixture inhibited 100% viral replication. The calculated selective indices are about 13 and 18, respectively. The obtained results make them promising for a preparation with anti-Coronavirus action.

Molecular Screening for High-Risk Human Papillomaviruses in Patients with Periodontitis.

Members of the Papillomaviridae family account for 27.9-30% of all infectious agents associated with human cancer. The aim of our study was to investigate the presence of high-risk HPV (human papilloma virus) genotypes in patients with periodontitis and a pronounced clinical picture. To achieve this goal, after proving the bacterial etiology of periodontitis, the samples positive for bacteria were examined for the presence of HPV. The genotype of HPV is also determined in samples with the presence of the virus proven by PCR (polymerase chain reaction). All positive tests for bacteria associated with the development of periodontitis indicated the presence of HPV. There was a statistically significant difference in HPV positive results between the periodontitis positive target group and the control group. The higher presence of high-risk HPV genotypes in the target group, which was also positive for the presence of periodontitis-causing bacteria, has been proven. A statistically significant relationship was established between the presence of periodontitis-causing bacteria and high-risk strains of HPV. The most common HPV genotype that tests positive for bacteria associated with the development of periodontitis is HPV58.

Tailoring acyclovir prodrugs with enhanced antiviral activity: rational design, synthesis, human plasma stability and in vitro evaluation.

Bile acid prodrugs have served as a viable strategy for refining the pharmaceutical profile of parent drugs through utilizing bile acid transporters. A series of three ester prodrugs of the antiherpetic drug acyclovir (ACV) with the bile acids cholic, chenodeoxycholic and deoxycholic were synthesized and evaluated along with valacyclovir for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The in vitro antiviral activity of the three bile acid prodrugs was also evaluated against Epstein-Barr virus (EBV). Plasma stability assays, utilizing ultra-high performance liquid chromatography coupled with tandem mass spectrometry, in vitro cytotoxicity and inhibitory experiments were conducted in order to establish the biological profile of ACV prodrugs. The antiviral assays demonstrated that ACV-cholate had slightly better antiviral activity than ACV against HSV-1, while it presented an eight-fold higher activity with respect to ACV against HSV-2. ACV-chenodeoxycholate presented a six-fold higher antiviral activity against HSV-2 with respect to ACV. Concerning EBV, the highest antiviral effect was demonstrated by ACV-chenodeoxycholate. Human plasma stability assays revealed that ACV-deoxycholate was more stable than the other two prodrugs. These results suggest that decorating the core structure of ACV with bile acids could deliver prodrugs with amplified antiviral activity.

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin.

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex(0-9) HexNAc(2-4) Hex(0-3) Pent(0-3) Fuc(0-3). A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(ß1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.

Synthesis, antioxidative and antiviral activity of hydroxycinnamic acid amides of thiazole containing amino acid.

The synthesis and the biological (antioxidant and antiviral) activities of novel hydroxycinnamic acid amides of a thiazole containing TFA.valine-4-carboxylic acid ethyl ester are reported. The amides have been synthesized from p-coumaric, ferulic and sinapic acids with the corresponding TFA.valine-thiazole-4-carboxylic acid ethyl ester using the coupling reagent N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 4-(dimethylamino) pyridine (DMAP) as a catalyst. The antioxidant properties of the newly synthesized amides have been studied for then antioxidative activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH)* test. The newly synthesized compounds have been tested against the replication in vitro of influenza virus A (H3N2) and human herpes virus 1 and 2 (HSV-1 and HSV-2).

Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties.

A thermophilic actinomycete strain Thermoactinomyces sp. 21E producing a highly thermostable serine collagenase was isolated from Bulgarian soil. The collagenase, produced extracellular by Thermoactinomyces sp. 21E, was purified to homogeneity by heat treatment, ultrafiltration, saturation with ammonium sulfate and gel filtration chromatography with a 101-fold increase in specific activity and 58% recovery. The collagenase has a relative molecular mass of 50000 by SDS-PAGE. The optimum temperature for the enzyme activity was 60-65 degrees C in the absence of Ca(2+) and 70-75 degrees C in the presence of Ca(2+). About 40% of the original activity remaining after incubation at 85 degrees C for 30 min in the presence of Ca(2+). The optimum pH for the enzyme activity was 9.0-9.5 and the enzyme was stable for 1h at 70 degrees C in the pH range from 7.5 to 12.5. The collagenase was strongly inhibited by active-site inhibitors of serine protease PMSF and DFP, which indicated that the enzyme is serine protease. The enzyme activity was completely inhibited by Hg(2+), Cu(2+) and Fe(2+). However, Ca(2+ )strongly activated the collagenase activity. The collagenase from Thermoactinomyces sp. 21E showed high activity toward type I collagen, acid-soluble collagen, gelatin and Pz-PLGPR. However, elastin for collagenase was inert as substrate. The properties of the collagenase from strain 21E suggest that this enzyme is a new collagenolytic protease that differs from the collagenases and serine proteases reported so far.