Synthesis of 1-(2-Hydroxyphenyl)- and (3,5-Dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives as Promising Scaffolds for the Development of Novel Antimicrobial and Anticancer Agents.

Increasing antimicrobial resistance among Gram-positive pathogens and pathogenic fungi remains one of the major public healthcare threats. Therefore, novel antimicrobial candidates and scaffolds are critically needed to overcome resistance in Gram-positive pathogens and drug-resistant fungal pathogens. In this study, we explored 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid and its 3,5-dichloro-2-hydroxyphenyl analogue for their in vitro antimicrobial activity against multidrug-resistant pathogens. The compounds showed structure-dependent antimicrobial activity against Gram-positive pathogens (S. aureus, E. faecalis, C. difficile). Compounds 14 and 24b showed promising activity against vancomycin-intermediate S. aureus strains, and favorable cytotoxic profiles in HSAEC-1 cells, making them attractive scaffolds for further development. 5-Fluorobenzimidazole, having a 3,5-dichloro-2-hydroxyphenyl substituent, was found to be four-fold, and hydrazone, with a thien-2-yl fragment, was two-fold stronger than clindamycin against methicillin resistant S. aureus TCH 1516. Moreover, hydrazone, bearing a 5-nitrothien-2-yl moiety, showed promising activity against three tested multidrug-resistant C. auris isolates representing major genetic lineages (MIC 16 µg/mL) and azole-resistant A. fumigatus strains harboring TR34/L98H mutations in the CYP51A gene. The anticancer activity characterization demonstrated that the 5-fluorobenzimidazole derivative with a 3,5-dichloro-2-hydroxyphenyl substituent showed the highest anticancer activity in an A549 human pulmonary cancer cell culture model. Collectively these results demonstrate that 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives could be further explored for the development of novel candidates targeting Gram-positive pathogens and drug-resistant fungi.

Application of Antiviral, Antioxidant and Antibacterial Glycyrrhiza glabra L., Trifolium pratense L. Extracts and Myristica fragrans Houtt. Essential Oil in Microcapsules.

Viruses and bacteria can disrupt normal human functions; therefore, ways to use the beneficial properties of plants to promote health are constantly being researched. Plant materials that accumulate biologically active compounds can be used to create a new pharmaceutical form. This study aimed to investigate the biological activity of selected plant extracts and essential oil and to produce microcapsules. The main compounds in extracts and essential oil were determined using chromatographic methods, antioxidant activity was evaluated spectrophotometrically, antimicrobial activity was assessed by monitoring the growth of nine pathogens, and the antiviral effect on infected bird cells with coronavirus was evaluated. Trifolium pratense L. extract had the highest antioxidant (26.27 ± 0.31 and 638.55 ± 9.14 µg TE/g dw by the DPPH and ABTS methods, respectively) and antiviral activity (56 times decreased titre of virus). Liquorice extract expressed antibacterial activity against Gram-positive pathogens and the highest antioxidant activity using the FRAP method (675.71 ± 4.61 mg FS/g dw). Emulsion stability depended on excipients and their amount. Microcapsules with extracts and essential oil were 1.87 mm in diameter, and their diameter after swelling was increased more than two times in intestinal media, while less than 0.5 times in gastric media.

In vitro antiviral activity of fifteen plant extracts against avian infectious bronchitis virus.

BACKGROUND: Avian infectious bronchitis (IB) is a disease that can result in huge economic losses in the poultry industry. The high level of mutations of the IB virus (IBV) leads to the emergence of new serotypes and genotypes, and limits the efficacy of routine prevention. Medicinal plants, or substances derived from them, are being tested as options in the prevention of infectious diseases such as IB in many countries. The objective of this study was to investigate extracts of 15 selected medicinal plants for anti-IBV activity. RESULTS: Extracts of S. montana, O. vulgare, M. piperita, M. officinalis, T. vulgaris, H. officinalis, S. officinalis and D. canadense showed anti-IBV activity prior to and during infection, while S. montana showed activity prior to and after infection. M. piperita, O. vulgare and T. vulgaris extracts had > 60 SI. In further studies no virus plaques (plaque reduction rate 100%) or cytopathogenic effect (decrease of TCID50 from 2.0 to 5.0 log10) were detected after IBV treatment with extracts of M. piperita, D. canadense and T. vulgaris at concentrations of extracts ≥0.25 cytotoxic concentration (CC50) (P < 0.05). Both PFU number and TCID50 increased after the use of M. piperita, D. canadense, T. vulgaris and M. officinalis extracts, the concentrations of which were 0.125 CC50 and 0.25 CC50 (P < 0.05). Real-time PCR detected IBV RNA after treatment with all plant extracts using concentrations of 1:2 CC50, 1:4 CC50 and 1:8 CC50. Delta cycle threshold (Ct) values decreased significantly comparing Ct values of 1:2 CC50 and 1:8 CC50 dilutions (P < 0.05). CONCLUSIONS: Many extracts of plants acted against IBV prior to and during infection, but the most effective were those of M. piperita, T. vulgaris and D. canadense .

Chromatographic Data Segmentation Method: A Hybrid Analytical Approach for the Investigation of Antiviral Substances in Medicinal Plant Extracts.

The methodology described in this article will significantly reduce the time required for understanding the relations between chromatographic data and bioactivity assays. The methodology is a hybrid of hypothesis-based and data-driven scientific approaches. In this work, a novel chromatographic data segmentation method is proposed, which demonstrates the capability of finding what volatile substances are responsible for antiviral and cytotoxic effects in the medicinal plant extracts. Up until now, the full potential of the separation methods has not been exploited in the life sciences. This was due to the lack of data ordering methods capable of adequately preparing the chromatographic information. Furthermore, the data analysis methods suffer from multidimensionality, requiring a large number of investigated data points. A new method is described for processing any chromatographic information into a vector. The obtained vectors of highly complex and different origin samples can be compared mathematically. The proposed method, efficient with relatively small sized data sets, does not suffer from multidimensionality. In this novel analytical approach, the samples did not need fractionation and purification, which is typically used in hypothesis-based scientific research. All investigations were performed using crude extracts possessing hundreds of phyto-substances. The antiviral properties of medicinal plant extracts were investigated using gas chromatography-mass spectrometry, antiviral tests, and proposed data analysis methods. The findings suggested that (i) ß- cis-caryophyllene, linalool, and eucalyptol possess antiviral activity, while (ii) thujones do not, and (iii) α-thujone, ß-thujone, cis- p-menthan-3-one, and estragole show cytotoxic effects.

Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines.

BACKGROUND: Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. Enzyme-linked immunosorbent assays (ELISA) are commercially available for detection of SBV-specific antibodies in bovine sera and milk. Here we describe the development and evaluation of an indirect ELISA based on a yeast derived recombinant SBV nucleocapsid protein (N) for the detection of SBV-specific antibodies in bovine saliva. Development of a non-invasive test to detect antibodies in individual bovine saliva samples could potentially provide a test suitable for calves and adult cattle. The aim of this study was to investigate the agreement between the levels of antibodies (IgG) measured in milk and sera, and the level of antibodies (IgG and IgA) in saliva, in comparison with the antibody levels detected in sera and milk with commercially available test. RESULTS: Serum, milk and saliva samples from 58 cows were collected from three dairy herds in Lithuania and tested for the presence of SBV-specific antibodies. The presence of IgG antibodies was tested in parallel serum and milk samples, while the presence of IgA and IgG antibodies was tested in saliva samples. The presence of SBV-specific IgG and IgA in saliva was tested using an indirect ELISA based on a yeast-derived recombinant N protein. The presence of SBV-specific IgG in milk and sera was tested in parallel using a commercial recombinant protein based test. The sensitivities of the newly developed tests were as follows: 96 % for the IgG serum assay and 94 % for the IgG milk assay and 85 % and 98 % for IgG and IgA in saliva tests, when compared with data generated by a commercial IgG assay. CONCLUSIONS: Data from testing the saliva IgG and IgA and also the milk and serum IgG with indirect SBV-specific ELISAs showed close agreement with the commercial serum and milk IgG assay data. The level of IgG in saliva was notably lower in comparison to IgA. The newly developed method exhibits the potential to serve as an easily transferable tool for epidemiological studies.

Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

BACKGROUND: Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production. RESULTS: In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test. CONCLUSIONS: We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies.

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

Generation of recombinant Schmallenberg virus nucleocapsid protein in yeast and development of virus-specific monoclonal antibodies.

Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

Raccoon dog rabies surveillance and post-vaccination monitoring in Lithuania 2006 to 2010.

BACKGROUND: Oral rabies vaccination (ORV) in rabies infected regions should target the primary rabies vector species, which in Lithuania includes raccoon dogs as well as red foxes. Specific investigations on ORV in raccoon dogs are needed e.g. evaluation of vaccine effectiveness under field conditions. The objective of the current study was to investigate the efficacy of the ORV programme 2006-2010 in Lithuania by examining the number of rabies cases and estimating the prevalences of a tetracycline biomarker (TTC) and rabies virus antibodies in raccoon dogs. METHODS: From 2006 to 2010, 12.5 million rabies vaccine-baits were distributed by aircraft. Baiting occurred twice per year (spring and autumn), targeting raccoon dogs and red foxes in a 63,000 km2 area of Lithuania. The mandibles of raccoon dogs found dead or killed in the vaccination area were analyzed by fluorescence microscopy for the presence of the TTC. Rabies virus sera neutralizing anti-glycoprotein antibody titres were determined using an indirect ELISA method and seroconversion (> 0.5 EU/ml) rates were estimated. RESULTS: During the study period, 51.5% of raccoon dog mandibles were positive for TTC. 1688 of 3260 tested adults and 69 of 175 tested cubs were TTC positive. Forty-seven percent of raccoon dog serum samples were positive for rabies virus antibodies. 302 of 621 investigated adults and 33 of 95 investigated cubs were seropositive. In the same time 302 of 684 and 43 of 124 tested samples were TTC and ELISA positive in spring; whereas 1455 of 2751 and 292 of 592 tested samples were TTC and ELISA positive in autumn. There was a positive correlation between the number of TTC and antibody positive animals for both adult and cub groups. CONCLUSIONS: ORV was effective in reducing the prevalence of rabies in the raccoon dog population in Lithuania. The prevalence of rabies cases in raccoon dogs in Lithuania decreased from 60.7% in 2006-2007 to 6.5% in 2009-2010.