The Effect of Municipal Biosolids on the Growth, Physiology and Synthesis of Phenolic Compounds in Ocimum basilicum L.

The continuous development of drinking water networks is leading to the production of increasing amounts of waste water and sewage sludge. Secondary-treated sewage sludge is called biosolids and can be used as fertilizers in agriculture due to its rich nutrient content. The aim of this study was to evaluate the effects of biosolids mixed with an eroded soil on the morphology, physiology and synthesis of bioactive compounds in basil. The study was performed in pots under laboratory-controlled conditions. In total, four substrates were tested: S1 biosolids 100%, S2 biosolids 15% + eroded soil 85%, S3 eroded soil 100% and S4 control (commercial growing substrate). At the morphological level, a significant increase in plant height, number of branches, fresh biomass and dry biomass was found in the S2 variant. At the physiological level, photosynthesis and chlorophyll content did not vary significantly, but the quantum yield of PSII (ΦPSII) was significantly higher at S1 and S2. The oxidative status evaluated by determining the activity of SOD, POD and CAT enzymes was better in S2 and S3 compared to S3. Regarding the synthesis of bioactive compounds (rosmarinic acid, caffeic acid and gallic acid), it was stimulated in S1 and S2. In conclusion, biosolids application stimulated the stress response mechanisms in basil plants by increasing the quantum yield chlorophyll fluorescence and catalase activity, alleviating the negative effects of eroded soil.

Long COVID: Molecular Mechanisms and Detection Techniques.

Long COVID, also known as post-acute sequelae of SARS-CoV-2 infection (PASC), has emerged as a significant health concern following the COVID-19 pandemic. Molecular mechanisms underlying the occurrence and progression of long COVID include viral persistence, immune dysregulation, endothelial dysfunction, and neurological involvement, and highlight the need for further research to develop targeted therapies for this condition. While a clearer picture of the clinical symptomatology is shaping, many molecular mechanisms are yet to be unraveled, given their complexity and high level of interaction with other metabolic pathways. This review summarizes some of the most important symptoms and associated molecular mechanisms that occur in long COVID, as well as the most relevant molecular techniques that can be used in understanding the viral pathogen, its affinity towards the host, and the possible outcomes of host-pathogen interaction.

Future Antimicrobials: Natural and Functionalized Phenolics.

With incidence of antimicrobial resistance rising globally, there is a continuous need for development of new antimicrobial molecules. Phenolic compounds having a versatile scaffold that allows for a broad range of chemical additions; they also exhibit potent antimicrobial activities which can be enhanced significantly through functionalization. Synthetic routes such as esterification, phosphorylation, hydroxylation or enzymatic conjugation may increase the antimicrobial activity of compounds and reduce minimal concentrations needed. With potent action mechanisms interfering with bacterial cell wall synthesis, DNA replication or enzyme production, phenolics can target multiple sites in bacteria, leading to a much higher sensitivity of cells towards these natural compounds. The current review summarizes some of the most important knowledge on functionalization of natural phenolic compounds and the effects on their antimicrobial activity.

Prenylated phenolics as promising candidates for combination antibacterial therapy: Morusin and kuwanon G.

Combination of antibiotics with natural products is a promising strategy for potentiating antibiotic activity and overcoming antibiotic resistance. The purpose of the present study was to investigate whether morusin and kuwanon G, prenylated phenolics in Morus species, have the ability to enhance antibiotic activity and reverse antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. Commonly used antibiotics (oxacillin, erythromycin, gentamicin, ciprofloxacin, tetracycline, clindamycin) were selected for the combination studies. Checkerboard and time-kill assays were used to investigate potential bacteriostatic and bactericidal synergistic interactions, respectively between morusin or kuwanon G and antibiotics. According to both fractional inhibitory concentration index and response surface models, twenty combinations (14 morusin-antibiotic combinations, six kuwanon G-antibiotic combinations) displaying bacteriostatic synergy were identified, with 4-512-fold reduction in the minimum inhibitory concentration values of antibiotics in combination. Both morusin and kuwanon G reversed oxacillin resistance of methicillin-resistant Staphylococcus aureus. In addition, morusin reversed tetracycline resistance of Staphylococcus epidermidis. At half of the minimum inhibitory concentrations, combinations of morusin with oxacillin or gentamicin showed bactericidal synergy against methicillin-resistant Staphylococcus aureus. Fluorescence and differential interference contrast microscopy and scanning electron microscopy showed an increase in the membrane permeability and massive leakage of cellular content in methicillin-resistant Staphylococcus aureus exposed to morusin or kuwanon G. Overall, our findings strongly indicate that both prenylated compounds are good candidates for the development of novel antibacterial combination therapies.

Graphene oxide effects in early ontogenetic stages of Triticum aestivum L. seedlings.

Nanomaterials are being used increasingly in various areas such as electronic devices manufacture, medicine, mechanical devices production, and even food industry. Therefore, the evaluation of their toxicity is mandatory. Graphene oxide (GO) has been shown to have both positive as well as negative impact on different crop plants, depending on species, dose, and duration of exposure. The current study evaluated the impact of GO sheets at different concentrations (500, 1000 and 2000 mg/L) on physiological, biochemical and genetic levels to determine the possible toxic action. Wheat caryopses were treated with GO for 48 h and 7 days. The germination rate and roots elongation decreased in a dose-response manner, except the sample treated with GO at a concentration of 1000 mg/L. Mitotic index has ascendant trend; its increase may be due to the accumulation of prophases GO induced significant accumulation of the cells with aberrations, their presence suggests a clastogenic/aneugenic effect of these carbon nanomaterials. Regarding enzymatic and non-enzymatic antioxidant system defence, the activity varied depending on the dose of GO. Thus, chlorophyll a pigments content decreased significantly at high dose (2000 mg/L), while the carotenoid pigments had lower content at 500 mg/L of GO, and no statistical difference encountered in case of chlorophyll b amount. The antioxidant enzyme activity (CAT, POD, and SOD) was higher at low dose of GO, indicating the presence of oxidative stress generated as a response to the GO treatment. Also, the free radical scavenging activity of the polyphenolic compounds was enhanced upon GO exposure. The GO accumulation has been identified by transmission electron microscopy only at plumules level, near the intercellular space.

Blue and Red LED Illumination Improves Growth and Bioactive Compounds Contents in Acyanic and Cyanic Ocimum basilicum L. Microgreens.

Microgreens are an excellent source of health-maintaining compounds, and the accumulation of these compounds in plant tissues may be stimulated by exogenous stimuli. While light quality effects on green basil microgreens are known, the present paper aims at improving the quality of acyanic (green) and cyanic (red) basil microgreens with different ratios of LED blue and red illumination. Growth, assimilatory and anthocyanin pigments, chlorophyll fluorescence, total phenolic, flavonoids, selected phenolic acid contents and antioxidant activity were assessed in microgreens grown for 17 days. Growth of microgreens was enhanced with predominantly blue illumination, larger cotyledon area and higher fresh mass. The same treatment elevated chlorophyll a and anthocyanin pigments contents. Colored light treatments decreased chlorophyll fluorescence ΦPSII values significantly in the green cultivar. Stimulation of phenolic synthesis and free radical scavenging activity were improved by predominantly red light in the green cultivar (up to 1.87 fold) and by predominantly blue light in the red cultivar (up to 1.73 fold). Rosmarinic and gallic acid synthesis was higher (up to 15- and 4-fold, respectively, compared to white treatment) in predominantly blue illumination. Red and blue LED ratios can be tailored to induce superior growth and phenolic contents in both red and green basil microgreens, as a convenient tool for producing higher quality foods.

INFLUENCE OF ORGANIC FOLIAR FERTILIZATION ON ANTIOXIDANT ACTIVITY AND CONTENT OF POLYPHENOLS IN OCIMUM BASILICUM L.

Basil is an important medicinal and culinary herb, cultivated on large areas in many countries. With the growing necessity of ecological products, organic crops need to be expanded, but a more complete characterization of such agriculture systems is required. The present paper aims to evaluate total phenolics and flavonoid contents, antioxidant activity of Ocimum basilicum L. under organic fertilization with four different foliar fertilizers (Fylo®, Geolino Plants&Flowers®, Cropmax®, Fitokondi®). The total content of phenolic compounds was stimulated by all foliar fertilizers used in the experiment. In the first year, the highest increase was obtained in plants fertilized with Fylo (29%) and Fitokondi (27%) while in the second year Fitokondi fertilizer treatment lead to the highest increase of total phenolics (28%) compared to the control plants. The production of total phenolics was enhanced in the second year probably because the experiment was started earlier on April compared to first year. Foliar fertilization of basil plants can thus be used to obtain increased yield and phenolic compounds synthesis with little effect on the physiological parameters that were analyzed, allowing better performance of basil under organic fertilization.

Micronutrient Patterns and Low Intake of Vitamin A, Vitamin D, Folate, Magnesium, and Potassium Among Prediabetes and Type 2 Diabetes Patients.

Background Assessing micronutrient intake is important in identifying deficiencies that may contribute to insulin resistance, poor glycemic control, and increased risk of diabetes-related complications. The study's objectives were to evaluate micronutrient intake in prediabetes (PD) and type 2 diabetes (T2DM) patients compared to recommended dietary intakes (RDI) and to determine the associations between the micronutrient patterns and both anthropometric measurements and biomarkers of diabetes. Methods This cross-sectional study was conducted on 349 patients with T2DM and 252 patients with PD. Micronutrient intake was evaluated using a validated food frequency questionnaire. Micronutrient patterns were extracted from factor analysis using principal component analysis with varimax rotation. Participants in the highest tertile were considered to have the highest adherence to the corresponding micronutrient pattern. Results T2DM patients had a significantly lower intake of vitamin E (9.4 ± 0.2 vs. 10.1 ± 0.3 mg; p = 0.048), vitamin D (44.3 ± 1.1 vs. 48.9 ± 1.7 IU; p = 0.020), and thiamin (1.3 ± 0.1 vs. 1.4 ± 0.1 mg; p = 0.013) compared to PD patients. All patients had a significantly lower intake of vitamin A, vitamin D, folate, magnesium, and potassium and a significantly higher intake of vitamin B12 and copper compared to RDI. Three distinct micronutrient patterns were identified within each group. In the PD group, the Fe-Mn-Se pattern correlated significantly with waist circumference (WC) and fasting plasma glucose (FPG). The Vit.C-K-Folate pattern showed significant associations with body fat (BF). The Vit.B2-P-Vit.B12 pattern was significantly linked to WC, body mass index (BMI), BF, FPG, and serum insulin (SI). For the T2DM patients, the K-Folate-Mg pattern displayed an inverse and significant association with weight and WC. The Iron-Se-Vit.B3 pattern showed a significant association with low-density lipoprotein (LDL) cholesterol, triglycerides, and total cholesterol. The Vit.B2-P-Ca pattern was significantly associated with fasting plasma glucose (FPG). Conclusion This study demonstrated that T2DM patients had significantly lower vitamin E, vitamin D, and thiamin intake than PD patients. Both T2DM and PD patients had a significantly lower intake of vitamin A, vitamin D, folate, magnesium, and potassium compared to the RDI. Among the identified micronutrient patterns, only the K-Folate-Mg pattern exhibited a significant association with reduced body weight and WC.

Biopolymers-Based Macrogels with Applications in the Food Industry: Capsules with Berry Juice for Functional Food Products.

The present study focused on the development of gel-based capsules from sodium alginate and the fresh juice from different berries: chokeberry, sea buckthorn, and blueberry. Obtained through the extrusion method, the macrocapsules were added into yogurt, a well-known and consumed dairy product. In order to establish the changes that can occur for the food product, the samples were tested over 7 and 15 days of storage in refrigeration conditions. According to the results, the antioxidant activity increased during storage and gels can represent a good option for bioactive substances' encapsulation. Sensorial analysis performed indicated that consumers are open to consuming yogurt berry capsules and, according to the results observed in the scientific literature, they no longer rejected the product due to the bitterness and sourness of sea buckthorn or aronia. Sea buckthorn capsules were brighter (L*) than chokeberry and blueberry capsules due to carotene content and dark colors. Minimal diameter variations and small standard deviations (SD = 0.25/0.33) suggest that extrusion methods and the Caviar box are good for gel capsule development. Yogurt luminosity varied with capsules; control had the highest, followed by sea buckthorn yogurt. Samples with chokeberry and blueberry (dark) capsules had lower luminosity. Over 8 and 15 days, luminosity slightly decreased, while a* and b* (hue and saturation) increased. Post-storage, the sample with chokeberry capsules showed a light purple color, indicating color transfer from capsules, with increased antioxidant activity. Differences between the samples and control were less pronounced in the sample with sea buckthorn capsules. Values for color differences between yogurt samples during the storage period revealed the most significant difference during the first storage period (day 1-8), with blueberries showing the lowest difference, indicating the stability of the blueberry capsules' wall during storage.

The knowns and unknowns of long COVID-19: from mechanisms to therapeutical approaches.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has been defined as the greatest global health and socioeconomic crisis of modern times. While most people recover after being infected with the virus, a significant proportion of them continue to experience health issues weeks, months and even years after acute infection with SARS-CoV-2. This persistence of clinical symptoms in infected individuals for at least three months after the onset of the disease or the emergence of new symptoms lasting more than two months, without any other explanation and alternative diagnosis have been named long COVID, long-haul COVID, post-COVID-19 conditions, chronic COVID, or post-acute sequelae of SARS-CoV-2 (PASC). Long COVID has been characterized as a constellation of symptoms and disorders that vary widely in their manifestations. Further, the mechanisms underlying long COVID are not fully understood, which hamper efficient treatment options. This review describes predictors and the most common symptoms related to long COVID's effects on the central and peripheral nervous system and other organs and tissues. Furthermore, the transcriptional markers, molecular signaling pathways and risk factors for long COVID, such as sex, age, pre-existing condition, hospitalization during acute phase of COVID-19, vaccination, and lifestyle are presented. Finally, recommendations for patient rehabilitation and disease management, as well as alternative therapeutical approaches to long COVID sequelae are discussed. Understanding the complexity of this disease, its symptoms across multiple organ systems and overlapping pathologies and its possible mechanisms are paramount in developing diagnostic tools and treatments.

Detection of Biological Molecules Using Nanopore Sensing Techniques.

Modern biomedical sensing techniques have significantly increased in precision and accuracy due to new technologies that enable speed and that can be tailored to be highly specific for markers of a particular disease. Diagnosing early-stage conditions is paramount to treating serious diseases. Usually, in the early stages of the disease, the number of specific biomarkers is very low and sometimes difficult to detect using classical diagnostic methods. Among detection methods, biosensors are currently attracting significant interest in medicine, for advantages such as easy operation, speed, and portability, with additional benefits of low costs and repeated reliable results. Single-molecule sensors such as nanopores that can detect biomolecules at low concentrations have the potential to become clinically relevant. As such, several applications have been introduced in this field for the detection of blood markers, nucleic acids, or proteins. The use of nanopores has yet to reach maturity for standardization as diagnostic techniques, however, they promise enormous potential, as progress is made into stabilizing nanopore structures, enhancing chemistries, and improving data collection and bioinformatic analysis. This review offers a new perspective on current biomolecule sensing techniques, based on various types of nanopores, challenges, and approaches toward implementation in clinical settings.

Nanopore Sequencing Assessment of Bacterial Pathogens and Associated Antibiotic Resistance Genes in Environmental Samples.

As seen in earlier and present pandemics, monitoring pathogens in the environment can offer multiple insights on their spread, evolution, and even future outbreaks. The present paper assesses the opportunity to detect microbial pathogens and associated antibiotic resistance genes, in relation to specific pathogen sources, by using nanopore sequencing in municipal waters and wastewaters in Romania. The main results indicated that waters collecting effluents from a meat processing facility exhibit altered communities' diversity and abundance, with reduced values (101-108 and 0.86-0.91) of Chao1 and, respectively, Simpson diversity indices and Campylobacterales as main order, compared with other types of municipal waters where the same diversity index had much higher values of 172-214 and 0.97-0.98, and Burkholderiaceae and Pseudomonadaceae were the most abundant families. Moreover, the incidence and type of antibiotic resistance genes were significantly influenced by the proximity of antibiotic sources, with either tetracycline (up to 45% of total reads) or neomycin, streptomycin and tobramycin (up to 3.8% total reads) resistance incidence being shaped by the sampling site. As such, nanopore sequencing proves to be an easy-to-use, accessible molecular technique for environmental pathogen surveillance and associated antibiotic resistance genes.

Design of Functional Polymer Systems to Optimize the Filler Retention in Obtaining Cellulosic Substrates with Improved Properties.

In the present work, the possibility of increasing the calcium carbonate (CaCO3) content in sheets of paper to optimize their properties was investigated. A new class of polymeric additives for papermaking is proposed as well as a method for their use in paper sheet containing the CaCO3 precipitated addition. Calcium carbonate precipitated (PCC) and fibers cellulose were adjusted with a cationic polyacrylamide flocculating agent (polydiallyldimethylammonium chloride (plyDADMAC) or cationic polyacrylamide (cPAM)). PCC was obtained in the laboratory by a double-exchange reaction between calcium chloride (CaCl2) and sodium carbonate (Na2CO3) suspension. After testing, the dosage of PCC was established at 35%. To improve the systems of additives studied, the materials obtained were characterized and their optical and mechanical properties were analysed. The PCC had a positive influence over all of the paper samples, but in the case of use of cPAM and polyDADMAC polymers the paper obtained had superior properties compared to the paper obtained without additives. Also, the samples obtained in the presence of cationic polyacrylamide exhibit superior properties to those obtained in the presence of polyDADMAC.

The Future Packaging of the Food Industry: The Development and Characterization of Innovative Biobased Materials with Essential Oils Added.

The need to replace conventional, usually single-use, packaging materials, so important for the future of resources and of the environment, has propelled research towards the development of packaging-based on biopolymers, fully biodegradable and even edible. The current study furthers the research on development of such films and tests the modification of the properties of the previously developed biopolymeric material, by adding 10, respectively 20% w/v essential oils of lemon, grapefruit, orange, cinnamon, clove, mint, ginger, eucalypt, and chamomile. Films with a thickness between 53 and 102 µm were obtained, with a roughness ranging between 147 and 366 nm. Most films had a water activity index significantly below what is required for microorganism growth, as low as 0.27, while all essential oils induced microbial growth reduction or 100% inhibition. Tested for the evaluation of physical, optical, microbiological or solubility properties, all the films with the addition of essential oil in the composition showed improved properties compared to the control sample.

The Use of Biopolymers as a Natural Matrix for Incorporation of Essential Oils of Medicinal Plants.

The benefits of using biopolymers for the development of films and coatings are well known. The enrichment of these material properties through various natural additions has led to their applicability in various fields. Essential oils, which are well-known for their beneficial properties, are widely used as encapsulating agents in films based on biopolymers. In this study, we developed biopolymer-based films and tested their properties following the addition of 7.5% and 15% (w/v) essential oils of lemon, orange, grapefruit, cinnamon, clove, chamomile, ginger, eucalyptus or mint. The samples were tested immediately after development and after one year of storage in order to examine possible long-term property changes. All films showed reductions in mass, thickness and microstructure, as well as mechanical properties. The most considerable variations in physical properties were observed in the 7.5% lemon oil sample and the 15% grapefruit oil sample, with the largest reductions in mass (23.13%), thickness (from 109.67 µm to 81.67 µm) and density (from 0.75 g/cm3 to 0.43 g/cm3). However, the microstructure of the sample was considerably improved. Although the addition of lemon essential oil prevented the reduction in mass during the storage period, it favored the degradation of the microstructure and the loss of elasticity (from 16.7% to 1.51% for the sample with 7.5% lemon EO and from 18.28% to 1.91% for the sample with 15% lemon EO). Although the addition of essential oils of mint and ginger resulted in films with a more homogeneous microstructure, the increase in concentration favored the appearance of pores and modifications of color parameters. With the exception of films with added orange, cinnamon and clove EOs, the antioxidant capacity of the films decreased during storage. The most obvious variations were identified in the samples with lemon, mint and clove EOs. The most unstable samples were those with added ginger (95.01%), lemon (92%) and mint (90.22%).

Fecal microbiota transplantation in non-communicable diseases: Recent advances and protocols.

Fecal microbiota transplant (FMT) is a therapeutic method that aims to restore normal gut microbial composition in recipients. Currently, FMT is approved in the USA to treat recurrent and refractory Clostridioides difficile infection and has been shown to have great efficacy. As such, significant research has been directed toward understanding the potential role of FMT in other conditions associated with gut microbiota dysbiosis such as obesity, type 2 diabetes mellitus, metabolic syndrome, neuropsychiatric disorders, inflammatory bowel disease, irritable bowel syndrome, decompensated cirrhosis, cancers and graft-versus-host disease. This review examines current updates and efficacy of FMT in treating conditions other than Clostridioides difficile infection. Further, protocols for administration of FMT are also discussed including storage of fecal samples in stool banks, inclusion/exclusion criteria for donors, fecal sample preparation and methods of treatment administration. Overall, understanding the mechanisms by which FMT can manipulate gut microbiota to provide therapeutic benefit as well as identifying potential adverse effects is an important step in clarifying its long-term safety and efficacy in treating multiple conditions in the future.

Assessment of the Fertilization Capacity of the Aquaculture Sediment for Wheat Grass as Sustainable Alternative Use.

Periodic removal of sediment from aquaculture ponds is practiced to maintain their productivity and animal welfare. The recovery of sediment as a plant fertilizer could alleviate the costs of sediment removal. The objective of this study was to test the effects of a dried sediment, extracted from an aquaculture pond used for common carp cultivation, on the growth and physiology of potted wheat grass and the quality of the juice obtained from wheat grass. The results showed that sediment application did not produce significant morphological changes, although the values for plant height (16.94-19.22 cm), leaf area (19.67-139.21 mm2), and biomass (3.39-4.26 g/plant) were higher in sediment-grown plants. However, at a physiological level, the effect was negative, decreasing photosynthesis (0.82-1.66 µmol CO2 m2s-1), fluorescence ΦPSII (0.737-0.782), and chlorophyll content (1.40-1.83 CCI). The juice yield was reduced in the sediment treatments (46-58 g/100 g), while the quality was improved by increasing the content of phenols (2.55-3.39 µg/mL gallic acid equivalent), flavonoids (1.41-1.85 µg/mL quercetin equivalent), and antioxidant activity (47.99-62.7% inhibition of; 2,2-diphenyl-1-picrylhydrazyl). The positive results obtained in this study can be attributed to the moderate nutrient content of the sediment and a negligible concentration of heavy metals.

A Conservative Replacement in the Transmembrane Domain of SARS-CoV-2 ORF7a as a Putative Risk Factor in COVID-19.

The ongoing COVID-19 pandemic follows an unpredictable evolution, driven by both host-related factors such as mobility, vaccination status, and comorbidities and by pathogen-related ones. The pathogenicity of its causative agent, SARS-CoV-2 virus, relates to the functions of the proteins synthesized intracellularly, as guided by viral RNA. These functions are constantly altered through mutations resulting in increased virulence, infectivity, and antibody-evasion abilities. Well-characterized mutations in the spike protein, such as D614G, N439K, Δ69-70, E484K, or N501Y, are currently defining specific variants; however, some less studied mutations outside the spike region, such as p. 3691 in NSP6, p. 9659 in ORF-10, 8782C > T in ORF-1ab, or 28144T > C in ORF-8, have been proposed for altering SARS-CoV-2 virulence and pathogenicity. Therefore, in this study, we focused on A105V mutation of SARS-CoV-2 ORF7a accessory protein, which has been associated with severe COVID-19 clinical manifestation. Molecular dynamics and computational structural analyses revealed that this mutation differentially alters ORF7a dynamics, suggesting a gain-of-function role that may explain its role in the severe form of COVID-19 disease.

Emergence of the First Strains of SARS-CoV-2 Lineage B.1.1.7 in Romania: Genomic Analysis.

BACKGROUND: The United Kingdom reported the emergence of a new and highly transmissible SARS-CoV-2 variant (B.1.1.7) that rapidly spread to other countries. The impact of this new mutation-which occurs in the S protein-on infectivity, virulence, and current vaccine effectiveness is still under evaluation. OBJECTIVE: The aim of this study is to sequence SARS-CoV-2 samples of cases in Romania to detect the B.1.1.7 variant and compare these samples with sequences submitted to GISAID. METHODS: SARS-CoV-2 samples were sequenced and amino acid substitution analysis was performed using the CoV-GLUE platform. RESULTS: We have identified the first cases of the B.1.1.7 variant in samples collected from Romanian patients, of which one was traced to the region of the United Kingdom where the new variant was originally sequenced. Mutations in nonstructural protein 3 (Nsp3; N844S and D455N) and ORF3a (L15F) were also detected, indicating common ancestry with UK strains as well as remote connections with strains from Nagasaki, Japan. CONCLUSIONS: These results indicate, for the first time, the presence and characteristics of the new variant B.1.1.7 in Romania and underscore the need for increased genomic sequencing in patients with confirmed COVID-19.

Introduction and Characteristics of SARS-CoV-2 in North-East of Romania During the First COVID-19 Outbreak.

Romania officially declared its first Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) case on February 26, 2020. The first and largest coronavirus disease 2019 (COVID-19) outbreak in Romania was recorded in Suceava, North-East region of the country, and originated at the Suceava regional county hospital. Following sheltering-in-place measures, infection rates decreased, only to rise again after relaxation of measures. This study describes the spread of SARS-CoV-2 in Suceava and other parts of Romania and analyses the mutations and their association with clinical manifestation of the disease during the period of COVID-19 outbreak. Sixty-two samples were sequenced via high-throughput platform and screened for variants. For selected mutations, putative biological significance was assessed, and their effects on disease severity. Phylogenetic analysis was conducted on Romanian genomes (n = 112) and on sequences originating from Europe, United Kingdom, Africa, Asia, South, and North America (n = 876). The results indicated multiple introduction events for SARS-CoV-2 in Suceava, mainly from Italy, Spain, United Kingdom, and Russia although some sequences were also related to those from the Czechia, Belgium, and France. Most Suceava genomes contained mutations common to European lineages, such as A20268G, however, approximately 10% of samples were missing such mutations, indicating a possible different arrival route. While overall genome regions ORF1ab, S, and ORF7 were subject to most mutations, several recurring mutations such as A105V were identified, and these were mainly present in severe forms of the disease. Non-synonymous mutations, such as T987N (Thr987Asn in NSP3a domain), associated with changes in a protein responsible for decreasing viral tethering in human host were also present. Patients with diabetes and hypertension exhibited higher risk ratios (RR) of acquiring severe forms of the disease and these were mainly related to A105V mutation. This study identified the arrival routes of SARS-CoV-2 in Romania and revealed potential associations between the SARS-CoV-2 genomic organization circulating in the country and the clinical manifestation of COVID-19 disease.