Impact of molecular weight on the formation of electrosprayed chitosan microcapsules as delivery vehicles for bioactive compounds

The molecular weight of chitosan is one of its most determinant characteristics, which affects its processability and its performance as a biomaterial. However, information about the effect of this parameter on the formation of electrosprayed chitosan microcapsules is scarce. In this work, the impact of chitosan molecular weight on its electrosprayability was studied and correlated with its effect on the viscosity, surface tension and electrical conductivity of solutions. A Discriminant Function Analysis revealed that the morphology of the electrosprayed chitosan materials could be correctly predicted using these three parameters for almost 85% of the samples. The suitability of using electrosprayed chitosan capsules as carriers for bioactive agents was also assessed by loading them with a model active compound, (−)-epigallocatechin gallate (EGCG). This encapsulation, with an estimated efficiency of around 80% in terms of preserved antioxidant activity, showed the potential to prolong the antiviral activity of EGCG against murine norovirus via gradual bioactive release combined with its protection against degradation in simulated physiological conditions.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Gloria Sanchez was supported by the “Ramón y Cajal” Young Investigator Program. The authors would like to thank the Spanish MINECO project AGL2015-63855-C2-1 and INIA grant RTA2014-00024-C04-03 for financial support.Peer reviewe

Optimization of electrospraying conditions for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion

Electrospraying has recently emerged as a novel microencapsulation technique with potential for the protection of probiotics. However, research efforts are still needed to minimize the viability loss observed during the processing of sensitive strains, and to maximize productivity. The aim of the present work was the optimization of the electrospraying conditions for the microencapsulation of a model probiotic microorganism, Lactobacillus plantarum, within a whey protein concentrate matrix. In a pre-optimization step, the convenience of encapsulating fresh culture instead of freeze-dried bacteria was established. Additionally, a surface response methodology was used to study the effect of the applied voltage, surfactant concentration, and addition of a prebiotic to the formulation on cell viability and productivity. Viability losses lower than 1 log10 CFU were achieved and the bacterial counts of the final products exceeded 8.5 log10 CFU/g. The protection ability of the developed structures during storage and in-vitro digestion was also evaluated.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Russell Cruz Morfin received a scholarship from the Mexican National Council for Science and Technology (CONACYT), Call 2014. Gloria Sánchez is supported by the “Ramón y Cajal” Young Investigator program of the MINECO. This work was financially supported by the Spanish MINECO project AGL2012-30647 and by the CSIC project 201470I002.Peer reviewe

Comparing analytical methods to detect SARS-CoV-2 in wastewater

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene glycol (PEG) precipitation protocol. Moreover, two different RNA extraction methods were compared in this study: a commercial manual spin column centrifugation kit and an automated protocol based on magnetic silica beads. Overall, the evaluated concentration methods did not impact the recovery of gamma-irradiated SARS-CoV-2 nor MgV, while extraction methods showed significant differences for PEDV. Mean recovery rates of 42.9 ± 9.5%, 27.5 ± 14.3% and 9.0 ± 2.2% were obtained for gamma-irradiated SARS-CoV-2, PEDV and MgV, respectively. Limits of detection (LoD95%) for five genomic SARS-CoV-2 targets (N1, N2, gene E, IP2 and IP4) ranged from 1.56 log genome equivalents (ge)/mL (N1) to 2.22 log ge/mL (IP4) when automated system was used; while values ranging between 2.08 (N1) and 2.34 (E) log ge/mL were observed when using column-based extraction method. Different targets were also evaluated in naturally contaminated wastewater samples with 91.2%, 85.3%, 70.6%, 79.4% and 73.5% positivity, for N1, N2, E, IP2 and IP4, respectively. Our benchmarked comparison study suggests that the aluminum precipitation method coupled with the automated nucleic extraction represents a method of acceptable sensitivity to provide readily results of interest for SARS-CoV-2 WBE surveillance.The study was funded by grants from CSIC (202070E101), Generalitat Valenciana (Covid_19-SCI), MICINN co-founded by AEI/FEDER, UE (AGL2017-82909), and MICINN/AEI (PID2019-105509RJ-I00). EC-F is recipient of a predoctoral contract from the MICINN, Call 2018. WR is holder of the APOSTD/2018/150 postdoctoral contract from Generalitat Valenciana.Peer reviewe

Antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems

Silver nanoparticles (AgNP) have strong broad-spectrum antimicrobial activity and gained increased attention for the development of AgNP based products, including medical and food applications. Initially, the efficacy of AgNP and silver nitrate (AgNO3) was evaluated for inactivating norovirus surrogates, the feline calicivirus (FCV) and the murine norovirus (MNV). These norovirus surrogates were exposed to AgNO3 and AgNP solutions for 24 h at 25 °C and then analyzed by cell-culture assays. Both AgNP and silver ions significantly decreased FCV and MNV infectivity in a dose-dependent manner between concentrations of 2.1 and 21 mg/L. Furthermore, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films were prepared by depositing a coating of thermally post-processed electrospun PHBV18/AgNP fiber mats over compression moulded PHBV3 films. After 24 h exposure at 37 °C and 100% RH, no infectious FCV were recovered when in contact with the AgNP films while MNV titers decreased by 0.86 log. The morphology of the PHBV18 and PHBV18/AgNP fibers studied by SEM showed smooth and continuous fibers in both cases and the EDAX analysis confirmed the homogeneously distribution of AgNP into the coating and onto the PHBV3/PHBV18 layer. This study showed, for the first time, the suitability of the PHBV18/AgNP electrospun coating for antiviral surfaces.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (RYC-2012-09950, RYC-2014-158, AGL2015-63855-C2-1-R and INIA grant RTA2014-00024-C04-03). GS and MJF were supported by the “Ramón y Cajal” Young Investigator from the MINECO (RYC-2012-09950 and RYC-2014-15842). JLC-M was supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of Colombian Government.Peer reviewe

Efficacy of Cinnamaldehyde Against Enteric Viruses and Its Activity After Incorporation Into Biodegradable Multilayer Systems of Interest in Food Packaging

Cinnamaldehyde (CNMA), an organic compound that gives cinnamon its flavor and odor, was investigated for its virucidal activity on norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), and hepatitis A virus (HAV). Initially, different concentrations of CNMA (0.1, 0.5 and 1 %) were individually mixed with each virus at titers of ca. 6–7 log10 TCID50/ml and incubated 2 h at 4 and 37 °C. CNMA was effective in reducing the titers of norovirus surrogates in a dose-dependent manner after 2 h at 37 °C, while HAV titers were reduced by 1 log10 after treatment with 1 % of CNMA. When incubation time was extended, HAV titers were reduced by 3.4 and 2.7 log10 after overnight incubation at 37 °C with 1 and 0.5 % of CNMA, respectively. Moreover, this paper analyzed, for the first time, the antiviral activity of adding an active electrospun interlayer based on zein and CNMA to a polyhydroxybutyrate packaging material (PHB) in a multilayer form. Biodegradable multilayer systems prepared with 2.60 mg/cm2 (~9.7 %) of CNMA completely inactivated FCV according to ISO 22196:2011, while MNV titers were reduced by 2.75 log10. When the developed multilayer films were evaluated after one month of preparation or at 25 °C, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37 °C. The results show the excellent potential of this system for food contact applications as well as for active packaging technologies in order to maintain or extend food quality and safety.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (RYC-2012-09950, AGL2015-63855-C2-1 and INIA Grant RTA2014-00024-C04-03). GS was supported by the “Ramón y Cajal” Young Investigator and MJF was recipient of a Juan de la Cierva contract from the MINECO. JLC-M was supported by the Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) of Colombian Government and WR by the “Student Mobility for Placement e SMP” Grant of the EU Life Learning Program.Peer reviewe

Epidemiological Surveillance of Norovirus and Rotavirus in Sewage (2016-2017) in Valencia (Spain)

The aim of the present study was to perform the molecular epidemiology of rotaviruses and noroviruses detected in sewage samples from a large wastewater facility from the city of Valencia, Spain. A total of 46 sewage samples were collected over a one-year period (September 2016 to September 2017). Norovirus and rotavirus were detected and quantified by RT-qPCR, genotyped by semi-nested RT-PCR and further characterized by sequencing and phylogenetic analyses. Noroviruses and rotaviruses were widely distributed in sewage samples (69.6% for norovirus GI, 76.0% norovirus GII, and 71.7% rotaviruses) and viral loads varied from 4.33 to 5.75 log PCRU/L for norovirus GI, 4.69 to 6.95 log PCRU/L for norovirus GII, and 4.08 to 6.92 log PCRU/L for rotavirus. Overall, 87.5% (28/32) of GI noroviruses could not be genotyped, 6.25% (2/32) of the samples contained GI.2 genotype, and another 6.25% (2/32) were positive for GI.4 genotype. The most common genotype of GII noroviruses was GII.2 (40%, 14/35), followed by GII.6 (8.6%, 3/35) and GII.17 (5.7%, 2/35) while the remaining GII strains could not be typed (45.7%, 16/35). Rotavirus VP4 genotype P[8] was the only one found in 19 out of 33 rotavirus-positive samples (57.7%). G2 was the most prevalent rotavirus VP7 genotype (15.2%, 5/33) followed by G3, G9, and G12, with two positive samples for each genotype (6.1%, 2/33). In one sample both G1 and G2 genotypes were detected simultaneously (3%). The results presented here show that the surveillance of noroviruses and rotaviruses in sewage is useful for the study of their transmission in the population and their molecular epidemiology

Combined kinetic analysis of SARS-CoV-2 RNAemia, N-antigenemia and virus-specific antibodies in critically ill adult COVID-19 patients

Combined kinetic analysis of plasma SARS‐CoV‐2 RNAemia, Nucleocapsid (N)‐antigenemia and virus‐specific antibodies may help ascertain the role of antibodies in preventing virus dissemination in COVID‐19 patients. We performed this analysis in a cohort of 71 consecutive critically ill COVID‐19 patients (49 male; median age, 65 years) using RT‐PCR assay, lateral flow immunochromatography method and receptor binding domain (RBD) and N‐based immunoassays. A total of 338 plasma specimens collected at a median of 12 days after symptoms onset were available for analyses. SARS‐ CoV‐2 RNAemia and N‐antigenemia were detected in 37 and 43 specimens from 26 (36.5%) and 30 (42.2%) patients, respectively. Free RNA was the main biological form of SARS‐CoV‐2 found in plasma. The detection rate for both viral components was associated with viral load at the upper respiratory tract. Median time to SARS‐CoV‐2‐RBD antibody detection was 14 days (range, 4-38) from onset of symptoms. Decreasing antibody levels were observed in parallel to increasing levels of both RNAemia and N‐antigenemia, yet overall a fairly modest inverse correlation (Rho = −0.35; P < 0.001) was seen between virus RNAemia and SARS‐CoV‐2‐RBD antibody levels. The data cast doubts on a major involvement of antibodies in virus clearance from the bloodstream within the timeframe examined

Early detection of SARS-CoV-2 infection cases or outbreaks at nursing homes by targeted wastewater tracking

Objectives Near-source tracking of SARS-CoV-2 RNA in the sewage drains serving particular buildings may allow rapid identification of SARS-CoV-2 infection cases or local outbreaks. In this pilot study, we investigated whether this was the case for nursing homes (NH). Methods The study involved five NH (from A to E) affiliated to the Clínico-Malvarrosa Health Department, Valencia (Spain). These were nursing or mixed nursing/care homes of different sizes, altogether providing care for 472 residents attended by a staff of 309. Near-source sewage samples were screened for presence of SARS-CoV-2 RNA by RT-qPCR at least 5 days per week during the study period. SARS-CoV-2 RNA testing in nasopharyngeal swabs from residents and staff was performed with the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Massachusetts, USA). Results SARS-CoV-2 RNA was detected in wastewater samples from four of the five NH. SARS-CoV-2 infection cases were documented in three of these four NH. Of the two NH without SARS-CoV-2 infection cases, no SARS-CoV-2 RNA was detected in sewer samples from one facility, while it was repeatedly detected in samples from the other. Presence of SARS-CoV-2 RNA in sewage preceded identification of isolated cases among residents or staff or outbreak declaration in two NH, with lag times ranging from 5 to 19 days. Conclusion Our study demonstrated that intermittent or persistent detection of SARS-CoV-2 RNA in NH sewers can provide an early warning of subsequent individual cases or outbreaks in these facilities.N