Different Behavior of Enteric Bacteria and Viruses in Clay and Sandy Soils after Biofertilization with Swine Digestate.

Enteric pathogens from biofertilizer can accumulate in the soil, subsequently contaminating water and crops. We evaluated the survival, percolation and leaching of model enteric pathogens in clay and sandy soils after biofertilization with swine digestate: PhiX-174, mengovirus (vMC0), Salmonella enterica Typhimurium and Escherichia coli O157:H7 were used as biomarkers. The survival of vMC0 and PhiX-174 in clay soil was significantly lower than in sandy soil (iT90 values of 10.520 ± 0.600 vs. 21.270 ± 1.100 and 12.040 ± 0.010 vs. 43.470 ± 1.300, respectively) and PhiX-174 showed faster percolation and leaching in sandy soil than clay soil (iT90 values of 0.46 and 2.43, respectively). S. enterica Typhimurium was percolated and inactivated more slowly than E. coli O157:H7 (iT90 values of 9.340 ± 0.200 vs. 6.620 ± 0.500 and 11.900 ± 0.900 vs. 10.750 ± 0.900 in clay and sandy soils, respectively), such that E. coli O157:H7 was transferred more quickly to the deeper layers of both soils evaluated (percolation). Our findings suggest that E. coli O157:H7 may serve as a useful microbial biomarker of depth contamination and leaching in clay and sandy soil and that bacteriophage could be used as an indicator of enteric pathogen persistence. Our study contributes to development of predictive models for enteric pathogen behavior in soils, and for potential water and food contamination associated with biofertilization, useful for risk management and mitigation in swine digestate recycling.

Propidium Monoazide Integrated with qPCR Enables the Detection and Enumeration of Infectious Enteric RNA and DNA Viruses in Clam and Fermented Sausages.

The increase of foodborne viral outbreaks highlights the need for a rapid and sensitive method for the prediction of viral infectivity in food samples. This study assesses the use of propidium monoazide (PMA) coupled with real-time PCR methods (RT-qPCR or qPCR for RNA or DNA viruses, respectively) in the determination of viral infectivity in complex animal-related food matrices. Clam and Spanish fermented sausage ("chorizo") samples were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) and mengovirus (vMC0). PMA-qPCR/RT-qPCR discriminated infective virus particles, with significant reductions (>2.7 log10 or 99.7%). Additionally, infectious HAdV-2 and vMC0 were quantified by plaque assay (in plaque forming units, PFU), and compared with those in virus genomes copies (GCs) quantified by PMA-qPCR/RT-qPCR. A consistent correlation (R2 > 0.92) was showed between PFU and GCs along serial 10-fold dilutions in both DNA and RNA virus and in both food matrices. This study shows the use of PMA coupled to qPCR/RT-qPCR as a promising alternative for prediction of viral infectivity in food samples in comparison to more expensive and time-consuming methods and for those viruses that are not able to grow under available cell culture techniques.

Effect of UV light on the inactivation of recombinant human adenovirus and murine norovirus seeded in seawater in shellfish depuration tanks.

Shellfish depuration is a process that aims to eliminate pathogens from mollusk tissues. Seawater disinfection during the depuration process is important and ultraviolet (UV) light treatment is the most used method worldwide. Viral models are usually employed as surrogates of fastidious viruses in viability studies. The aim of this study was to employ methods based on green fluorescent protein (GFP) fluorescence and plaque forming units to detect, respectively, recombinant adenovirus (rAdV-GFP) and murine norovirus (MNV) artificially seeded in environmental matrices. These assays were applied to assess the inactivation of rAdV-GFP and MNV in seawater in recirculation shellfish depuration tanks with and without UV light treatment. Kinetics of rAdV GFP-expression was previously measured by UV-spectrophotometer. Flow cytometry (FC), fluorescence microscopy (FM), and plaque assay were used to determine virus titer and detection limits. The influence of the environmental matrix on the performance of the methods was prior determined using either drinking water or filtered seawater seeded with rAdV-GFP. Disinfection of seeded seawater was evaluated with and without UV treatment. The time of 24-h post-infection was established as ideal for fluorescence detection on rAdV-GFP infected cells. FC showed lower sensitivity, when compared to FM, which was similar to plaque assay. Seawater disinfection on depuration tanks was promising and rAdV-GFP declined 99.99 % after 24 and 48 h with and without UV treatment, respectively. MNV was completely inactivated after 24 h in both treatments. As conclusion, the depuration tanks were effective to inactivate rAdV-GFP and MNV and the UV disinfection treatment accelerated the process.

Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays.

BACKGROUND: Human adenoviruses (HAdVs) are the second-leading cause of childhood gastroenteritis worldwide. This virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially UV light inactivation. Molecular techniques, such as PCR-based methods (Polymerase Chain Reaction), are commonly used to detect and identify viral contamination in water, although PCR alone does not allow the discrimination between infectious and non-infectious viral particles. A combination of cell culture and PCR has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture. This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system. METHODS: Water samples were collected, concentrated and HAdV quantified by real-time PCR. Viral integrity was evaluated by enzymatic assay (DNase I) and infectivity by plaque assay (PA) and integrated cell culture using transcribed mRNA (ICC-RT-qPCR). Samples containing particles of infectious HAdV were selected for sequencing and molecular characterization. RESULTS: The analyzed sites contained 83, 66 and 58% undamaged HAdV particles (defined as those in which the genetic material is protected by the viral capsid) at Peri Lagoon, spring source water and public supply system water, respectively. Of these, 66% of the particles (by PA) and 75% (by ICC-RT-qPCR) HAdV were shown to be infectious, due to being undamaged in Peri Lagoon, 33% (by PA) and 58% (by ICC-RT-qPCR) in spring source water and 8% (by PA) and 25% (by ICC-RT-qPCR) in the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 102 HAdV genome copies per milliliter of infectious viral particles in the environmental water samples. The molecular characterization studies indicated that HAdV-2 was the prevalent serotype. CONCLUSIONS: These results indicate a lack of proper public health measures. We suggest that HAdV can be efficiently used as a marker of environmental and drinking water contamination and ICC-RT-qPCR demonstrated greater sensitivity and speed of detection of infectious viral particles compared to PA.

Effects of Ilex paraguariensis A. St. Hil. (yerba mate) on herpes simplex virus types 1 and 2 replication.

The antiherpes effects of the crude extract obtained from Ilex paraguariensis leaves (yerba mate) and their purified fractions were investigated. The most active fraction was selected and assayed to determine the viral multiplication steps upon which it acted. In order to detect the major components of this fraction, thin layer chromatography (TLC) analysis was performed. The antiviral activity was evaluated against HSV-1 and HSV-2 by a viral plaque number reduction assay (IC(50) ) and the cytotoxicity by a MTT assay (CC(50) ). According to the obtained results, all tested samples showed antiherpes activity at noncytotoxic concentrations, and the ethyl acetate fraction was the most active (SI = CC(50) /IC(50) = 188.7 and 264.7 for HSV-1 and HSV-2, respectively). The results also demonstrated that this fraction exerts antiviral activity by the reduction of viral infectivity, the inhibition of virus entry into cells and cell-to-cell virus spread, as well as by the impaired levels of ICP27, ICP4, gD and gE proteins of HSV-1. The TLC analysis showed that this fraction contains monodesmosidic triterpenoid saponins, matesaponin-1 (a bidesmosidic one), caffeic and chlorogenic acids and rutin, which suggests that they could act synergistically and be responsible for the detected antiherpes activity.

Evidence for zoonotic transmission of group C rotaviruses among children in Belém, Brazil.

The prevalence and potential zoonotic transmission of group C rotavirus (RVC) were examined by testing fecal samples collected from children during a longitudinal study that was carried out in the outskirts of Belém, Brazil, from December 1982 to March 1986. The study involved a group of 30 children who were followed from birth to 3 years. Of the 77 samples tested from 29 children, 5 (6.5%) were positive for human and 3 (4%) for porcine RVC by using nested PCR assay with primers specific for VP6 gene of human or porcine RVC and by Southern hybridization using a probe specific for VP6 gene of both human and porcine RVC. In addition, a total of 59 fecal specimens from the 30th child were tested, 1 (1.7%) and 14 (23.7%) were positive for human and porcine RVC, respectively. Partial nucleotide sequences of VP6 gene demonstrated that the six human strains detected in Brazil were homologous with other human RVC, and 14 of the 17 porcine RVC strains examined showed a complete homology among themselves but differed slightly from the porcine Cowden strain, suggesting that a single porcine RVC strain was circulating in Belém. This study is the first to provide evidence for transmission of RVC from swine to human. They also indicate that both human and porcine RVC were endemic in Belém.

Evaluation of antiviral activity of South American plant extracts against herpes simplex virus type 1 and rabies virus.

This paper describes the screening of different South American plant extracts and fractions. Aqueous and organic extracts were prepared and tested for antiherpetic (HSV-1, KOS and 29R strains) and antirabies (PV strain) activities. The evaluation of the potential antiviral activity of these extracts was performed by using an MTT assay for HSV-1, and by a viral cytopathic effect (CPE) inhibitory method for rabies virus (RV). The results were expressed as 50% cytotoxicity (CC(50)) for MTT assay and 50% effective (EC(50)) concentrations for CPE, and with them it was possible to calculate the selectivity indices (SI = CC(50)/EC(50)) of each tested material. From the 18 extracts/fractions tested, six extracts and four fractions showed antiviral action. Ilex paraguariensis, Lafoensia pacari, Passiflora edulis, Rubus imperialis and Slonea guianensis showed values of SI > 7 against HSV-1 KOS and 29-R strains and Alamanda schottii showed a SI of 5.6 against RV, PV strain.

Relationship between the contamination of gulls (Larus dominicanus) and oysters (Crassostrea gigas) with Salmonella serovar Typhimurium by PCR-RFLP.

Shellfish can bioaccumulate in their tissues pathogenic contaminants present in water and they have been related with several outbreaks of food-borne diseases worldwide. With their increased population in urban areas, gulls have been reported as an important source of water environment contamination. During a 10-month period, water, gulls feces and oyster samples were collected in a shellfish harvesting site and analyzed for total and fecal coliform counts (water) and Salmonella presence (gull feces and oyster meat). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to differentiate Salmonella species detected in gulls and oysters. Salmonella presence was detected in 3/10 of oyster samples and in 6/10 of gull feces samples by PCR. There was a relationship between Salmonella presence in oysters and fecal contamination in water. Restriction profiles of both gulls and oyster samples were similar to Salmonella Typhimurium profile by RFLP. These findings indicate strong evidence that gulls can contribute to Salmonella contamination of harvested oysters.

Detection of Torque teno virus in Epstein-Barr virus positive and negative lymph nodes of patients with Hodgkin lymphoma.

The age-specific incidence of Hodgkin lymphoma (HL) is bimodal with peaks occurring among young adults (15 - 34 years old) and people older than 45 years. Epstein-Barr virus (EBV) is associated with only one-third of HL cases. This study sought to determine if Torque teno virus (TTV) might be independently associated with HL. The presence of EBV was appraised by in situ hybridization and immunohistochemistry in lymph node biopsies from 46 patients (3 - 81 years old) with HL. TTV DNA was assessed by PCR amplification. EBV was detected in 22 (48%) patients. TTV DNA was detected in 24/46 (52%) patients, as well as in 12/20 (60%) control patients with lymphoid unspecific hyperplasia. TTV DNA was not significantly more frequent in EBV negative (54%) than in EBV positive (50%) nodes. However, it was observed that the group of young adults (15 - 34 years, n = 19) showed the lowest EBV frequency (21%) but the highest TTV occurrence (60%). This may suggest an involvement of TTV infection in the pathogenesis of HL in young adults. Further large population-based studies are required to confirm our findings.

Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus.

Human rabies is a viral disease with a great impact on public health, mainly on account of its fatal course in the majority of cases. Despite the well-established prophylaxis by immunization, rabies is believed to be responsible for 40,000-70,000 human deaths per year, mostly in endemic areas. Palliative support and experimental protocols to avoid death have been employed with no expressive results, with the exception of a recent human case of recovery from rabies. No antiviral drugs are currently available to fight against this infection. In combination with the prophylaxis, an antiviral drug would be useful for human rabies treatment, providing enhanced protection against the encephalitis caused by the virus. Phenolic compounds are derived from the secondary plant metabolism, although they can also be obtained by synthetic processes. Many studies have shown a great range of pharmacological effects for these substances, including vasodilatation, antiallergenic, antiinflammatory and antiviral properties, among others. In this study, the potential in-vitro anti-rabies activity of 24 synthetic phenolic compounds was evaluated using McCoy cells and PV rabies strain. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the inhibition of viral cytopathic effects. Isoprinosine and ketamine were used as positive controls. The tested compounds showed selectivity indices (SI=CC50/IC50) ranging from 1.0 to 3.9. Six phenolic compounds failed to inhibit the cytopathic effect to any degree, and four showed SI > or = 3.0. According to these results, some probable structure-activity relationships are suggested. It was observed that the presence of free hydroxyl and ether groups influenced the anti-rabies activity. However, additional studies are required to establish these relationships.

Evaluation of antiherpetic activity and genotoxic effects of tea catechin derivatives.

This study evaluated the antiherpetic activity and genotoxicity of catechin and some of its derivatives using the MTT colorimetric and comet assays, respectively. The results showed that all compounds have antiviral activity with selective indices varying from 1.3 to 13, depending on the tested HSV-1 strain. It was observed that the same concentration of the compounds that protects the Vero cells against the viral infection induces genotoxicity. It was also observed that the molecules containing three hydroxyl groups on the B ring caused less DNA damage and showed better antiviral effect than those with two hydroxyls on the same ring, but if there is an additional galloyl group, these results can be altered. The bioavailability and stereochemistry could be related to the antiviral and genotoxic effects detected.

Detection of poliovirus type 2 in oysters by using cell culture and RT-PCR

Devido ao hábito alimentar filtrante, os moluscos bivalves são contaminados por vírus presentes em águas contaminadas por esgoto. Os enterovírus são geralmente usados como modelos para a detecção de vírus em moluscos bivalves devido a sua importância em saúde pública. No presente estudo, ostras foram colocadas em aquários de vidro contendo água do mar adicionada de algas unicelulares. Dois tipos de experimentos foram realizados: a) ostras bioacumulando quatro diferentes concentrações de poliovírus: 5 x 104, 2,5 x 104, 5 x 103, 5 x 102 PFU/mL durante 20h; b) tecidos de ostras inoculados diretamente com 6,0 x 105 e 1,0 x 105 PFU/mL. Após a semeadura, os tecidos foram processados por um método de adsorção-eluição-precipitação. Controles positivos foram realizados por inoculação de 6,0 x 105 PFU/mL de poliovírus diretamente nos tecidos processados das ostras. Os extratos teciduais foram testados para presença do vírus por ensaios de placa de lise (PFU), RT-PCR e cultura celular integrada ao PCR (ICC/PCR). Este último consistiu na inoculação das amostras sobre monocamadas de células VERO seguida de RT-PCR do fluido celular infeccioso. No primeiro experimento (ensaio de bioacumulação por 20h), foram detectados até 5 x 103 PFU de poliovírus, após 24 e 48h de replicação nas células. Os ensaios de RT-PCR e ICC/PCR foram capazes de detectar 3 e 0,04 PFU de poliovírus, respectivamente nos ensaios de bioacumulação. Quando os extratos teciduais processados foram semeados, os ensaios de placa de lise demonstraram recuperação de vírus infecciosos em todas as concentrações testadas. Pudemos concluir que partículas viáveis de poliovírus podem ser detectadas em ostras após bioacumulação e que estas técnicas podem ser diretamente aplicadas na detecção de vírus em amostras ambientais.

In vitro testing for genotoxicity of violacein assessed by Comet and Micronucleus assays.

Chromobacterium violaceum is a Gram (-) bacteria found in water samples and soils from tropical and subtropical regions of the world. Violacein, the major pigment produced by these bacteria, has been shown to have antibiotic, antitumoral and trypanocidal activities. In the present work, the genotoxicity of violacein was investigated in four different cell lines by using the alkaline Comet assay and in VERO cells using the Micronucleus test. In the alkaline Comet assay, violacein, when tested at concentrations ranging from 0.19 to 1.5 microM, did not induce a significant increase in DNA damage in HEp-2 and MA104 cells. However, violacein was positive for DNA damage in FRhK-4 cells and for both DNA damage and micronuclei in VERO cells, in a concentration-response relationship. The results of this study indicated that violacein is genotoxic in VERO and FRhK-4 cells. These findings contribute to a comprehensive evaluation of the pharmacological potential of violacein.

Evaluation of anti-herpetic and antioxidant activities, and cytotoxic and genotoxic effects of synthetic alkyl-esters of gallic acid.

The n-alkyl esters of gallic acid (CAS 13857-8) have a diverse range of uses as antioxidants in food, cosmetics and pharmaceutical industries. Pharmaceutical studies performed with these compounds have found that they have many therapeutic potentialities including anti-cancer, antiviral and antimicrobial properties. However, more interest has been devoted to their antioxidant activity due to the ability to scavenge and reduce reactive oxygen species (ROS) formation. In this study, gallic acid and 14 different alkyl gallates were tested. The cytotoxicity and anti-herpetic (HSV-1, KOS and 29-R strains) activity were studied by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay and the cell viability by using the Trypan blue dye exclusion method. The genotoxicity was studied by the Comet assay and the antioxidant activity by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and microsomal lipid peroxidation-inhibiting activities. The results showed that all the tested compounds have anti-herpetic activity at non cytotoxic concentrations with selectivity indices (SI = CC50/EC50) varying from 0.89 to 18.34, depending on the used HSV-1 strain. It was observed that all tested alkyl gallates showed some degree of genotoxicity, at the tested concentrations, except cetyl gallate, at 256.60 micromol/L (p <0.05, t-Student test), probably induced by ROS released by infected cells and/or by the alkyl gallates that were not antioxidants, at the tested concentrations, in which they demonstrated anti-herpetic activity. The hydroxyl groups can induce DNA damage due interactions with some metal ions, which are naturally present in the culture medium supplemented with fetal bovine serum, probably explaining the genotoxicity detected. However, the obtained results showed considerable antioxidant activity at smaller concentrations, when compared to quercetin which is considered as a reference drug due to its already described antioxidant potential: DPPH radical scavenging activity with IC50 values varying from 17 to 31 micromol/L; and microsomal lipid peroxidation-inhibiting activity with IC50 values varying from 21 to 59 micromol/L. It was observed that the presence of hydroxyl groups in these molecules is important for their pharmacological profile, but the length of the lateral carbonic chain does not have considerable influence.

Characterization of rotavirus P genotypes circulating among paediatric inpatients in northern Brazil

De novembro de 1992 a agosto de 1993, 28 amostras fecais positivas para rotavirus, obtidas de pacientes pediatricos hospitalizados em Belem, Brasil, com idades inferiores a 4 anos, foram testadas por RT-PCR visando a determinacao dos genotipos P. Com excecao de 7 criancas nao diarreicas, todos os pacientes apresentavam diarreia a admissao ou a desenvolviam enquanto internados no hospital. Cepas de rotavirus com especifidades antigenicas P correspondentes aos genotipos P1B[4] e P1A[8]...