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.

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.

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]...