Quantification and molecular characterization of intact rotavirus species A (RVA) in municipal solid waste leachate.

AIMS: Leachate comprises a solid waste decomposition product found fresh in collection trucks or as an effluent in landfills. This study aimed to assess the occurrence, concentrations, and genetic diversity of intact rotavirus species A (RVA) in solid waste leachate. METHODS AND RESULTS: Leachate samples were concentrated by ultracentrifugation, treated with propidium monoazide (PMA), and exposed to LED photolysis. Treated and untread samples were extracted using the QIAamp Fast DNA Stool mini kit, and nucleic acids were screened for RVA employing a Taqman® Real-time PCR. The PMA RT-qPCR method detected RVA in eight out of nine truck samples and in 15.40% (2/13) of the landfill leachate samples. The RVA concentrations in the PMA-treated samples ranged from 4.57 × 103 to 2.15 × 107 genomic copies (GC) 100 mL-1 in truck leachate and from 7.83 × 103 to 1.42 × 104 GC 100 mL-1 in landfill samples. Six truck leachate samples were characterized as RVA VP6 genogroup I2 by partial nucleotide sequencing. CONCLUSIONS: The high intact RVA detection rates and concentrations in truck leachate samples indicate potential infectivity and comprise a warning for solid waste collectors concerning hand-to-mouth contact and the splash route.

Use of chicken immunoglobulin Y in general virology.

Immunoglobulin Y (IgY), an antibody present in birds, reptiles, and amphibians, is actively transported from the serum to egg yolks, where it is stored in large quantities. The use of chicken polyclonal IgY instead of mammalian IgG antibodies for biomedical applications has ethical and economic advantages, such as the lack of a need for animal bleeding because the antibodies are extracted from eggs after hen immunization and the low cost of the production and purification methods. This article reviews the latest IgY applications in diagnostic virology and the therapeutic use of IgY in viral gastroenteritis.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections.

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

SARS-CoV-2 surveillance-based on municipal solid waste leachate in Brazil.

Municipal solid waste leachate-based epidemiology is an alternative viral tracking tool that applies fresh truck leachate as an early warning of public health emergencies. This study aimed to investigate the potential of SARS-CoV-2 surveillance based on solid waste fresh truck leachate. Twenty truck leachate samples were ultracentrifugated, nucleic acid extracted, and real-time RT-qPCR SARS-CoV-2 N1/N2 applied. Viral isolation, variant of concern (N1/N2) inference, and whole genome sequencing were also performed. SARS-CoV-2 was detected on 40% (8/20) of samples, with a concentration from 2.89 to 6.96 RNA Log10 100 mL-1. The attempt to isolate SARS-CoV-2 and recover the whole genome was not successful; however, positive samples were characterized as possible pre-variant of concern (pre-VOC), VOC Alpha (B.1.1.7) and variant of interest Zeta (P.2). This approach revealed an alternative tool to infer SARS-CoV-2 in the environment and may help the management of local surveillance, health, and social policies.

Quantification of infectious Human mastadenovirus in environmental matrices using PMAxx-qPCR.

Molecular methodologies providing data on viral concentration and infectivity have been successfully used in environmental virology, supporting quantitative risk assessment studies. The present study aimed to assess human mastadenovirus (HAdV) intact particles using a derivative of propidium monoazide associated with qPCR (PMAxx-qPCR) in aquatic matrices. Initially, different concentrations of PMAxx were evaluated to establish an optimal protocol for treating different naturally contaminated matrices, using 10 min incubation in the dark at 200 rpm at room temperature and 15 min of photoactivation in the PMA-Lite™ LED photolysis device. There was no significant reduction in the quantification of infectious HAdV with increasing concentration of PMAxx used (20 µM, 50 µM, and 100 µM), except for sewage samples. In this matrix, a reduction of 5.01 log of genomic copies (GC)/L was observed from the concentration of 50 µM and revealed 100% HAdV particles with damaged capsids. On the other hand, the mean reduction of 0.51 log in stool samples using the same concentration mentioned above demonstrated 83% of damaged particles eliminated in the stool. Following, 50 µM PMAxx-qPCR protocol revealed a log reduction of 0.91, 0.67, and 1.05 in other samples of raw sewage, brackish, and seawater where HAdV concentration reached 1.47 × 104, 6.81 × 102, and 2.33 × 102 GC/L, respectively. Fifty micrometers of PMAxx protocol helped screen intact viruses from different matrices, including sea and brackish water.

Human adenovirus in municipal solid waste leachate and quantitative risk assessment of gastrointestinal illness to waste collectors.

Leachate is a variable effluent from waste management systems generated during waste collection and on landfills. Twenty-two leachate samples from waste collection trucks and a landfill were collected from March to December 2019 in the municipality of Rio de Janeiro (Brazil) and were analyzed for Human Adenovirus (HAdV), bacterial indicators and physico-chemical parameters. For viral analysis, samples were concentrated by ultracentrifugation and processed for molecular analysis using QIAamp Fast DNA Stool mini kit® for DNA extraction followed by nested-PCR and qPCR/PMA-qPCR TaqMan® system. HAdV was detected by nested-PCR in 100% (9/9) and 83.33% (12/13) of the truck and landfill leachate samples, respectively. Viral concentrations ranged from 8.31 × 101 to 6.68 × 107 genomic copies per 100 ml by qPCR and PMA-qPCR. HAdV species A, B, C, and F were characterized using nucleotide sequencing. HAdV were isolated in A549 culture cells in 100% (9/9) and 46.2% (6/13) from truck and landfill leachate samples, respectively. Regardless of the detection methods, HAdV concentration was predicted by the quantity of total suspended solids. A quantitative microbial risk assessment was performed to measure the probability of gastrointestinal (GI) illness attributable to inadvertent oral ingestion of truck leachate, revealing the higher probability of disease for the direct splashing into the oral cavity (58%) than for the gloved hand-to-mouth (33%). In a scenario where waste collectors do not wear gloves as protective personal equipment, the risk increases to 67%. This is the first study revealing infectious HAdV in solid waste leachate and indicates a potential health risk for waste collectors.

Production and Evaluation of Chicken Egg Yolk Immunoglobulin (IgY) against Human and Simian Rotaviruses.

Producing specific antibodies in chickens is an attractive approach for diagnosis or therapeutic applications. Besides the high immunoglobulin Y (IgY) yield transferred to the egg yolk and its suitability for large-scale production, such an approach is more bioethical for animal maintenance. The IgY technology offers new possibilities for application in human and veterinary diagnostics and therapeutics, including strategies for treating severe intestinal diseases in children, particularly in emerging countries. Herein, we describe the production and purification of polyclonal antibodies against rotavirus group A (RVA) in immunised hens aiming at its application in prophylaxis and treatment of rotavirus-induced diarrhoea. For this purpose, we inoculated Rhodia laying chickens (Gallus gallus domesticus) with two or three doses of RVA combined with adjuvants or only adjuvants (control group). As the egg-laying period began, the yolk protein purification processes yielded a high concentration of specific IgY, the highest titre resulting from the group of hens that received three doses of the immunogen. The purified IgY blocked the functional activity of RVA in MA-104 cells, thus confirming the neutralisation ability. Therefore, anti-RVA IgY could be a promising candidate for pre- and post-exposure prevention or treatment of rotavirus-induced diarrhoea.

Assessment of Viral Contamination of Five Brazilian Artisanal Cheese Produced from Raw Milk: a Randomized Survey.

Enteric viruses have been described as important contaminants in fresh and ready-to-eat foods such as sandwiches, deli meat and dairy products. This is a cross-sectional randomized survey to estimate the prevalence of norovirus and human adenovirus (HAdV) from 100 Brazilian artisanal raw milk cheese samples (Minas and Coalho) obtained from different agroindustries in four producing regions in the states of Minas Gerais and one in Piauí, respectively. From October 2017 to April 2018, norovirus genogroups I and II and HAdV were investigated in these cheese samples by RT-qPCR and qPCR, respectively. Viruses were detected in 43 samples, being 26 norovirus GI strains, 14 HAdV, and 3 both viruses. Norovirus GII strains were not detected. Viral concentrations ranged from 6.17 × 104 to 1.44 × 107 genome copies/L-1 and murine norovirus 1 used as internal process control showed 100% success rate of recovery with efficiency of 10%. There was a trend towards a higher positivity rate for both viruses in the rainy season, and HAdV were more commonly found among samples with higher fecal coliform counts. This study is a first step in assessing the risk that this contamination may pose to the consumer of raw products as well as emphasizing the need for good manufacturing practices, quality control systems in the dairy industry and markets. As a randomized survey, we established baseline figures for viruses' prevalence in five types of ready-to-eat raw milk artisanal Brazilian cheese, to allow any monitoring trends, setting control targets and future local risk analyses studies.

Evaluation of Viral Recovery Methodologies from Solid Waste Landfill Leachate.

Leachate from solid waste landfill is a dark liquid of variable composition and possible source of contamination of groundwater and surface waters. This study aims to assess skimmed milk flocculation and ultracentrifugation as viral concentration methods associated to different nucleic acid extraction protocols in order to establish a methodology for virus recovery from sanitary landfill leachate. Spiking experiments using human adenovirus (HAdV) and bacteriophage PP7 revealed the association of QIAamp Fast DNA Stool mini kit® nucleic acid extraction and ultracentrifugation as an effective method for recovering HAdV (346.18%) and PP7 (523.97%) when compared to organic flocculation method (162.64% for HAdV and 0.61% for PP7) that presented PCR inhibition in all undiluted samples. Ultracentrifugation applied in three landfill samples confirm efficiency of the methodology detecting HAdV in all samples with a mean of 3.44E + 06 ± 1.56E + 06 genomic copies/mL. Nucleotide sequencing characterized HAdV as belonging to group B and F. JC polyomavirus (JCPyV) was also investigated in those samples; however, detection was not observed. Methodologies for detection of viruses in leachate can be useful to generate data for future health risk analysis of workers who have contact with solid urban waste, as well as populations exposed to different environmental matrices contaminated by these effluents.

Cynomolgus Monkeys (Macaca fascicularis) as an Experimental Infection Model for Human Group A Rotavirus.

Group A rotaviruses (RVA) are one of the most common causes of severe acute gastroenteritis in infants worldwide. Rotaviruses spread from person to person, mainly by faecal⁻oral transmission. Almost all unvaccinated children may become infected with RVA in the first two years of life. The establishment of an experimental monkey model with RVA is important to evaluate new therapeutic approaches. In this study, we demonstrated viral shedding and viraemia in juvenile⁻adult Macaca fascicularis orally inoculated with Wa RVA prototype. Nine monkeys were inoculated orally: seven animals with human RVA and two control animals with saline solution. During the study, the monkeys were clinically monitored, and faeces and blood samples were tested for RVA infection. In general, the inoculated animals developed an oligosymptomatic infection pattern. The main clinical symptoms observed were diarrhoea in two monkeys for three days, associated with a reduction in plasmatic potassium content. Viral RNA was detected in seven faecal and five sera samples from inoculated animals, suggesting virus replication. Cynomolgus monkeys are susceptible hosts for human Wa RVA infection. When inoculated orally, they presented self-limited diarrhoea associated with presence of RVA infectious particles in faeces. Thus, cynomolgus monkeys may be useful as animal models to evaluate the efficacy of new antiviral approaches.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

Detecção de rotavírus do grupo A em amostras fecais: adaptação do teste de aglutinação em látex para o emprego de anticorpos IgY anti-Rotavírus A / Detection of group a rotavirus in fecal samples: adaptation of latex agglutination test for anti-rotavirus a igy antibody employment

A infecção pelo rotavírus A (RVA) é responsável por cerca de 453.000 mortes anualmente e aproximadamente 40 por cento das hospitalizações por diarreia em crianças menores de cinco anos em todo o mundo, sendo o principal causador de gastroenterite aguda nesse grupo populacional. O desenvolvimento de um método de diagnóstico rápido, barato, sensível e específico para a detecção de RVA é importante do ponto de vista epidemiológico porque permite identificar surtos no local de ocorrência. O uso da imunoglobulina Y (IgY), anticorpo purificado a partir da gema de ovo, vem crescendo nos últimos anos, devido às características vantajosas quando comparada com a imunoglobulina G (IgG), como a obtenção de anticorpos de forma não invasiva e a produção de anticorpos em grandes concentrações. Este trabalho objetivou a adaptação de um teste de diagnóstico através da substituição do anticorpo de captura IgG pela IgY específica para o antígeno de RVA (LATEXY-ROTA). Para isto 09 frangas poedeiras foram imunizadas com o RVA, os ovos foram coletados e a IgY purificada a partir da gema do ovo por polietileno glicol 6.000, seguida da purificação adicional por cromatografia de troca iônicaA IgY anti-RVA purificada foi ligada covalentemente à partículas de poliestireno e usada como insumo na adaptação de um teste de aglutinação em látex, sendo testada em um painel de amostras fecais sabidamente positivas e negativas previamente selecionadas pelo Centro Regional de Referência para Rotaviroses do Laboratório de Virologia Comparada e Ambiental (LVCA/IOC-FIOCRUZ)...

Foi obtida uma sensibilidade de 75 por cento e uma especificidade de 87,5 por cento quando o LATEXY-ROTA foi comparado com um teste imunoenzimático comercial disponível (padrão ouro). Quando comparado com dois testes comerciais de aglutinação em látex testados no painel de amostras utilizando a IgG, o LATEXY-ROTA obteve uma sensibilidade de 100 por cento e especificidade de 88,24 por cento. Baseado nos dados obtidos, sugerimos a viabilidade da substituição da IgG por IgY no ensaio de aglutinação pelo látex...

The infection by rotavirus (RV) is responsible for approximately 453,000 deaths annually and approximately 40 percent of hospitalizations by diarrhea in children under five years worldwide, being the major cause of acute gastroenteritis in this populationgroup. The development of a rapid method, inexpensive, sensitive and specific for rotavirus diagnosis is important from the disease because it allows the identification of outbreaks in the site of occurrence. The use of Immunoglobulin Y (IgY) antibodypurified from egg yolk, has been grown in recent years, due to the advantageous features compared to immunoglobulin G (IgG), as a noninvasive recovery of antibodies and production in high concentrations. The aim of this method was to adapt a diagnostic test by replacing the IgG capture antibody by specific IgY for RVAantigen (LATEXY-ROTA). For that, 09 laying hens were immunized with RVA, the eggs were collected and IgY purified from egg yolk by polyethylene glycol 6,000, followed by purification by ion exchange chromatography. The purified anti-IgY RVA was covalently bound to polystyrene particles, being tested in a panel of positive and negative fecal samples previously determined by the Rotavirus Regional ReferenceCenter of Comparative and Environmental Virology Laboratory (LVCA/IOCFIOCRUZ). A sensitivity of 75% and specificity of 85,7% was observed when the adapted test was compared to a commercial available enzyme immunoassay (goldenstandard). When compared to two commercial latex agglutination tests using the IgG tested on the panel of samples, the LATEXY-ROTA had a sensitivity of 100 percent and specificity of 88.2 percent. Based on the obtained data, we suggest the feasibility of replacing the IgG by IgY in the latex agglutination assay...