Microbiome and virome on indoor surfaces of an Antarctic research ship

BACKGROUND Few studies have focused on microbial diversity in indoor environments of ships, as well as the role of the microbiome and its ecological interconnections. In this study, we investigated the microbiome and virome present on the internal surfaces of a polar ship in different stages (beginning, during, and at the end) of the Brazilian Antarctic expedition in order to evaluate abundance of microorganisms in different periods. OBJECTIVES AND METHODS We used shotgun metagenomic analysis on pooled samples from sampling surfaces in the ship's interior to track the microbial diversity. FINDINGS Considering the total fraction of the microbiome, the relative abundance of bacteria, eukaryotes, viruses, and archaea was 83.7%, 16.2%, 0.04%, and 0.002%, respectively. Proteobacteria was the most abundant bacterial phyla, followed by Firmicutes, Actinobacteria, and Bacteroidetes. Concerning the virome, the greatest richness of viral species was identified during the middle of the trip, including ten viral families after de novo assembly: Autographiviridae, Chrysoviridae, Genomoviridae, Herelleviridae, Myoviridae, Partitiviridae, Podoviridae, Potyviridae, Siphoviridae, and Virgaviridae. MAIN CONCLUSIONS This study contributed to the knowledge of microbial diversity in naval transportation facilities, and variations in the abundance of microorganisms probably occurred due to factors such as the number of passengers and activities on the ship.

Detection of salivirus in raw sewage samples in Rio de Janeiro, Brazil

Gastroenteric viruses are important pathogens related to cases of acute gastroenteritis, affecting millions of people worldwide with a major impact on children under five in developing countries. The introduction of metagenomic approach techniques in the 2000s has allowed the description of new viruses, among them Salivirus, which has been associated worldwide with cases of diarrhea. This study aimed to detect salivirus in raw sewage samples from a wastewater treatment plant (WWTP) collected between June 2013 and May 2014 in Rio de Janeiro, Brazil. Fifty-two samples collected weekly were tested by using a real-time quantitative PCR (qPCR). Salivirus genome was detected in 71.1% (37/52) of the samples, with viral concentration ranging from 7.56 x 104 to 7.20 x 106 genomic copies per liter. Higher viral loads were detected in the summer and fall of 2014, although these data were not sufficient to infer seasonality for this virus. The high prevalence of salivirus in sewage samples highlights the importance of viral research in wastewater to generate data on salivirus circulation, increasing understanding regarding its dissemination in the population.

Evaluation of skimmed milk flocculation method for virus recovery from tomatoes

Abstract This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety.

Evaluation of skimmed milk flocculation method for virus recovery from tomatoes

Abstract This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety.

A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil

A gastroenteritis outbreak that occurred in 2013 in a low-income community in Rio de Janeiro was investigated for the presence of enteric viruses, including species A rotavirus (RVA), norovirus (NoV), astrovirus (HAstV), bocavirus (HBoV), aichivirus (AiV), and adenovirus (HAdV). Five of nine stool samples (83%) from patients were positive for HAdV, and no other enteric viruses were detected. Polymerase chain reaction products were sequenced and subjected to phylogenetic analysis, which revealed four strains and one strain of non-enteric HAdV-A12 and HAdV-F41, respectively. The HAdV-A12 nucleotide sequences shared 100% nucleotide similarity. Viral load was assessed using a TaqMan real-time PCR assay. Stool samples that were positive for HAdV-A12 had high viral loads (mean 1.9 X 107 DNA copies/g stool). All four patients with HAdV-A12 were < 25 months of age and had symptoms of fever and diarrhoea. Evaluation of enteric virus outbreaks allows the characterisation of novel or unique diarrhoea-associated viruses in regions where RVA vaccination is routinely performed.

Virologia ambiental e saneamento no Brasil: uma revisão narrativa / Environmental virology and sanitation in Brazil: a narrative review / Virología ambiental y saneamiento en Brasil: una revisión narrativa

Os serviços de saneamento básico têm papel fundamental no controle da transmissão de diversos agentes patogênicos de veiculação hídrica, especialmente vírus responsáveis por causar gastroenterites agudas e hepatites. Entre os agentes virais de maior impacto para a saúde pública, podem ser destacados os vírus das hepatites A, os rotavírus e norovírus, adenovírus e enterovírus, os quais são responsáveis pela contaminação de diversos ecossistemas aquáticos brasileiros. A alta circulação de vírus no ambiente vem sendo relacionada às condições sanitárias inadequadas das comunidades, incluindo a falta na cobertura de serviços ou ineficácia de tecnologias convencionais na eliminação ou redução da carga viral presente na água ou no esgoto. Este estudo aborda uma revisão das relações entre virologia, saúde e saneamento, enfatizando a epidemiologia das infecções virais de transmissão hídrica e o impacto na saúde pública.

Sanitation services play a critical role in controlling transmission of numerous waterborne pathogens, especially viruses that cause acute gastroenteritis and hepatitis. The viral agents with the greatest public health impact are hepatitis A virus, rotaviruses and noroviruses, adenoviruses, and enteroviruses, contaminating many Brazilian aquatic ecosystems. Heavy circulation of viruses in the environment has been related to inadequate local sanitary conditions, including incomplete coverage of services or inefficacy of conventional technologies in eliminating or reducing the viral load in water or sewage. This study reviews the relations between virology, health, and sanitation, emphasizing the epidemiology of waterborne viral infections and their public health impact.

El servicio de saneamiento posee un rol en el control de la transmisión de muchos patógenos transmitidos por el agua, especialmente aquellos virus responsables de causar gastroenteritis aguda y hepatitis. Entre los agentes virales de mayor impacto sobre la salud pública se pueden destacar los virus de la hepatitis A, rotavirus, norovirus, adenovirus y enterovirus, los cuales son responsables de la contaminación de diversos ecosistemas acuáticos brasileños. Una alta circulación del virus en el medio ambiente se relaciona con condiciones sanitarias inadecuadas de las comunidades, como la falta de cobertura de los servicios o la ineficacia de las tecnologías convencionales en eliminar la carga viral del agua. Esta revisión está enfocada en las relaciones entre la virología, la salud y el saneamiento, con énfasis en la epidemiología de las infecciones virales transmitidas por el agua y el impacto en la salud pública.

Detection of enteric viruses in activated sludge by feasible concentration methods

Human enteric viruses are responsible to cause several diseases, including gastroenteritis and hepatitis, and can be present in high amounts in sewage sludge. This study compared virus recovery efficiency of two feasible concentration methods used for detecting human adenovirus (HAdV), rotavirus species A (RV-A), norovirus genogroup II (NoV GII) and hepatitis A virus (HAV) in sewage sludge from an activated sludge process. Twelve sewage sludge samples were collected bi-monthly from January to July, 2011. Ultracentrifugation was compared with a simplified protocol based on beef extract elution for recovering enteric viruses. Viruses were quantified by quantitative real-time PCR assays and virus recovery efficiency and limits of detection were determined. Methods showed mean recovery rates lower than 7.5%, presenting critical limits of detection (higher than 102 103 genome copies -GC L-1 for all viruses analyzed). Nevertheless, HAdV were detected in 90% of the analyzed sewage sludge samples (range: 1.8 x 104 to 1.1 x 105 GC L-1), followed by RV-A and NoV (both in 50%) and HAV (8%). Results suggesting that activated sludge is contaminated with high viral loads and HAdV are widely disseminated in these samples. The low virus recovery rates achieved, especially for HAV, indicate that other feasible concentration methods could be developed to improve virus recovery efficiency in these environmental matrices.

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.

Genotyping of gastroenteric viruses in hospitalised children: first report of norovirus GII.21 in Brazil

This retrospective study (April-September 2003) was designed to investigate the roles of the main viruses responsible for cases of acute infantile gastroenteritis in hospitalised children up to two years of age. The viruses were identified in 64.7% (88/136) of the cases and the detection rates of rotavirus A (RVA), norovirus (NoV) and astrovirus were 41.9% (57/136), 30.3% (24/79) and 12.7% (7/55), respectively. RVA and NoV were detected in 20 of the 24 reported nosocomial infection cases. This study identified the first circulation of the genotype NoV GII.21 in Brazil and highlights the need to establish differential diagnoses through active laboratorial surveillance.

Detection of enteric viruses in recreational waters of an urban lagoon in the city of Rio de Janeiro, Brazil

The aim of this study was to determine the occurrences of the group A rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV) in the surface waters of an urban lagoon (Rodrigo de Freitas Lagoon) in the city of Rio de Janeiro, Brazil. During one year of surveillance, water samples were obtained from the lagoon and other interconnected ecosystems (river and beach). The samples were concentrated using an adsorption-elution method with a negatively charged membrane and tested by qualitative and quantitative polymerase chain reaction assays. RVA was the most prevalent virus detected (24.3%) with a viral load ranging from 3.0 x 10¹-5.6 x 10(4) genome copies/L, followed by NoV (18.8%) and HAdV (16.7%). Considering water samples suitable for bathing, according to Escherichia coli criterion (< 2,000 most probable number/100 mL), viruses were detected in 50% (57/114) of them. Physicochemical parameters were also measured and showed possible correlations between turbidity and RVA presence and between pH and NoV presence. These data demonstrate the importance of considering viral parameters to ensure water quality and the utilisation of these parameters as additional tools for the characterisation of environmental contamination.

Genetic diversity of noroviruses in Brazil

Norovirus (NoV) infections are a major cause of acute gastroenteritis outbreaks around the world. In Brazil, the surveillance system for acute diarrhoea does not include the diagnosis of NoV, precluding the ability to assess its impact on public health. The present study assessed the circulation of NoV genotypes in different Brazilian states by partial nucleotide sequencing analysis of the genomic region coding for the major capsid viral protein. NoV genogroup II genotype 4 (GII.4) was the prevalent (78 percent) followed by GII.6, GII.7, GII.12, GII.16 and GII.17, demonstrating the great diversity of NoV genotypes circulating in Brazil. Thus, this paper highlights the importance of a virological surveillance system to detect and characterize emerging strains of NoV and their spreading potential.

Molecular detection of human astrovirus in an urban sewage treatment plant in Rio de Janeiro, Brazil

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7 percent of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2 percent and 4.3 percent was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

Hospitalization due to norovirus and genotypes of rotavirus in pediatric patients, state of Espírito Santo

Viruses are the leading cause for hospitalization due to gastroenteritis worldwide. Group A rotaviruses (RV) are the most prevalent and are assorted in glycoproteins (G) and protease sensitive (P) dual genotypes based on polymorphic genes that encode the external VP7 and VP4 capsid proteins, respectively. Noroviruses (NoV) have increasingly answered by sporadic gastroenteritis. This study aimed to determine the prevalence of NoV and RV in 68 hospitalized children, between July 2004 and November 2006, at a pediatric hospital in Vitória city, state of Espírito Santo, Southeastern Brazil. Nucleic acid was extracted from fecal suspension following the guanidine-silica procedure. Reverse transcriptase-polymerase chain reaction (RT-PCR) and polyacrylamide gel electrophoresis were employed for NoV and RV detection, respectively. RV genotyping was accomplished using RT-PCR followed by heminested multiplex PCR with specific primers for the most prevalent types of G and P. Fecal samples were positive for NoV and RV in 39.7 percent (27/68) and 20.5 percent (14/68), respectively and together were responsible for 60 percent (41/68) of the cases. RV genotypes were: 50 percent G9P[8], 28.7 percent G2P[4], 7.1 percent G1P[8], G2P[8] and G?P[8]. Vomit was a prominent manifestation observed in 92 percent and 85 percent of the NoV and RV cases, respectively. The median hospitalization was 5 and 5.5 days for the patients infected with NoV and RV, respectively. The data showed that NoV prevailed over RV and it also corroborated the emergence of RV G9 genotype followed by G2P[4], reinforcing the need for RV genotype surveillance.

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7 percent) and transmission (83.3 percent) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60 percent, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1 percent of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.

Dengue viruses activity in Piauí, Brazil

The present paper reports a laboratory investigation performed between the years of 2000 and 2002 to stydy a virological surveillance program introduced in the state of Piauí to support an epidemiological survey of the disease. Dengue virus type 3 (DENV-3) existence in the state was detected in May 2002 when a high number of dengue cases due to DENV-1 and DENV-2 were reported. An assessment on the population knowledge about the disease and its transmission showed that almost 50 percent of the population were still unaware of the epidemiological features of dengue.

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain

Molecular epidemiology of dengue viruses in Brazil

Os vírus dengue (DEN) apresentam propriedades antigênicas distintas que caracterizam quatro sorotipos denominados DEN-1, 2, 3 e 4. Desde a década de 70, evidências laboratoriais têm demonstrado a ocorrência de variaçäo intratípica entre os vírus DEN; entretanto, somente com o avanço das metodologias moleculares foi possível estabelecer variantes genéticas para cada sorotipo. A identificaçäo genotípica tem sido uma importante abordagem para determinar a origem e a dispersäo de epidemias e para tentar estabelecer correlaçäo de virulência entre as variantes dos vírus DEN. Apresenta resultados obtidos através de estudos epidemiologia molecular realizados com amostras de vírus DEN-1 e DEN-2, que causaram epidemias no Brasil, na última década.

Dengue in the State of Rio de Janeiro, Brazil, 1986-1998

This paper presents epidemiological, laboratory, and clinical data on 12 years of dengue virus activity in the State of Rio de Janeiro from the time the disease was first confirmed virologically in April 1986 through April 1998. DEN-1 and DEN-2 viruses are the serotypes circulating in the state and were responsible for the epidemics reported during the last 12 years. The results published here show both the impact of dengue virus infections on the population and laboratory advances that have improved dengue diagnosis.