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.

Molecular characterization of feline calicivirus variants from multicat household and public animal shelter in Rio de Janeiro, Brazil

ABSTRACT The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.

Molecular characterization of feline calicivirus variants from multicat household and public animal shelter in Rio de Janeiro, Brazil.

The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.

Surveillance of Noroviruses in Rio De Janeiro, Brazil: Occurrence of New GIV Genotype in Clinical and Wastewater Samples.

Genogroup (G) IV norovirus (NoV) has been described in the literature as infectious agents in humans, although there are few reports regarding the frequency and spread of this virus, resulting in insufficient epidemiological data. The aim of this study was to investigate the occurrence of GIV norovirus in the State of Rio de Janeiro, Brazil in order to evaluate frequency, concentration, and genetic diversity using clinical and environmental approaches. For this purpose, 316 stool samples were collected from acute gastroenteritis cases reported over a period of three years. Wastewater samples were also obtained from the main wastewater treatment plant (WWTP) located in Rio de Janeiro throughout one year, totalizing 156 samples. All samples were submitted to quantitative analysis by TaqMan™ real-time PCR for GIV norovirus. Three out of 316 clinical samples were positive (0.9%) for GIV, with viral load ranging from 104 to 106 genome copies (CG) per gram. Regarding wastewater samples, GIV were detected in 52% of raw sewage, with viral load ranging from 104 to 106 CG per liter. Phylogenetic analysis revealed the circulation of a new GIV genotype in both clinical and environmental samples. To our knowledge, this is the first description of GIV norovirus in clinical samples in Brazil. These results demonstrate the importance of performing laboratory surveillance of clinical and environmental samples, assisting the comprehension of the epidemiology pattern of viruses with neglected diagnosis and indefinite impact in the population.

Detection and Molecular Characterization of Gemycircularvirus from Environmental Samples in Brazil.

Gemycircularvirus (GemyCV) is a group of viruses which has been recently proposed as a new viral genus detected in fecal and environmental samples around the world. GemyCVs have been detected in human blood, brain tissue, cerebrospinal fluid, and stool sample. In the present study, we demonstrate for the first time, through molecular detection and characterization, the presence of GemyCVs in environmental samples from Brazil. Our results show a percentage of positivity ranging from 69 (25/36) to 97 % (35/36) in river water samples collected in Manaus, Amazon region, and wastewater from a wastewater treatment plant located in Rio de Janeiro, respectively, revealing GemyCVs as an important environmental contaminant.

Noroviruses associated with outbreaks of acute gastroenteritis in the State of Rio Grande do Sul, Brazil, 2004-2011.

BACKGROUND: Acute gastroenteritis norovirus (NoV) in a country of continental dimensions like Brazil has resulted in under-reporting of the number of outbreaks, as well as the genotypes associated. OBJECTIVES: To demonstrate the role of NoV in outbreaks occurring in the State of Rio Grande do Sul, Southern Brazil, we determined its prevalence, as well as the genotypes associated, and evaluated clinical and epidemiological aspects. STUDY DESIGN: NoV investigation was carried out in rotavirus group A negative stool samples from 2265 patients from 741 outbreaks that occurred in the State of Rio Grande do Sul, Brazil, during a period of eight years (2004-2011). NoV detection and nucleotide sequencing for genotype characterization was carried by using sets of primers targeting a conservative Rd-Rp polymerase genome region and the viral capsid gene, respectively. RESULTS: NoVs were detected in 817 stool samples (36.1%) and associated with 327 outbreaks (44.1%). NoV GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, GII.15, GII.17, GII.21; and GI.1 and GI.3 were characterized. GII.4 was the most frequently detected (72.3%), with five variants identified (Asia_2003, Hunter_2004, Yerseke_2006a, Den_Haag_2006b, New Orleans_2009). This study describes the first detection of GI.1 and GII.13 and GII.15 in Brazil and demonstrates NoV winter-spring seasonality in this region of the country. CONCLUSIONS: NoVs were responsible for almost 50% of outbreaks, with about 70% of them resulting from genotype GII.4 and its variants. The seasonality observed could help health authorities to establish a system of active surveillance in order to reduce NoV impact especially in congregate settings.

Temporal dynamics of norovirus GII.4 variants in Brazil between 2004 and 2012.

Noroviruses (NoVs) are the major cause of acute gastroenteritis outbreaks, and, despite a wide genetic diversity, genotype II.4 is the most prevalent strain worldwide. Mutations and homologous recombination have been proposed as mechanisms driving the epochal evolution of the GII.4, with the emergence of new variants in 1-3-year intervals causing global epidemics. There are no data reporting the dynamics of GII.4 variants along a specific period in Brazil. Therefore, to improve the understanding of the comportment of these variants in the country, the aim of this study was to evaluate the circulation of NoV GII.4 variants during a 9-year period in 3 out of 5 Brazilian regions. A total of 147 samples were sequenced, and a phylogenetic analysis of subdomain P2 demonstrated the circulation of six GII.4 variants, Asia_2003, Hunter_2004, Den Haag_2006b, Yerseke_2006a, New Orleans_2009, and Sydney_2012, during this period. The most prevalent variant was Den Haag_2006b, circulating in different Brazilian regions from 2006 to 2011. A Bayesian coalescent analysis was used to calculate the mean evolutionary rate of subdomain P2 as 7.3 × 10(-3) (5.85 × 10(-3) -8.82 × 10(-3)) subst./site/year. These analyses also demonstrated that clade Den Haag_2006b experienced a rapid expansion in 2005 and another in 2008 after a period of decay. The evaluation of the temporal dynamics of NoV GII.4 in Brazil revealed a similar pattern, with few exceptions, to the worldwide observation. These data highlight the importance of surveillance for monitoring the emergence of new strains of NoV GII.4 and its impact on cases of acute gastroenteritis.

Norovirus: genotipagem e caracterização molecular de variantes GII.4 no Brasil, 2005-2010 / Norovirus: genotyping and molecular characterization of GII.4 variants in Brazil, 2005-2010

O gênero Norovirus pertence à família Caliciviridae e está dividido em cinco genogrupos (G), os quais GI, GII e GIV são descritos infectando humanos. O GII é o mais prevalente, contendo 21 genótipos, sendo o GII.4 o responsável pela grande maioria dos surtos mundialmente. Os norovirus (NoV) são vírus não envelopados, de simetria icosaédrica e seu genoma é composto por ssRNA de aproximadamente 7,5 kb, dividido em 3 regiões abertas de leitura (ORF). A ORF1 codifica para proteínas não estruturais, incluindo a RNA polimerase RNA dependente, a ORF2 codifica para proteína estrutural VP1, que compõe o capsídeo viral e a ORF3 para a proteína estrutural VP2. A VP1, utilizada para a classificação em genótipos, é dividida em três domínios denominados S, P1 e P2, estando este último localizado na região mais exposta do capsídeo viral e considerado hipervariável, acumulando maior número de mutações não-sinônimas. O objetivo deste estudo foi caracterizar os diferentes genótipos de NoV e variantes de NoV GII.4 circulantes em diferentes estados das cinco regiões brasileiras no período de 2005-2010. Com este propósito foram selecionadas 337 amostras de fezes provenientes de casos de gasrenterite aguda recebidos no Laboratório de Virologia Comparada e Ambiental, por demanda espontânea, e previamente diagnosticados como NoV pela reação em cadeia da polimerase precedida de transcrição reversa (RT-PCR). Para a caracterização molecular dos NoV foi realizado o sequenciamento parcial de duas regiões diferentes do gene que codifica para a VP1: a região D, para genotipagem, e a região do domínio P2, para caracterização das variantes de GII.4. Com o sequenciamento da região D de 265 amostras, 80,4(por cento) (213/265) foram caracterizadas como GII e 1,5(por cento) (4/265) como GI. Não foi possível a caracterização de 18,1(por cento) (48/265) das amostras. Os genótipos GI.2, GI.5, GI.8, GII.4, GII.6, GII.7, GII.12, GII.16, GII.17 e GII.20 foram detectados, sendo o mais prevalente o GII.4 (79(por cento)), encontrado em 13 dos 15 estados avaliados, seguido do GII.6 (13(por cento)). Esta foi a primeira descrição da circulação dos genótipos GI.5, GII.12, GII.16, GII.17 e GII.20 no Brasil. Com o sequenciamento da região P2 de 114 amostras GII.4 foram caracterizadas cinco variantes denominadas 2003, 2004, 2006a, 2006b e 2010, sendo a variante 2006b a mais prevalente (54,4(por cento)), seguida da 2010 (21,9(por cento)) e 2006a (17,5(por cento)). A caracterização de um subclado formado por 22 amostras dentro da variante 2006b sugere a emergência de uma nova variante a partir desta. A grande diversidade genética dos NoV circulando no Brasil, assim como a possibilidade da emergência de novas variantes demonstra a necessidade do estabelecimento de uma rede nacional de vigilância que disponibilizaria informações referentes à dispersão geográfica e temporal destes vírus como ocorre outros países.

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.

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%) 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.

Bayesian coalescent inference reveals high evolutionary rates and expansion of Norovirus populations.

Noroviruses (NoV) are a leading cause of outbreaks of nonbacterial acute gastroenteritis in humans worldwide and have become an important cause of hospitalization of children in South America. NoV belong to the family Caliciviridae and are non-enveloped single stranded, positive sense, RNA viruses. NoV of genotype GII/4 have emerged worldwide, causing four epidemic seasons of viral gastroenteritis during which four novel variants emerged. Despite the importance of NoV outbreaks, little is known about the evolutionary rates, viral spread and population dynamics of NoV populations. In order to gain insight into these matters, a Bayesian Markov chain Monte Carlo (MCMC) approach was used to analyze region D or full-length VP1 gene sequences of GII/4 NoV populations isolated in Brazil or Japan, respectively. The results of these studies revealed that the expansion population growth model was the best to fit the data in both datasets. The dates of the most common recent ancestors revealed that these viruses can quickly emerge in a geographical location. A mean evolutionary rate of 1.21 x 10(-2) nucleotide substitution/site/year (s/s/y) was obtained for the VP1 gene using full-length sequences. This rate is higher than the rates reported for other rapidly evolving RNA. Roughly similar rates (1.44 x 10(-2)s/s/y) were found using region D sequences, revealing the suitability of this region for evolutionary studies, in agreement with previous reports. High evolutionary rates and fast population growth may have contributed to the vigorous initial transmission dynamics of the GII/4 NoV populations studied.