Human norovirus in Brazil: an update of reports in different settings.

Norovirus is an important etiologic agent of acute gastroenteritis and has become even more relevant in Brazil after the implementation of the monovalent rotavirus vaccine in 2006 through the public health system, now representing a significant portion of the etiology of acute diarrheal diseases. Although diagnosing acute gastroenteritis caused by norovirus is a relatively simple process, and the infection tends to be self-limited, the virus can be considerably harmful to vulnerable populations, such as children, the elderly, and immunocompromised individuals. The spread of norovirus is also particularly favorable among such groups due to its mode of transmission, favored by cluttered environments such as in hospitals and densely populated regions. Additionally, norovirus' ability to spread through water and food creates the need for measures to ensure adequate sanitation and the development of effective measures to prevent outbreaks and severe manifestations of the disease. This review aims to address the main reports of human norovirus detected in Brazil over the years, focusing on clinical-hospital, food-related, and urban conglomerate contexts, including the circulating strains.

Molecular Characterization of Enteric Viruses Causing Acute Gastroenteritis among Children under 5 Years Old in Distrito Central, Honduras.

INTRODUCTION: Diarrheal diseases constitute a significant public health problem in terms of mortality and morbidity. In Honduras and around the world, RVs have consistently emerged as the single most important etiologic agent in acute childhood diarrhea. However, other viruses, such as NoVs and HAstVs, have also been shown to be responsible for viral gastroenteritis. Unfortunately, the country has limited information concerning the etiologic role of these viral agents in acute gastroenteritis. This study investigated the frequency, genotypes, and epidemiological characteristics of RV-A, NoVs, and HAstVs among children under 5 years old in Distrito Central, Honduras. METHODS: Stool samples and their corresponding epidemiological data were collected from children with acute gastroenteritis in three healthcare centers in Distrito Central. All samples were screened by immunoassays for RV-A and HAstVs. RV-A-positive samples were molecularly characterized by RT-PCR and genotyping assays. RT-PCR was also applied to confirm HAstVs positivity and to detect NoVs, followed by nucleotide sequencing to assign their genotypes. RESULTS: Our results show that at least one viral agent was detected in 31% of the children. The frequency of RV-A, NoVs, and HAstVs was 14%, 13%, and 5%, respectively. The most frequent RV-A genotype was G2P[4], occurring in 93% of cases. 92.3% of NoVs-positive samples belonged to genogroup II, with GII.4 and GII.16 being the most common. HAstVs were clustered into three genotypes: HAstV-1, HAstV-2, and HAstV-8. Only one sample showed coinfection with NoVs and HAstVs. CONCLUSION: This comprehensive molecular and epidemiological characterization of enteric viruses demonstrates the vast diversity of these agents and describes for the first time NoVs and HAstVs as causative agents of acute childhood gastroenteritis in Distrito Central, Honduras. This suggests that further in-depth studies of the pediatric population are necessary to develop and implement effective preventive and control measures in the country.

Analysis of viral diversity in dogs with acute gastroenteritis from Brazilian Amazon.

Viral gastroenteritis is commonly reported in dogs and involves a great diversity of enteric viruses. In this research, viral diversity was investigated in dogs with diarrhea in Northern Brazil using shotgun metagenomics. Furthermore, the presence of norovirus (NoV) was investigated in 282 stool/rectal swabs of young/adult dogs with or without diarrhea from two public kennels, based on one-step reverse transcription polymerase chain reaction (RT-PCR) for genogroup VI and VII (GVI and GVII) and real-time RT-PCR for GI, GII, and GIV. Thirty-one viral families were identified, including bacteriophages. Phylogenetic analyses showed twelve complete or nearly complete genomes belonging to the species of Protoparvovirus carnivoran1, Mamastrovirus 5, Aichivirus A2, Alphacoronavirus 1, and Chipapillomavirus 1. This is the first description of the intestinal virome of dogs in Northern Brazil and the first detection of canine norovirus GVII in the country. These results are important for helping to understand the viral groups that circulate in the canine population.

Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis.

Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10-3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Retrospective molecular analysis of norovirus recombinant strains in the amazon region, Brazil.

BACKGROUND: Noroviruses are enteric viruses that cause acute gastroenteritis worldwide. Over two decades, GII.4 genotype was responsible for most cases. However, recombinant strains have emerged and changed the epidemiological context of these infections. OBJECTIVES: The aim of this study was to identify the recombinant genetic strains of norovirus causing gastroenteritis in Brazilian children from the Amazon region. METHODS: We analyzed 534 cases of gastroenteritis between 2015 and 2016. Genotypic characterization was performed by partial sequencing of ORF1 and ORF2. Evolutionary history was inferred by Bayesian inference using MrBayes. Recombinant strains were confirmed by Simplot and RDP4 analysis. FINDINGS: We performed viral detection tests and identified a norovirus frequency of 31.8% (175/534). Based on viral RdRp and VP1 genes, nine genotypes were identified: GIIP31/GII.4, GII·P16/GII.4, GII·P7/GII.6, GII·P21/GII.13, GII·P33/GII.1, GII·P17/GII.17, GI·P7/GI.7, GII·P4/NT, and GII.7/NT. The phylogenetic tree showed evolutionary relationships among the genotypes, including the recombinant strains. This is the first description of GII·P33/GII.1 and GII·P21/GII.13 genotypes in Brazil. CONCLUSION: Norovirus evolution has been characterized by the continuous replacement of variants that have new antigenic properties. In recent years, recombinant strains have displaced GII.4, improving the viral fitness and influencing the viral transmissibility and pathogenicity.

Virological and Epidemiological Features of Norovirus Infections in Brazil, 2017-2018.

Noroviruses are considered an important cause of acute gastroenteritis (AGE) across all age groups. Here, we investigated the incidence of norovirus, genotypes circulation, and norovirus shedding in AGE stool samples from outpatients in Brazil. During a two-year period, 1546 AGE stool samples from ten Brazilian states were analyzed by RT-qPCR to detect and quantify GI and GII noroviruses. Positive samples were genotyped by dual sequencing using the ORF1/2 junction region. Overall, we detected norovirus in 32.1% of samples, with a massive predominance of GII viruses (89.1%). We also observed a significant difference between the median viral load of norovirus GI (3.4×105 GC/g of stool) and GII (1.9×107 GC/g). The most affected age group was children aged between 6 and 24 m old, and norovirus infection was detected throughout the year without marked seasonality. Phylogenetic analysis of partial RdRp and VP1 regions identified six and 11 genotype combinations of GI and GII, respectively. GII.4 Sydney[P16] was by far the predominant genotype (47.6%), followed by GII.2[P16], GII.4 Sydney[P31], and GII.6[P7]. We detected, for the first time in Brazil, the intergenogroup recombinant genotype GIX.1[GII.P15]. Our study contributes to the knowledge of norovirus genotypes circulation at the national level, reinforcing the importance of molecular surveillance programs for future vaccine designs.

EHEC O111:H8 strain and norovirus GII.4 Sydney [P16] causing an outbreak in a daycare center, Brazil, 2019.

BACKGROUND: This study describes the investigation of an outbreak of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) at a daycare center in southeastern Brazil, involving fourteen children, six staff members, six family members, and one nurse. All bacterial and viral pathogens detected were genetically characterized. RESULTS: Two isolates of a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O111:H8 were recovered, one implicated in a case of HUS and the other in a case of uncomplicated diarrhea. These isolates had a clonal relationship of 94% and carried the stx2a and eae virulence genes and the OI-122 pathogenicity island. The EHEC strain was determined to be a single-locus variant of sequence type (ST) 327. EHEC isolates were resistant to ofloxacin, doxycycline, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole and intermediately resistant to levofloxacin and ciprofloxacin. Rotavirus was not detected in any samples, and norovirus was detected in 46.7% (14/30) of the stool samples, three of which were from asymptomatic staff members. The noroviruses were classified as the recombinant GII.4 Sydney [P16] by gene sequencing. CONCLUSION: In this outbreak, it was possible to identify an uncommon stx2a + EHEC O111:H8 strain, and the most recent pandemic norovirus strain GII.4 Sydney [P16]. Our findings reinforce the need for surveillance and diagnosis of multiple enteric pathogens by public health authorities, especially during outbreaks.

Norovirus strains in patients with acute gastroenteritis in rural and low-income urban areas in northern Brazil.

From 2010-2016, a total of 251 stool samples were screened for norovirus using next-generation sequencing (NGS) followed by phylogenetic analysis to investigate the genotypic diversity of noroviruses in rural and low-income urban areas in northern Brazil. Norovirus infection was detected in 19.9% (50/251) of the samples. Eight different genotypes were identified: GII.4_Sydney[P31] (64%, 32/50), GII.6[P7] (14%, 7/50), GII.17[P17] (6%, 3/50), GII.1[P33] (6%, 3/50), GII.3[P16] (4%, 2/50), GII.2[P16] (2%, 1/50), GII.2[P2] (2%, 1/50), and GII.4_New Orleans[P4] (2%, 1/50). Distinct GII.6[P7] variants were recognized, indicating the presence of different co-circulating strains. Elucidating norovirus genetic diversity will improve our understanding of their potential health burden, in particular for the GII.4_Sydney[P31] variant.

Detection, risk factors and molecular diversity of norovirus GIII in cattle in Uruguay.

Uruguay is a leading exporter of bovine meat and dairy products, and cattle production is one of the principal economic backbones in this country. A main clinical problem faced by livestock farmers is neonatal calf diarrhea (NCD); however, causes of NCD have not been extensively studied in Uruguay. Bovine norovirus (BoNoV) has been proposed as one of the possible etiologies of NCD as experimentally infected calves developed diarrhea and enteropathy, although limited information is available from field surveys. The aims of this study were to determine the frequency of infection, to investigate possible risk factors, and to determine the molecular diversity of BoNoV in Uruguay. A total of 761 samples of feces or intestinal contents from dairy and beef calves were analyzed through RT-qPCR. The overall frequency of detection of BoNoV was 66.1% with higher frequency in dairy (70.5%) than beef (15.9%) calves (p < 0.01). BoNoV was detected similarly in diarrheic (78.8%) and non-diarrheic (76.2%) dairy calves (p = 0.50). Calves ≤2 weeks of age (84%) were infected more often than older (62.7%) calves (p < 0.01). Phylogenetic analysis confirmed the presence of GIII.1 and GIII.2 genotypes. In addition, we reported the circulation of recombinant strains and the detection of a strain with the recently described novel VP1 genotype. This study represents the first report describing the circulation, the associated risk factors, and the molecular diversity of BoNoV in Uruguay.

Norovirus compared to other relevant etiologies of acute gastroenteritis among families from a semirural county in Chile.

OBJECTIVE: To determine the dynamics of norovirus disease, a major cause of acute gastroenteritis (AGE), compared to other relevant etiologies, among families living in a lower middle income area. STUDY DESIGN: Families with three or more members and with one or more healthy children <24 months of age were followed for 1-2 years to detect any AGE. Stool samples were tested for viral and bacterial pathogens and a questionnaire was completed for those with norovirus or rotavirus AGE. RESULTS: Between April and June 2016, 110 families were enrolled, with 103 of them completing ≥12 months of follow-up. A total of 159 family AGE episodes were detected, mostly affecting one individual (92%). At least one pathogen was detected in 56% (94/169) of samples, of which 75/94 (80%) were sole infections. Norovirus was most common (n=26), followed closely by enteropathogenic Escherichia coli (EPEC) (n=25), rotavirus (n=24), and astrovirus (n=23). The annual incidence of family AGE was 0.77, and 0.12 for norovirus. Most norovirus AGE occurred in children <4 years old (96%). Only 13/159 (8%) index AGE cases resulted in a secondary case, of which four were associated with norovirus. The majority of norovirus strains were GII (85%), with a mild predominance of GII.4 (9/26; 35%); most norovirus isolates (69%) were recombinants. CONCLUSIONS: The family incidence of AGE in this lower middle income community was nearly one episode per year, mostly caused by viruses, specifically norovirus closely followed by rotavirus and astrovirus. Norovirus infections primarily affected children <4 years old and secondary cases were uncommon.

Molecular Characterization of Norovirus Circulating in Northwest Mexico During 2013-2014.

Norovirus (NoV) is an important etiological agent of diarrhea in children and adults. In Mexico, NoV screening is not routinely performed. NoV is highly infectious and is responsible for massive outbreaks due to the consumption of contaminated food. The study was a cross-sectional design. Samples of diarrheal stools were collected from (62) children and (38) adults with acute gastroenteritis during 2013-2014. The circulating genogroups of NoV were detected by amplifying the RdRp gene fragment, and for the genotyping, the capsid and polymerase fragments were sequenced. Seventy-seven percent of the analyzed samples were positive for NoV. Genotyping was possible for 51 samples; for polymerase GII.P2, GII.P31, GII.P4, GII.P7, GII.P40, and GI.P14 were identified, whereas for capsid, genotypes GI.3, GII.2, GII.4, GII.5, GII.14, and GII.17. In conclusion, there is a high prevalence of gastroenteritis due to NoV in the northwest of Mexico, including genotypes that have not been reported previously in Mexico.

Gastroenteric Viruses Detection in a Drinking Water Distribution-to-Consumption System in a Low-Income Community in Rio de Janeiro.

The availability of drinking water is one of the main determinants of quality of life, disease prevention and the promotion of health. Viruses are important agents of waterborne diseases and have been described as important markers of human faecal contamination. This study aimed to investigate viruses' presence as an indicator of drinking water quality in low-income communities in the Manguinhos area, Rio de Janeiro, Brazil. Three hundred and four drinking water samples (2L/each) were collected along the drinking water distribution-to-consumption pathway in households, as well as healthcare and school units. Water samples were collected both directly from the water supply prior to distribution and after storage in tanks and filtration units. Using qPCR, viruses were detected 50 times in 45 water samples (15%), 19 of these being human adenovirus, 17 rotavirus A and 14 norovirus GII. Viral loads recovered ranged from 5E+10 to 8.7E+106 genome copies/Liter. Co-detection was observed in five household water samples and there was no difference regarding virus detection across sampling sites. Precarious and inadequate environmental conditions characterized by the lack of local infrastructure regarding basic sanitation and waste collection in the territory, as well as negligent hygiene habits, could explain viral detection in drinking water in regions with a water supply system.

Molecular epidemiology of norovirus outbreaks in Argentina, 2013-2018.

Noroviruses are a leading cause of endemic and epidemic acute gastroenteritis in all age groups. However, in Latin America, there are limited and updated data regarding circulating genotypes. The aim of this study was to assess the prevalence and genetic diversity of norovirus outbreaks in Argentina from 2013 to 2018. Stool samples from 29 acute gastroenteritis (AGE) outbreaks were available for viral testing. Norovirus was detected in samples from 18 (62.1%) outbreaks (2 GI and 16 GII). Both GI outbreaks were typed as GI.6[P11] whereas 10 different GII genotypes were detected, in which GII.4 viruses were the most frequently detected (29.4%, associated with GII.P31 and GII.P16) followed by GII.1[P33] and GII.6[P7] (17.6% each). Like GII.4 viruses, GII.2 viruses were also detected in association with different polymerases (GII.P2 and GII.P16). Our findings underscore the importance of dual RNA-dependent RNA polymerase-VP1 typing since recombinant strains with new polymerase sequences emerge frequently suggesting a possible role in improved fitness of these viruses. This study represents the most recent multi-year assessment of the molecular epidemiology of norovirus strains associated with AGE outbreaks in Argentina. Molecular surveillance of norovirus has to be considered to monitor possible changes in dominant genotypes which may assist to inform the formulation of future vaccines.

Molecular and evolutionary characterization of norovirus GII.17 in the northern region of Brazil.

BACKGROUND: Currently, norovirus (NoV) is associated with one-fifth of all acute gastroenteritis (AGE) cases worldwide. The NoV GII.17_2014 variant has been associated with gastroenteritis outbreaks in several Asian countries, replacing the previously dominant Sydney 2012 variant. There is limited data about circulation of this new strain in Brazil. This study aimed to describe the phylogenetic and evolutionary characteristics of the GII.17_2014 strains in the Northern region of Brazil. METHODS: NoV was detected by enzyme immunoassay (EIA) in 645 stool samples of AGE cases that were reported in Pará and Amazonas states during 2015-2016. All positive samples were tested for NoV GI and GII by reverse transcription polymerase chain reaction (RT-PCR) and the amplicons were subjected to genome sequencing. The GII.17-positive samples were retested by PCR using different sets of designed primers, which target a highly conserved capsid gene region. Next, the amplicons were sequenced and phylogenetically analyzed using Bayesian inferences. RESULTS: Of the 645 samples tested, 208 (32.2%) tested were positive for NoV by EIA, among which 95 (45.7%) were genotyped. Among the genotyped samples, 12 (12.6%) were characterized as GII.17_2014 with the first case detected in November 2015 (1/30, 3.3%) and the others in 2016 (11/65, 16.9%). All strains found in our study were clustered in clade D (epidemic strain). The uncorrelated log-normal model estimations calculated the rate of evolution for GII-17 strains as 1.95 × 10- 3 (1.28 × 10- 3-2.63 × 10- 3). In total, 36 nucleotide changes were observed after analyzing the VP1 sequence, among which 28 occurred in the P2 region. CONCLUSIONS: These data demonstrate the evolutionary dynamics in NoV GII.17_2014 strains, which indicated high mutation rates with nucleotide substitutions and indels that are related to the elevated levels of antigenic diversity. This partly explains the increase in viral prevalence.

Norovirus and human adenovirus occurrence and diversity in recreational water in a karst aquifer in the Yucatan Peninsula, Mexico.

AIMS: To determine the seasonal occurrence and diversity of norovirus (NoV) and human adenovirus (HAdV) in groundwater from sinkholes, and brackish water used for recreational activities in the karst aquifer of the Yucatan Peninsula, Mexico. METHODS AND RESULTS: Hollow fibre ultrafiltration was used to concentrate viruses and standard plaque assay methods were used to enumerate somatic and F+ specific coliphages as viral indicators. Real-time quantitative polymerase chain reaction assays were used to estimate the number of genome copies for NoV strains GI, and GII, and HAdVs. The predominant NoV genotypes and HAdV serotypes were identified by comparative sequence analysis. Somatic and male F+ specific coliphages were detected at concentrations up to 94 and 60 plaque-forming units per 100 ml respectively. The NoV genogroup I (GI) was associated with 50% of the sampled sites during the rainy season only, at concentrations ranging from 120 to 1600 genome copies per litre (GC l-1 ). The NoV genogroup II (GII) was detected in 30 and 40% of the sampled sites during the rainy and dry seasons, respectively, at concentrations ranging from 10 to 290 GC l-1 . During the rainy and dry seasons, HAdVs were detected in 20% of the sites, at concentrations ranging from 24 to 690 GC l-1 . Identification of viral types revealed the presence of NoV GI.2, GII.Pe, GII.P16 and GII.P17, and HAdV F serotypes 40 and 41. CONCLUSIONS: These findings demonstrate that NoVs and HAdVs are prevalent as virus contaminants in the karst aquifer, representing potential health risks particularly during the rainy season, in one of the most important areas used for tourism in Mexico. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few studies conducted in karst aquifers that provide a foundational baseline of the distribution, concentrations and diversity of NoVs and HadVs in these particular environments.

High prevalence of enteric viruses associated with acute gastroenteritis in pediatric patients in a low-income area in Vitória, Southeastern Brazil.

Acute gastroenteritis (AGE) is a significant cause of child mortality worldwide. In Brazil, despite the reduction in infant mortality achieved in recent years, many children still die because of undiagnosed AGE. The prevalence, viral load, and circulating genotypes of rotavirus A (RVA), human adenovirus (HAdV), and norovirus GII (NoV GII) were investigated in children with AGE during 12 months in Vitoria, Espírito Santo, Southeastern Brazil. Enteric viruses were detected in stool samples, quantified by quantitative polymerase chain reaction, sequenced, and compared phylogenetically. The overall prevalence was 93.3% (125/134). Cases of single infection (41.8%) and mixed infection (51.5%) were observed; in 21.6% of cases, all the three viruses were detected. RVA had the highest number of copies in all infections. Phylogenetic analysis revealed predominantly the presence of RVA genotype G3, followed by G2 and G9. HAdV clustered within subgroup C, but some samples harbored subgroups A, D, or F. All sequenced NoV-positive samples clustered within the prevalent genotype GII.4. The high prevalence of RVA, HAdV, and NoV in diarrheal feces clarifies the etiology of AGE in this population, and the presence of RVA in vaccinated children reinforces the importance of monitoring programs to identify the causes of gastroenteritis and contribute to the reliability of diagnosis.

Recombinant noroviruses detected in Mid-West region of Brazil in two different periods 2009-2011 and 2014-2015: Atypical breakpoints of recombination and detection of distinct GII.P7-GII.6 lineages.

Noroviruses are an important cause of acute gastroenteritis. The high incidence of norovirus is a reflection of its great genomic and antigenic variability resultant of evolutionary mechanisms, such as recombination. Herein, the main objective of this study was to characterize partially two regions of norovirus genome (RdRp and VP1) from fecal samples, collected in two different time periods (2009-2011 and 2014-2015) in the Mid-West region of Brazil. Twenty samples were sequenced and characterized (GI.P5-GI.5, GII.P16-GII.3, GI.P7-GI.7, GII.Pe-GII.4 and GII.P7-GII.6). Sequences of GII.Pe-GII.4 genotype were also characterized as Sydney 2012 variant. Genotypes GII.P7-GII.6, GII.P16-GII.3 and GII.Pe-GII.4 (16/20-80%) were identified as norovirus recombinants by phylogeny and bioinformatic analyzes. The GII.P7-GII.6 (62.5%) and GII.Pe-GII.4 (25%) genotypes had recombination point's upstream ORF1/2 overlapping region, whereas GII.P16-GII.3 (12.5%) genotype had the recombination point in the overlapping region. Furthermore, the GII.P7-GII.6, from samples collected in 2009-2011 had different recombinant points than the GII.P7-GII.6 from samples obtained in 2014-2015, forming two different clusters in the phylogenetic analysis. Our study brings information on the circulation of recombinant norovirus genotypes in Mid-West of Brazil, including recombinants with atypical recombination breakpoints, and provides evidence for the circulation of different lineages of the same recombinant genotype.

Assessment of viral and bacterial contamination of fresh and ripened semi-hard cheeses.

This study aimed to evaluate viral and bacterial contamination from typical Brazilian cheeses, such as Minas (fresh) and Prato (ripened), commercially obtained in the Greater Metropolitan Region of the State of Rio de Janeiro, Brazil. Minas [30], Prato [30] and sliced Prato [30] cheese samples were investigated for norovirus genogroup I and II (NoV GI-II) and human adenovirus (HAdV) by direct nucleic acid extraction using TRIzol and amplification by TaqMan based quantitative polymerase chain reaction. Listeria monocytogenes, Salmonella spp., coagulase-positive staphylococci (CPS) and fecal coliforms were also assessed by using standard counting methods. NoV GI and GII were detected in one sample (1.1%) each and HAdV in nine samples (10.0%) while bacteriological analysis revealed five samples (5.5%) contaminated with L. monocytogenes, 27 (30.0%) with fecal coliforms and 10 (11.1%) with CPS. Salmonella spp. was not detected in any sample. Viruses were detected in 11 samples (12.2%), of which 9 met the microbiological criteria used to evaluate the microbiological quality of the cheeses, stressing the importance of considering virological parameters for monitoring this food matrix.

Norovirus RNA in serum associated with increased fecal viral load in children: Detection, quantification and molecular analysis.

Worldwide, norovirus (NoV) is a major cause of acute gastroenteritis (AGE) responsible for pandemics every ~3 years, and over 200,000 deaths per year, with the majority in children from developing countries. We investigate the incidence of NoV in children hospitalized with AGE from Belém, Pará, Brazil, and also correlated viral RNA levels in their blood and stool with clinical severity. For this purpose, paired stool and serum samples were collected from 445 pediatric patients, ≤9 years between March 2012 and June 2015. Enzyme-linked immunosorbent assay (EIA) was used to detect NoV in stool and reverse transcription quantitative PCR (RT-qPCR) used to quantify NoV RNA levels in sera (RNAemia) and in the positive stool. Positives samples were characterized by the partial ORF1/2 region sequence of viral genome. NoV antigen was detected in 24.3% (108/445) of stool samples, with RNAemia also present in 20.4% (22/108). RNAemia and a high stool viral load (>107 genome copies/gram of faeces) were associated with longer hospitalizations. The prevalent genotypes were GII.4 Sydney_2012 (71.6%-58/81) and New Orleans_2009 (6.2%-5/81) variants. Eight other genotypes belonging to GII were detected and four of them were recombinant strains. All sera were characterized as GII.4 and shared 100% similarity with their stool. The results suggest that the dissemination of NoV to the blood stream is not uncommon and may be related to increased faecal viral loads and disease severity.