Relative frequency of genomic mutations in SARS-CoV-2 recovered from southern Brazilian cases of COVID-19 through the Gamma, Delta and Omicron waves.

The presence of different mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can be related to changes in coronavirus disease (COVID-19) infection. Besides, these viral alterations associated with factors such as massive number of positive cases, vaccination and reinfections can be important in the viral evolution process. As well as, mutations found at low frequencies may have a more neutral action and consequently be less inclined to negative selection, facilitating their spread through the population. Related to that, we aimed to present mutations that are possibly relevant in the process of viral evolution found in 115 SARS-CoV-2 sequences from samples of individuals residing in the metropolitan region of Porto Alegre in the state of Rio Grande do Sul, Brazil. The genome from clinical samples was sequenced using High-Throughput Sequencing (HTS) and analyzed using a workflow to map reads and find variations/SNPs. The samples were separated into 3 groups considering the sample lineage. Of the total number of analyzed sequences, 35 were from the Gamma lineage, 35 from Delta and 45 from Omicron. Amino acid changes present in frequencies lower than 80% of the reads in the sequences were evaluated. 11 common mutations among the samples were found in the Gamma lineage, 1 in the ORF1ab gene, 7 in the S gene, 2 in the ORF6 gene and 1 in the ORF7a gene. While in the Delta lineage, a total of 11 mutations distributed in the ORF1ab, S, ORF7a and N genes, 2, 7, 1 and 1 mutation were found in each gene, respectively. And finally, in the Omicron, 16 mutations were identified, 2 in the ORF1ab gene, 12 in the S gene and 2 in the M gene. In conclusion, we emphasize that genomic surveillance can be a useful tool to assess how mutations play a key role in virus adaptation, and its process of susceptibility to new hosts showing the possible signs of viral evolution.

Diversity of Omicron sublineages and clinical characteristics in hospitalized patients in the southernmost state of Brazil.

BACKGROUND: Omicron has become the dominant variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since first reported in November 2021. From the initially detected Wuhan lineage, sublineages BA.2, BA.4, BA.5, BQ, XAG, and XBB have emerged over time and are dominant in many countries. Therefore, the aim is to evaluate which variants are circulating and the clinical characteristics of inpatients infected with the Omicron variant. METHODS: This retrospective cohort study selected hospitalized patients admitted with respiratory symptoms to a hospital in the state of Rio Grande do Sul, Brazil, between June and July 2022. SARS-CoV-2 results were analyzed together with clinical outcomes and vaccination status. A viral genome library was prepared and forwarded to the Illumina MiSeq Platform for sequencing. RESULTS: In total, 37 genomes were sequenced. Concerning the Omicron sublineages, our study detected: BA.1 (21 K), BA.2 (21 L), BA.4 (22A), BA.5 (22B), BA.2.12.1 (22C), BQ.1 (22E), XBB (22F), and XAG recombinant. Omicron BA.5 (30%), BA.2 (19%), and BQ.1 (19%) were the most frequent sublineages, respectively. In total, 38% of patients present hypertension, and the most common symptoms were coughing (62%). Analyzing the COVID-19 vaccination, 30% of patients were fully vaccinated, 49% had a partial vaccination status, and 21% were unvaccinated (no dose). CONCLUSIONS: BA.5 was the most prevalent sublineage in our study and surpassed the predominance of BA.2, as reported by the national genomic surveillance program. BQ.1 was diagnosed earlier in this study than it was officially reported in the state. Current data have demonstrated that the Omicron variant causes less severe infections, with the high rate of transmissibility and mutational landscape causing the rapid emergence of new sublineages.

DENV-1 genotype V linked to the 2022 dengue epidemic in Southern Brazil.

Even though Brazil is a country where the dengue virus (DENV) is endemic, until recently, Southern states did not have significant viral circulation, such as Rio Grande do Sul (RS), and some municipalities were even considered dengue-free. During 2022, these places have shown a sharp increase in the incidence of the disease, apparently following a worldwide growth pattern. Therefore, in this study, we monitor and characterize the genetic diversity of DENV circulating in southern Brazil through next-generation sequencing during an outbreak in 2022. We generated 70 DENV-1 genome sequences, all characterized as genotype V, divided into two clade clusters in the L1 lineage. Furthermore, unique mutations have been described in each clade of L1 lineage. Our results are essential in managing outbreaks since these data provide important information during the emergence of DENV circulation in RS. Since the south of Brazil has a lower viral circulation when compared to other Brazilian states, RS still lacks data that can help in understanding the transmission, dissemination, and evolution of the dengue virus. Hence, genomic surveillance efforts are essential to increase the accuracy of preventive actions and to control viral dissemination.

Reinfection by SARS-CoV-2 by divergent Omicron sublineages, 16 days apart.

Since the beginning of the SARS-CoV-2 pandemic, studies on the variants and sublineages stand out, mainly in the cases of reinfection in a short period. In this study, we describe a case of infection by BA.1.1 sublineage in an individual from Southern Brazil. The same patient acquired reinfection with sublineage BA.2 within 16 days after the first detection. The viral extraction and RT-qPCR were performed on the samples LMM72045 (collected in May 2022) and LMM72044 (collected in June 2022). After the confirmation of SARS-CoV-2 infection, we conducted the sequencing and viral genome analysis. This case of reinfection affected a 52-year-old male patient, without comorbidities, with three doses of vaccines against COVID-19, showing symptoms on May 19. These symptoms lasted for approximately six days. The patient returned to work activities on May 30. However, on June 4, the patient felt a new round of clinical signs that lasted for approximately seven days. Analysis of the viral genomes recovered from patients' clinical samples revealed that the two COVID-19 episodes were related to two divergent VOC Omicron sublineages, namely, BA.1.1 for the first round of symptoms and BA.2 for the second infection. Based on our findings, we can say that the present case of reinfection is the shortest described so far.

Prolonged SARS-CoV-2 Infection and Intra-Patient Viral Evolution in an Immunodeficient Child.

BACKGROUND: With the progression of the Coronavirus disease pandemic, the number of mutations in the viral genome has increased, showing the adaptive evolution of severe acute respiratory syndrome coronavirus 2 in humans and intensification in transmissibility. Long-term infections also allow the development of viral diversity. In this study, we report the case of a child with severe combined immu presenting a prolonged severe acute respiratory syndrome coronavirus 2 infection. We aimed to analyze 3 naso-oropharyngeal swab samples collected between August and December 2021 to describe the amino acid changes present in the sequence reads that may have a role in the emergence of new viral variants. METHODS: The whole genome from clinical samples was sequenced through high throughput sequencing and analyzed using a workflow to map reads and then find variations/single-nucleotide polymorphisms. In addition, the samples were isolated in cell culture, and a plaque forming units assay was performed, which indicates the presence of viable viral particles. RESULTS: The results obtained showed that the virus present in all samples is infectious. Also, there were 20 common mutations among the 3 sequence reads, found in the ORF1ab and ORF10 proteins. As well, a considerable number of uncommon mutations were found. CONCLUSIONS: In conclusion, we emphasize that genomic surveillance can be a useful tool to assess possible evolution signals in long-term patients.

Genomic and epidemiologic surveillance of SARS-CoV-2 in Southern Brazil and identification of a new Omicron-L452R sublineage.

Recently, SARS-CoV-2 Omicron variant (B.1.1.529) was first identified in Botswana in November 2021. In a short period of time, this highly mutated variant replaced the previous dominant Delta variant, causing an exponential increase in the number of COVID-19 cases, resulting in a new wave of pandemic. This current research article aims to analyze and summarize information about the genetic characteristics, amino acid mutations and epidemiological data providing scientific findings to enrich the SARS-CoV-2 knowledge. More importantly, we describe here, for the first time, the identification of a new Omicron variant of concern: Omicron-L452R in Brazil.

Viral isolation allows characterization of early samples of SARS-CoV-2 lineage B1.1.33 with unique mutations (S: H655Y and T63N) circulating in Southern Brazil in 2020.

Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil.

Genome Sequence of a Brazilian Bovine Enterovirus.

We report the nearly complete genome sequence of a Brazilian bovine enterovirus (genus Enterovirus, family Picornavirus). This enterovirus was isolated from an enteric and respiratory disease outbreak in a beef cattle herd in southern Brazil. Phylogeny indicates that this isolate belongs to the species Enterovirus E.

SARS-CoV-2 and COVID-19: A perspective from environmental virology.

December 2019 marked the beginning of the current Coronavirus disease pandemic (COVID-19). Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) was identified as the causative agent of a viral pneumonia outbreak in Wuhan, Hubei Province, China. The alarming spread levels and clinical severity elevated the status of COVID-19 to the global pandemic by the World Health Organization. In 6 months, more than 25 million cases of infected people and more than 890,000 deaths by COVID-19 had been reported worldwide. The main goal of this review is to shed light upon the current COVID-19 epidemic situation in Brazil with a health approach highlighting some unique environmental, animal and epidemiological aspects.

Quantitative microbial risk assessment of SARS-CoV-2 for workers in wastewater treatment plants.

Faecal-oral transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be validated, but it is a critical issue and additional research is needed to elucidate the risks of the novel coronavirus in sanitation systems. This is the first study that investigates the potential health risks of SARS-CoV-2 in sewage to wastewater treatment plant (WWTP) workers. A quantitative microbial risk assessment (QMRA) is applied for three COVID-19 scenarios (moderate, aggressive and extreme) to study the effects of different stages of the pandemic in terms of percentage of infected population on the probability of infection to WWTP workers. A dose-response model for SARS-CoV-1 (as a surrogate pathogen) is assumed in the QMRA for SARS-CoV-2 using an exponential model with k = 4.1 × 102. Literature data are incorporated to inform assumptions for calculating the viral load, develop the model, and derive a tolerable infection risk. Results reveal that estimates of viral RNA in sewage at the entrance of WWTPs ranged from 4.14 × 101 to 5.23 × 103 GC·mL-1 (viable virus concentration from 0.04 to 5.23 PFU·mL-1, respectively). In addition, estimated risks for the aggressive and extreme scenarios (2.6 × 10-3 and 1.3 × 10-2, respectively) were likely to be above the derived tolerable infection risk for SARS-CoV-2 of 5.5 × 10-4 pppy, thus reinforcing the concern of sewage systems as a possible transmission pathway of SARS-CoV-2. These findings are helpful as an early health warning tool and in prioritizing upcoming risk management strategies, such as Emergency Response Plans (ERPs) for water and sanitation operators during the COVID-19 and future pandemics.

Detection, Quantification, and Microbial Risk Assessment of Group A Rotavirus in Rivers from Uruguay.

The aim of this study was to detect, quantify, and assess the risk of infection and illness for Group A Rotavirus (RVA) in the watersheds of the Santa Lucia and Uruguay rivers in Uruguay. Monthly sampling was carried out for one year in six sites in the watershed of the Santa Lucía River and four in the Uruguay River. All the collection sites are used for recreational activities. Viral concentration was performed with the adsorption-elution method, and detection and quantification of RVA was carried out by TaqMan quantitative PCR (qPCR). Quantitative microbial risk assessment was applied to estimate the daily and annual risk of RVA infection, as well as the daily risk of illness considering direct exposure through recreational activity. RVA was detected in 42% (20/48) of the analyzed samples in the Uruguay River and 40% (29/72) in the Santa Lucía River. The virus was present in all the analyzed points in both watersheds. A pattern of seasonality, characterized by a higher detection frequency of the virus during coldest month of the year, was observed in both basins. The mean concentration for RVA was 1.3 × 105 genomic copies/L. The microbiological risk assessment shows that Santa Lucía watershed presented the highest daily risk of infection (6.41E-01) and illness (3.20E-01) estimated for the point downstream of Florida City; meanwhile for Uruguay River, the highest probabilities of infection (6.82E-01) and illness (3.41E-01) were estimated for the collection site for drinking water intake in Salto city. These results suggest that RVA contamination of these important rivers negatively impact on their microbiological quality since they are used for recreation and drinking water intake, demonstrating that the disposal of waste from cities located in their riverside confers a constant threat of infection for the general population, especially for children.

Correction to: Temporal dynamics of Human mastadenovirus species in cases of respiratory illness in southern Brazil.

The original version of this article unfortunately contained a mistake. The term "mortality rate" should be rather read as "morbidity rate" in the following fragment of the "Discussion" section.

Temporal dynamics of human mastadenovirus species in cases of respiratory illness in southern Brazil

Human mastadenovirus (HAdV) genus is related to several diseases, among them upper and lower respiratory tract illness. HAdV species B, C, D, and E are mainly associated with respiratory infections. The goal of this work was to identify the HAdV species associated with respiratory infections in hospitalized patients from southern Brazil. Samples were collected from 1996 to 2004 and 2011 to 2017. During this period, 28,524 samples were collected, and 9983 were positive for respiratory viruses, being 435 for HAdV. From these 435 samples, 57 were selected for characterization of HAdV species. For screening the presence of HAdV, a partial sequence of the DNA polymerase gene (DNApol gene) was amplified by nested PCR. Partial nucleotide sequencing was performed in positive samples, and HAdV (DNApol gene) was detected in 53 samples: species B (28;49.1%), C (16;8.0%), D (2; 3.5%), E (5; 8.7%), and untyped (2; 3.5%). Specie D was found only in 2017 and specie E in 2011 and 2012. The age of the patients ranged from < 1 to 81 years old, and 62.3%were male. No relationship between gender orage and identified HAdV species were observed. In addition, in the period of 2013­2017, 18 samples from patients who died were analyzed: 11 were related to species B, 4 to C, and 2 to D and 1 remained untyped. Circulation of HAdV species D and Evaried over the years, but species B and C were present throughout the evaluated period. In addition, respiratory infections by HAdVaffect elderly and children mainly. (AU)

Human mastadenovirus in water, sediment, sea surface microlayer, and bivalve mollusk from southern Brazilian beaches.

Anthropogenic contamination of beaches in the south of Brazil was assessed by detection of Escherichia coli, human mastadenovirus species C (HAdV-C) and F (HAdV-F) and hepatitis E virus (HEV). Sampling was carried out in October (2016), and in January, April and July (2017). Water, sediment, sea surface microlayer (SML), bivalves, and air sentinel samples were evaluated. Quantitative microbiological risk assessment (QMRA) was used to estimate the probability of swimmer infection. HAdV-C was present in 26% of the samples, for both qPCR and viral isolation. The highest rates of detection in genomic copies (GC) were in water (2.42E+10 GC/L), SML (2.08E+10 GC/L), sediment (3.82E+08 GC/g) and bivalves (3.91E+07 GC/g). QMRA estimated daily and annual risks with a maximum value (9.99E-01) in almost all of the samples. Viable HAdV-C was often detected in the SML, pointing that this is a source of infection for people bathing in these waters.

Temporal dynamics of Human mastadenovirus species in cases of respiratory illness in southern Brazil.

Human mastadenovirus (HAdV) genus is related to several diseases, among them upper and lower respiratory tract illness. HAdV species B, C, D, and E are mainly associated with respiratory infections. The goal of this work was to identify the HAdV species associated with respiratory infections in hospitalized patients from southern Brazil. Samples were collected from 1996 to 2004 and 2011 to 2017. During this period, 28,524 samples were collected, and 9983 were positive for respiratory viruses, being 435 for HAdV. From these 435 samples, 57 were selected for characterization of HAdV species. For screening the presence of HAdV, a partial sequence of the DNA polymerase gene (DNApol gene) was amplified by nested PCR. Partial nucleotide sequencing was performed in positive samples, and HAdV (DNApol gene) was detected in 53 samples: species B (28; 49.1%), C (16; 28.0%), D (2; 3.5%), E (5; 8.7%), and untyped (2; 3.5%). Specie D was found only in 2017 and specie E in 2011 and 2012. The age of the patients ranged from < 1 to 81 years old, and 62.3% were male. No relationship between gender or age and identified HAdV species were observed. In addition, in the period of 2013-2017, 18 samples from patients who died were analyzed: 11 were related to species B, 4 to C, and 2 to D and 1 remained untyped. Circulation of HAdV species D and E varied over the years, but species B and C were present throughout the evaluated period. In addition, respiratory infections by HAdV affect elderly and children mainly.

Microbial risk assessment in recreational freshwaters from southern Brazil.

In this study, total coliforms (TC), Escherichia coli, enterovirus (EV), rotavirus (RV), and human mastadenovirus species C and F (HAdV-C and HAdV-F) were evaluated in water samples from Belo Stream. For HAdV-C and F, the infectivity was assessed by integrated cell culture quantitative real-time polymerase chain reaction (ICC-qPCR). Samples were collected monthly (May/2015 to April/2016) at four sites. Viral analyses were performed for both ultracentrifuge-concentrated and unconcentrated samples. For site P4 (used for recreational purposes), QMRA was applied to estimate health risks associated with exposure to E. coli and HAdV-C and F. TC and E. coli were present throughout the collection period. EV and RV were not detected. HAdV-C were present in 8.51% (1.89E + 06 to 2.28E + 07 GC (Genomic Copies)/L) and 21.27% (2.36E + 05 to 1.29E + 07 GC/L) for unconcentrated and concentrated samples, respectively. For HAdV-F were 12.76% (2.77E + 07 to 3.31E + 08 GC/L) and 48.93% (1.10E + 05 to 4.50E + 08 GC/L) for unconcentrated and concentrated samples, respectively. For unconcentrated samples, infectivity for HAdV-C was detected in 37.20% (1st ICC-qPCR) and 25.58% (2nd ICC-qPCR). For HAdV-F, infectivity was detected in 6.97% (1st ICC-qPCR) and 6.97% (2nd ICC-qPCR). For concentrated samples, HAdV-C infectious was observed in 17.02% (1st ICC-qPCR) and in 8.51% (2nd ICC-qPCR). For HAdV-F, were present in 8.51% for both 1st and 2nd ICC-qPCR. Statistical analyzes showed significant difference between the collection sites when analyzed the molecular data of HAdV-F, data of TC and E. coli. Correlation tests showed direct correlation between HAdV-F with E. coli and TC. E. coli concentrations translated to the lowest estimates of infection risks (8.58E-05 to 2.17E-03). HAdV-F concentrations were associated with the highest infection risks at 9.99E-01 and for group C, 1.29E-01 to 9.99E-01. These results show that commonly used bacterial indicators for water quality may not infer health risks associated with viruses in recreational freshwaters.

Effects of pH, Temperature and Agitation on the Decolourisation of Dyes by Laccase-Containing Enzyme Preparation from Pleurotus sajor-caju

(1) Background: In this study, the effects of different pH values ​​(2.4, 3.2, 4.4 and 5.0), temperatures (30, 35, 40, 45 and 50°C) and agitation (100 rpm) on the enzymatic decolourisation of twenty-two dyes belonging to the chromophore groups anthraquinone, azo and triphenylmethane were assessed. (2) Methods: In all conditions, it was used a crude enzyme broth containing 30 U mL-1 laccases produced by Pleurotus sajor-caju PS-2001 in submerged process. (3) Results: Regarding the effects of pH values, the best results were obtained at pH 3.2 and 30°C, in which bleaching was observed for all dyes evaluated. In assays conducted at different temperatures, highest levels of decolourisation were observed at 35°C and pH 3.2 for nineteen of the dyes assessed. Thirteen dyes presented colour reduction exceeding 50% after the enzymatic treatment, including all acid and all disperse dyes evaluated. The reciprocal agitation of 100 rpm promoted negative effect on decolourisation. (4) Conclusion: From the results achieved, one can conclude that the laccase-containing preparation of P. sajor-caju PS-2001 has potential for the decolourisation of some dyes widely used in different industrial sectors, especially in the textile industry, and therefore could be used in future strategies for the biotreatment of coloured wastes.

Caffeine levels as a predictor of Human mastadenovirus presence in surface waters-a case study in the Sinos River basin-Brazil.

The presence of caffeine in environmental water samples is almost entirely human-related, given that there are virtually no industrial or agricultural releases. Caffeine has already been proposed as an anthropogenic marker for wastewater contamination of surface waters. The aim of this study was to evaluate if caffeine concentrations in water can be a predictor of virological and bacteriological contamination. Water samples were taken at three sampling sites from urban water streams from the hydrographic basin of the Sinos River (Brazil) monthly in the period of May 9th, 2016 to April 11th, 2017 (n = 36). Concentrations of Human mastadenovirus (HAdV-F and HAdV-C), fecal coliforms, and caffeine were measured in all collected samples. Concentrations of caffeine in water were strongly correlated with HAdV-F (rs = 0.704, p = 0.000). This study, for the first time, characterized caffeine concentrations in water as predictors of virus presence, with cut-off values presenting 92.9% specificity and 95.5% sensitivity for HAdV-F and 66.7% specificity and 80% sensitivity for HAdV-C. Considering its marked chemical stability and ease of quantification, caffeine concentrations can be used as a comprehensive marker of human contamination of water resources, also being predictive of bacteriological and virological concentrations.

Assessment of diversity of adenovirus DNA polymerase gene in recreational waters facilitated by ultracentrifugal concentration.

Adenoviruses (AdV) are related to respiratory and gastrointestinal diseases in animals and human beings. Their wide genetic diversity in water bodies and their resistance to environmental conditions allow the use of AdV as a reliable marker for detection of fecal contamination. In this work, the diversity of AdV along Belo Stream - in the city of Caxias do Sul, Rio Grande do Sul, Brazil - was evaluated. Samples were compared in both concentrated and unconcentrated forms. The identification of different AdV species was performed by amplifying a partial sequence of the DNA polymerase gene. AdV was detected in 24 out of 55 concentrated samples (43.6%) and the following species were identified: human adenovirus (HAdV) species C (4/55; 7.2%), D (6/55; 10.9%), E (2/55; 3.6%), and F (9/55; 16.3%). AdV related to other mammalian hosts, such as bovine adenovirus (1/55, 1.8%) and murine adenovirus (2/55, 3.6%), have also been identified; 23.6% (13/55) of the unconcentrated samples were positive, and identified as HAdV species C (6/55, 10.9%), D (1/55, 1.8%), and F (6/55, 10.9%). Results obtained evidenced the presence and the great diversity of AdV, mainly of human origin, circulating in Belo Stream. As expected, the concentration step performed helped to detect AdV in more samples.

Enteric viruses and adenovirus diversity in waters from 2016 Olympic venues.

Rio de Janeiro's inner and coastal waters are heavily impacted by human sewage pollution for decades. Enteric viruses, including human adenoviruses (HAdV), human enterovirus (EV), group A rotavirus (RV) and hepatitis A virus (HAV) are more likely to be found in contaminated surface waters. The present work aimed to assess the frequency and loads of EV, HAdV-C and -F species, RV and HAV in sand and water samples from venues used during the 2016 Summer Olympics and by tourists attending the event. Sixteen monthly collections were carried out from March 2015 to July 2016 in 12 different sites from Rio de Janeiro, Brazil. Total and thermotolerant coliform counting was performed along molecular detection of virus was performed using quantitative polymerase chain reaction (qPCR). Analyses of all samples were further investigated by integrated cell culture PCR to check about the presence of HAdV infectious virus particles. The results show that 95.9% of water samples showed contamination with at least one type of virus. Regarding the viruses individually (% for water and sand respectively): HAdV-C (93.1%-57.8%), HAdV-F (25.3%-0%), RV (12.3%-4.4%), EV (26.7%-8.8%) and HAV (0%). The viral loads ranged from 103gc/L up to 109gc/L (water), and 103gc/g to 106gc/g (sand). In the phylogenetic tree, were classified into four main clusters, referring to species C, D, F and BAdV. And up to 90% of sites studied presented at least once presence of infectious HAdV-C. The most contaminated points were the Rodrigo de Freitas Lagoon, where Olympic rowing took place, and the Marina da Glória, the starting point for the sailing races, demonstrating serious problem of fecal contamination of water resources and threatens the health of Olympic athletes, tourists and residents.