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Background: The human immunodeficiency virus 1 (HIV-1) infections in Brazil are predominantly caused by two subtypes, B and C., Objectives: Here we present the characterisation of a novel HIV-1 recombinant form, indicating a new Brazilian CRF_BC, named CRF146_BC., Methods: RDP, JphMM and Simplot recombination tools were used to evaluate the mosaic pattern., Findings: In this work, we identified three HIV-1 nucleotide sequences previously classified as unique recombinant forms (URFs), plus one new partial genome sharing the same BC recombination pattern. The mosaic genome is almost entirely represented by the subtype C sequence, with a small subtype B recombination region in the pol gene, at the Integrase level. The phylogenetic analyses strongly indicate a common origin between the strains, which were isolated in Rio Grande do Sul, Rio de Janeiro and Bahia states., Main Conclusions: Thus, the new HIV-1 CRF146_BC is circulating in three different Brazilian regions: South, Southeast and Northeast.

Mitoviruses are cryptic capsidless viruses belonging to the family Mitoviridae that replicate and are maintained in the mitochondria of fungi. Complete mitovirus-like sequences were recently assembled from plant transcriptome data and plant leaf tissue samples. Passion fruit (Passiflora spp.) is an economically important crop for numerous tropical and subtropical countries worldwide, and many virus-induced diseases impact its production. From a large-scale genomic study targeting viruses infecting Passiflora spp. in Brazil, we detected a de novo-assembled contig with similarity to other plant-associated mitoviruses. The contig is ∼2.6 kb long, with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRP). This contig has been named "passion fruit mitovirus-like 1" (PfMv1). An alignment of the predicted amino acid sequence of the RdRP of PfMv1 and those of other plant-associated mitoviruses revealed the presence of the six conserved motifs of mitovirus RdRPs. PfMv1 has 79% coverage and 50.14% identity to Humulus lupulus mitovirus 1. Phylogenetic analysis showed that PfMV1 clustered with other plant-associated mitoviruses in the genus Duamitovirus. Using RT-PCR, we detected a PfMv1-derived fragment, but no corresponding DNA was identified, thus excluding the possibility that this is an endogenized viral-like sequence. This is the first evidence of a replicating mitovirus associated with Passiflora edulis, and it should be classified as a member of a new species, for which we propose the name "Duamitovirus passiflorae"., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Since the emergence of SARS-CoV-2 in December 2019, more than 12,000 mutations in the virus have been identified. These could cause changes in viral characteristics and directly impact global public health. The emergence of variants is a great concern due to the chance of increased transmissibility and infectivity. Sequencing for surveillance and monitoring circulating strains is extremely necessary as the early identification of new variants allows public health agencies to make faster and more effective decisions to contain the spread of the virus. In the present study, we identified circulating variants in samples collected in Belo Horizonte, Brazil, and detected a recombinant lineage using the Sanger method. The identification of lineages was done through gene amplification of SARS-CoV-2 by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). By using these specific fragments, we were able to differentiate one variant of interest and five circulating variants of concern. We were also able to detect recombinants. Randomly selected samples were sequenced by either Sanger or Next Generation Sequencing (NGS). Our findings validate the effectiveness of Sanger sequencing as a powerful tool for monitoring variants. It is easy to perform and allows the analysis of a larger number of samples in countries that cannot afford NGS., (© 2024. The Author(s).)

Mosquitoes can transmit several pathogenic viruses to humans, but their natural viral community is also composed of a myriad of other viruses such as insect-specific viruses (ISVs) and those that infect symbiotic microorganisms. Besides a growing number of studies investigating the mosquito virome, the majority are focused on few urban species, and relatively little is known about the virome of sylvatic mosquitoes, particularly in high biodiverse biomes such as the Brazilian biomes. Here, we characterized the RNA virome of 10 sylvatic mosquito species from Atlantic forest remains at a sylvatic-urban interface in Northeast Brazil employing a metatranscriptomic approach. A total of 16 viral families were detected. The phylogenetic reconstructions of 14 viral families revealed that the majority of the sequences are putative ISVs. The phylogenetic positioning and, in most cases, the association with a high RNA-dependent RNA polymerase amino acid divergence from other known viruses suggests that the viruses characterized here represent at least 34 new viral species. Therefore, the sylvatic mosquito viral community is predominantly composed of highly divergent viruses highlighting the limited knowledge we still have about the natural virome of mosquitoes in general. Moreover, we found that none of the viruses recovered were shared between the species investigated, and only one showed high identity to a virus detected in a mosquito sampled in Peru, South America. These findings add further in-depth understanding about the interactions and coevolution between mosquitoes and viruses in natural environments., Importance: Mosquitoes are medically important insects as they transmit pathogenic viruses to humans and animals during blood feeding. However, their natural microbiota is also composed of a diverse set of viruses that cause no harm to the insect and other hosts, such as insect-specific viruses. In this study, we characterized the RNA virome of sylvatic mosquitoes from Northeast Brazil using unbiased metatranscriptomic sequencing and in-depth bioinformatic approaches. Our analysis revealed that these mosquitoes species harbor a diverse set of highly divergent viruses, and the majority comprises new viral species. Our findings revealed many new virus lineages characterized for the first time broadening our understanding about the natural interaction between mosquitoes and viruses. Finally, it also provided several complete genomes that warrant further assessment for mosquito and vertebrate host pathogenicity and their potential interference with pathogenic arboviruses., Competing Interests: The authors declare no conflict of interest.

Background: Amazonas was one of the most impacted Brazilian states by the COVID-19 pandemic. Mortality rates were high, and the health systems collapsed. It is important to identify possible intermediate reservoirs to avoid animal-to-human contamination. Several tropical fish are of commercial interest and are sold in large open-air markets in the region, representing a large economic and dietary importance., Objectives: This study aimed to verify if fish species of commercial importance, aerosols, and fish wastewater in local open-air markets, at a major capital city in the western Brazilian Amazon, are contaminated by SARS-CoV-2., Methods: 488 fish, 50 aerosol, and 45 wastewater samples were analyzed for the presence of SARS-CoV-2. The samples were subjected to extraction using the BIOGENE Viral DNA/RNA Extraction kit, and the molecular diagnosis was tested for SARS-CoV-2 using the Bio-Manguinhos SARS-CoV-2 (EDx) Molecular Kit., Results: It was not possible to detect the virus (Ct≤40, for Gene E) in these samples, however, in 181 samples of fish it was possible to detect the human RP gene (Ct≤35, for the RP Gene), indicating human contact. There was a high number of COVID-19 diagnoses in all city districts in which the samples were collected, showing that SARS-CoV-2 was circulating., Conclusion: This study indicates that fish of local commercial importance do not carry SARS-CoV-2 viral particles, despite circulation of SARS-CoV-2, and are not an important source of animal-to-human contamination. Despite these results, the human RP gene was found detectable in fish, air, and fish wastewater, showing that such places may carry human pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Sá Leitão et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We aimed to describe the landscape, including molecular, epidemiological, and clinical aspects of CHIKV infections in the Ribeirao Preto region, an area endemic to dengue. We randomly screened 3744 plasma samples that had undergone DENV diagnosis to evaluate CHIKV-RNA using an in-house RT-PCR assay. Positive samples were followed clinically, and RNA samples were submitted to whole genome sequencing. Seventeen cases (0.5 %) were positive for CHIKV-RNA despite being negative for DENV-RNA. Notably, half of the patients experienced prolonged arthralgia lasting more than 90 days. Compared with the healthy control group, leukopenia and thrombocytopenia were observed in all CHIKV-positive individuals with statistically significant P values (P < 0.0001 and P = 0.0003, respectively). The genomic analysis revealed that the CHIKV strains being studied are classified within the East-Central-South-African (ECSA) genotype. This analysis identified new mutations, E1: K211E and E2: V264A, while the previously known mutation E1: A226V was not detected among these strains. This study highlights the need for epidemiological surveillance and preparedness for potential CHIKV epidemics in Brazil, particularly where other arboviruses co-circulate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

In this study, we provide a genomic description of the first isolation of the Umattila virus (UMAV) in Brazil. The virus was obtained from the blood of a bird ( Turdus fumigatus ) and isolated in a C6/36 cell culture. The viral genome contains ten segments, and its organization is characteristic of viruses of the genus Orbivirus (family Sedoreoviridae ). The coding region of each segment was sequenced, demonstrating the nucleotide identity with UMAV. The phylogenetic inference results were in line with these findings and demonstrated the formation of two distinct monophyletic clades containing strains isolated around the world, where our isolate, belonging to the same clade as the prototype strain, was allocated to a different subclade, highlighting the genetic divergence between them. This work reports the first isolation of UMAV in Brazil, and due to the scarcity of information on this viral agent in the scientific literature, it is essential to carry out further studies to better understand its epidemiology, dispersion, and, in particular, its interactions with vertebrate hosts, vectors, and the environment.

Background: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits., Methods: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples., Results: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values., Conclusions: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Marseilleviruses (MsV) are a group of viruses that compose the Marseilleviridae family within the Nucleocytoviricota phylum. They have been found in different samples, mainly in freshwater. MsV are classically organized into five phylogenetic lineages (A/B/C/D/E), but the current taxonomy does not fully represent all the diversity of the MsV lineages. Here, we describe a novel strain isolated from a Brazilian saltwater sample named Marseillevirus cajuinensis. Based on genomics and phylogenetic analyses, M. cajuinensis exhibits a 380,653-bp genome that encodes 515 open reading frames. Additionally, M. cajuinensis encodes a transfer RNA, a feature that is rarely described for Marseilleviridae. Phylogeny suggests that M. cajuinensis forms a divergent branch within the MsV lineage A. Furthermore, our analysis suggests that the common ancestor for the five classical lineages of MsV diversified into three major groups. The organization of MsV into three main groups is reinforced by a comprehensive analysis of clusters of orthologous groups, sequence identities, and evolutionary distances considering several MsV isolates. Taken together, our results highlight the importance of discovering new viruses to expand the knowledge about known viruses that belong to the same lineages or families. This work proposes a new perspective on the Marseilleviridae lineages organization that could be helpful to a future update in the taxonomy of the Marseilleviridae family., Importance: Marseilleviridae is a family of viruses whose members were mostly isolated from freshwater samples. In this work, we describe the first Marseillevirus isolated from saltwater samples, which we called Marseillevirus cajuinensis . Most of M. cajuinensis genomic features are comparable to other Marseilleviridae members, such as its high number of unknown proteins. On the other hand, M. cajuinensis encodes a transfer RNA, which is a gene category involved in protein translation that is rarely described in this viral family. Additionally, our phylogenetic analyses suggested the existence of, at least, three major Marseilleviridae groups. These observations provide a new perspective on Marseilleviridae lineages organization, which will be valuable in future updates to the taxonomy of the family since the current official classification does not capture all the Marseilleviridae known diversity., Competing Interests: The authors declare no conflict of interest.

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 10 1 GC/g and 10 3 GC/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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil., Methods: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases., Results: Our RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in -1.18 PFU (confidence interval [CI] ranging from -2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20-30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97-100.00%), clinical specificity of 96.72% (95% CI, 88.65-99.60%), and overall accuracy of 98.00% (95% CI, 92.96-99.76%)., Discussion: Taken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

The hepatitis E virus is a major etiological agent of chronic hepatitis in immunosuppressed individuals. Seroprevalence in the liver transplantation setting varies according to the seroprevalence of the general population in different countries. This was a prospective cohort study of liver transplant recipients in southeastern Brazil. Recipients were systematically followed for one year, with the objective of determining the prevalence, incidence, and natural history of HEV infection in this population. We included 107 liver transplant recipients and 83 deceased donors. Positivity for anti-HEV IgG was detected in 10.2% of the recipients and in 9.7% of the donors. None of the patients tested positive for HEV RNA at baseline or during follow-up. There were no episodes of reactivation or seroconversion, even in cases of serological donor-recipient mismatch or in recipients with acute hepatitis. Acute and chronic HEV infections seem to be rare events in the region studied. That could be attributable to social, economic, and environmental factors. Our data indicate that, among liver transplant recipients, hepatitis E should be investigated only when there are elevated levels of transaminases with no defined cause, as part of the differential diagnosis of seronegative hepatitis after transplantation.

We examined the asymptomatic rates of SARS-CoV-2 infection during the Delta and Omicron waves in the city of São Paulo. Nasopharyngeal swabs were collected at strategic points of the city (open-air markets, bus terminals, airports) for SARS-CoV-2 RNA testing. Applying the questionnaire, the symptomatic individuals were excluded, and only asymptomatic cases were analyzed. During the Delta wave, a total of 4315 samples were collected, whereas 2372 samples were collected during the first Omicron wave. The incidence of the asymptomatic SARS-CoV-2 infection was 0.6% during the Delta wave and 0.8% during the Omicron wave. No statistical differences were found in the threshold amplification cycle. However, there was a statistical difference observed in the sublineage distribution between asymptomatic and symptomatic individuals. Our study determined the incidence of asymptomatic infection by monitoring individuals who remained symptom-free, thereby providing a reliable evaluation of asymptomatic SARS-CoV-2 carriage. Our findings reveal a relatively low proportion of asymptomatic cases, which could be attributed to our rigorous monitoring protocol for the presence of clinical symptoms. Investigating asymptomatic infection rates is crucial to develop and implement effective disease control strategies.

Introduction. Brazil was one of the most affected countries by the COVID-19 pandemic. Instituto Adolfo Lutz (IAL) is the reference laboratory for COVID-19 in São Paulo, the most populous state in Brazil. In April 2020, a secondary diagnostic pole named IAL-2 was created to enhance IAL's capacity for COVID-19 diagnosis. Hypothesis/Gap Statement. Public health laboratories must be prepared to rapidly respond to emerging epidemics or pandemics. Aim. To describe the design of IAL-2 and correlate the results of RT-qPCR tests for COVID-19 with secondary data on suspected cases of SARS-CoV-2 infection in the São Paulo state. Methodology. This is a retrospective study based on the analysis of secondary data from patients suspected of infection by SARS-CoV-2 whose clinical samples were submitted to real-time PCR after reverse transcription (RT-qPCR) at IAL-2, between 1 April 2020 and 8 March 2022. RT-qPCR Ct results of the different kits used were also analysed. Results. IAL-2 was implemented in April 2020, just over a month after the detection of the first COVID-19 case in Brazil. The laboratory performed 304,250 RT-qPCR tests during the study period, of which 98 319 (32.3 %) were positive, 205827 (67.7 %) negative, and 104 (0.03 %) inconclusive for SARS-CoV-2. RT-qPCR Ct values≤30 for E/N genes of SARS-CoV-2 were presented by 79.7 % of all the samples included in the study. Conclusion. IAL was able to rapidly implement a new laboratory structure to support the processing of an enormous number of samples for diagnosis of COVID-19, outlining strategies to carry out work with quality, using different RT-qPCR protocols.

Hepatitis D virus (HDV) is classified into 8 genotypes (1 to 8) and several subgenotypes. In Brazil, HDV-3 and HDV-1 predominate; however, most of the diagnosis efforts and molecular studies are directed to the area of endemicity of the Amazon Basin. Here, we determined the molecular epidemiological profile of circulating HDV in Brazilian HBsAg-positive patients between 2013 and 2015 in areas of endemicity and non-areas of endemicity. From 38 anti-HDV-positive individuals, 13 (34.2%) had detectable HDV-RNA and 11 (28.9%) were successfully sequenced. Partial HDAg (~320 nt) sequencing followed by phylogenetic analysis with reference sequences resulted in the identification of HDV-3 (9/11; 81.8%), HDV-5 (1/11; 9.1%), and HDV-8 (1/11; 9.1%). Most HDV-3 samples (8/9; 88.9%) were found in the endemic North region, while one was found in Central-West Brazil, a non-area of endemicity. HDV-5 and 8, genotypes native from African countries, were found in São Paulo, a cosmopolitan city from Southeast Brazil with a high circulation of immigrants. Phylogenetic analysis of HDV-8 strains indicated that the sample determined in our study, along with previously reported sequences from Brazil, formed a highly supported monophyletic clade, likely representing a putative novel HDV-8 subgenotype. IMPORTANCE Considered a neglected pathogen until the last 2 decades, an increase in the availability of genetic data of hepatitis D virus (HDV) strains around the world has been noticed recently, resulting in the proposition of different classifications. Our study aimed to determine the molecular epidemiological profile of HDV isolates circulating in areas of endemicity and non-areas of endemicity in Brazil. Based on the analyzed fragment, HDV-8 sequences clustered out of the clades formed by subgenotypes 8a and 8b might suggest the identification of a novel subgenotype, putatively designated subgenotype 8c. Our findings demonstrate the importance of continuous epidemiological surveillance to map HDV spread pathways and the introduction of imported variants. It also reinforces that as the amount of HDV genomes generated and reported increases, we will have changes in viral classification and, consequently, in our understanding of the dynamics of variability of this viral agent., Competing Interests: The authors declare no conflict of interest.

The main objective of this study was to investigate the dynamic of SARS-CoV-2 viral excretion in rectal swab (RS), saliva, and nasopharyngeal swab (NS) samples from symptomatic patients and asymptomatic contacts. In addition, in order to evaluate the replication potential of SARS-CoV-2 in the gastrointestinal (GI) tract and the excretion of infectious SARS-CoV-2 from feces, we investigated the presence of subgenomic nucleoprotein gene (N) mRNA (sgN) in RS samples and cytopathic effects in Vero cell culture. A prospective cohort study was performed to collect samples from symptomatic patients and contacts in Rio de Janeiro, Brazil, from May to October 2020. One hundred and seventy-six patients had samples collected at home visits and/or during the follow up, resulting in a total of 1633 RS, saliva, or NS samples. SARS-CoV-2 RNA was detected in 130 (73.9%) patients who had at least one sample that tested positive for SARS-CoV-2. The presence of replicating SARS-CoV-2 in RS samples, measured by the detection of sgN mRNA, was successfully achieved in 19.4% (6/31) of samples, whilst infectious SARS-CoV-2, measured by the generation of cytopathic effects in cell culture, was identified in only one RS sample. Although rare, our results demonstrated the replication capacity of SARS-CoV-2 in the GI tract, and infectious viruses in one RS sample. There is still a gap in the knowledge regarding SARS-CoV-2 fecal-oral transmission. Additional studies are warranted to investigate fecal or wastewater exposure as a risk factor for transmission in human populations.

Dengue fever is among the most significant public health concerns in Brazil. To date, the highest number of Dengue notifications in the Americas has been reported in Brazil, with cases accounting for a total number of 3,418,796 reported cases as of mid-December 2022. Furthermore, the northeastern region of Brazil registered the second-highest incidence of Dengue fever in 2022. Due to the alarming epidemiological scenario, in this study, we used a combination of portable whole-genome sequencing, phylodynamic, and epidemiological analyses to reveal a novel DENV-1 genotype V clade and the persistence of DENV-2 genotype III in the region. We further report the presence of non-synonymous mutations associated with non-structural domains, especially the NS2A (non-structural protein 2A), as well as describe synonymous mutations in envelope and membrane proteins, distributed differently between clades. However, the absence of clinical data at the time of collection and notification, as well as the impossibility of monitoring patients in order to observe worsening or death, restricts our possibility of correlating mutational findings with possible clinical prognoses. Together, these results reinforce the crucial role of genomic surveillance to follow the evolution of circulating DENV strains and understand their spread across the region through inter-regional importation events, likely mediated by human mobility, and also the possible impacts on public health and outbreak management.

Rotavirus (RVA) G8 is frequently detected in animals, but only occasionally in humans. G8 strains, however, are frequently documented in nations in Africa. Recently, an increase in G8 detection was observed outside Africa. The aims of the study were to monitor G8 infections in the Brazilian human population between 2007 and 2020, undertake the full-genotype characterization of the four G8P[4], six G8P[6] and two G8P[8] RVA strains and conduct phylogenetic analysis in order to understand their genetic diversity and evolution. A total of 12,978 specimens were screened for RVA using ELISA, PAGE, RT-PCR and Sanger sequencing. G8 genotype represented 0.6% (15/2434) of the entirely RVA-positive samples. G8P[4] comprised 33.3% (5/15), G8P[6] 46.7% (7/15) and G8P[8] 20% (3/15). All G8 strains showed a short RNA pattern. All twelve selected G8 strains displayed a DS-1-like genetic backbone. The whole-genotype analysis on a DS-1-like backbone identified four different genotype-linage constellations. According to VP7 analysis, the Brazilian G8P[8] strains with the DS-1-like backbone strains were derived from cattle and clustered with newly DS-1-like G1/G3/G9/G8P[8] strains and G2P[4] strains. Brazilian IAL-R193/2017/G8P[8] belonged to a VP1/R2.XI lineage and were grouped with bovine-like G8P[8] strains with the DS-1-like backbone strains detected in Asia. Otherwise, the Brazilian IAL-R558/2017/G8P[8] possess a "Distinct" VP1/R2 lineage never previously described and grouped apart from any of the DS-1-like reference strains. Collectively, our findings suggest that the Brazilian bovine-like G8P[8] strains with the DS-1-like backbone strains are continuously evolving and likely reassorting with local RVA strains rather than directly relating to imports from Asia. The Brazilian G8P[6]-DS-1-like strains have been reassorted with nearby co-circulating American strains of the same DS-1 genotype constellation. However, phylogenetic analyses revealed that these strains have some genetic origin from Africa. Finally, rather than being African-born, Brazilian G8P[4]-DS-1-like strains were likely imported from Europe. None of the Brazilian G8 strains examined here exhibited signs of recent zoonotic reassortment. G8 strains continued to be found in Brazil according to their intermittent and localized pattern, thus, does not suggest that a potential emergence is taking place in the country. Our research demonstrates the diversity of G8 RVA strains in Brazil and adds to the understanding of G8P[4]/P[6]/P[8] RVA genetic diversity and evolution on a global scale.

In the present study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was monitored in environmental samples from rural and vulnerable areas (a presidio, worker accommodation units, and river waters upstream and downstream of a rural community) from Minas Gerais State region, Southern Brazil, in August 2020. The sampling was performed prior to official declaration of the coronavirus disease (COVID-19) cases in those sites. SARS-CoV-2 RNA was detected in the presidio and workers accommodation units (3.0 × 10 4 virus genome copies (GC)/mL and 4.3 × 10 4 GC/mL of sewage, respectively). While SARS-CoV-2 was not detected in the river water upstream of the rural community, SARS-CoV-2 RNA was detected in downstream river waters (1.1 × 10 2 SARS-CoV-2 GC/mL). The results obtained in this study highlight the utility of SARS-CoV-2 monitoring in wastewater and human sewage as a non-invasive early warning tool to support health surveillance in vulnerable and remote areas, particularly in development countries., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Wastewater-based epidemiology (WBE) is a tool involving the analysis of wastewater for chemicals and pathogens at the community level. WBE has been shown to be an effective surveillance system for SARS-CoV-2, providing an early-warning-detection system for disease prevalence in the community via the detection of genetic materials in the wastewater. In numerous nation-states, studies have indicated the presence of SARS-CoV-2 in wastewater. Herein, we report the primary time-course monitoring of SARS-CoV-2 RNA in wastewater samples in São José do Rio Preto-SP/Brazil in order to explain the dynamics of the presence of SARS-CoV-2 RNA during one year of the SARS-CoV-2 pandemic and analyze possible relationships with other environmental parameters. We performed RNA quantification of SARS-CoV-2 by RT-qPCR using N1 and N2 targets. The proportion of positive samples for every target resulted in 100% and 96.6% for N1 and N2, respectively. A mean lag of -5 days is observed between the wastewater signal and the new SARS-CoV-2-positive cases reported. A correlation was found between the air and wastewater temperatures and therefore between the SARS-CoV-2 viral titers for N1 and N2 targets. We also observed a correlation between SARS-CoV-2 viral titers and media wastewater flow for the N1 target. In addition, we observed higher viral genome copies within the wastewater samples collected on non-rainy days for the N1 target. Thus, we propose that, based on our results, monitoring raw wastewater may be a broadly applicable strategy that might contribute to resolving the pressing problem of insufficient diagnostic testing; it may represent an inexpensive and early-warning method for future COVID-19 outbreaks, mainly in lower- and middle-income countries.

From 2016 to 2019, the largest outbreak caused by the Yellow Fever virus (YFV) in the 21st century in the Americas occurred in southeastern Brazil. A sylvatic cycle of transmission was reported near densely populated areas, such as the large metropolitan area of the city of São Paulo. Here, we describe the origin, spread, and movement of the YFV throughout the state of São Paulo. Whole-genome sequences were obtained from tissues of two patients who died due to severe yellow fever, during 2018-2019. Molecular analysis indicated that all analyzed tissues were positive for YFV RNA, with the liver being the organ with the highest amount of viral RNA. Sequence analysis indicates that genomes belonged to the South American genotype I and were grouped in the epidemic clade II, which includes sequences from the states of Goiás, Minas Gerais, and São Paulo of previous years. The analysis of viral dispersion indicates that the outbreak originated in Goiás at the end of 2014 and reached the state of São Paulo through the state of Minas Gerais after 2016. When the virus reached near the urban area, it spread towards both the east and south regions of the state, not establishing an urban transmission cycle in the metropolitan region of São Paulo. The virus that moved towards the east met with YFV coming from the south of the state of Rio de Janeiro, and the YFV that was carried to the south reached the Brazilian states located in the south region of the country., Competing Interests: The authors have declared that no competing interests exist.

We adopted the reverse-transcriptase-loop-mediated isothermal amplification (RT-LAMP) to detect severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) in patient samples. Two primer sets for genes N and Orf1ab were designed to detect SARS-CoV-2, and one primer set was designed to detect the human gene Actin. We collected prospective 138 nasopharyngeal swabs, 70 oropharyngeal swabs, 69 salivae, and 68 mouth saline wash samples from patients suspected to have severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 to test the RT-LAMP in comparison with the gold standard technique reverse-transcription quantitative polymerase chain reaction  (RT-qPCR). The accuracy of diagnosis using both primers, N5 and Orf9, was evaluated. Sensitivity and specificity for diagnosis were 96% (95% confidence interval [CI]: 87-99) and 85% (95% CI: 76-91) in 138 samples, respectively. Accurate diagnosis results were obtained only in nasopharyngeal swabs processed via extraction kit. Accurate and rapid diagnosis could aid coronavirus disease 2019 (COVID-19) pandemic management by identifying, isolating, and treating patients rapidly., (© 2022 Wiley Periodicals LLC.)

RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario., (© 2022. The Author(s).)

Background: Dozens of commercial RT-qPCR kits for SARS-CoV-2 detection are available with or without Emergency Use Authorization (EUA) by FDA or other regulatory agencies., Objective: We evaluated the clinical performance of two SARS-CoV-2 RT-PCR kits designed and produced in South America, "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador), for RT-qPCR SARS-CoV2 detection using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique., Results: We report a great clinical performance and analytical sensitivity for the two South American kits with sensitivity values of 96.4 and 100%, specificity of 100% and limit of detection in the range of 10 copies/uL of RNA extraction., Conclusions: "COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits are reliable SARS-CoV-2 tests made in South America that have been extensively used in Uruguay, Argentina, Brazil, Bolivia and Ecuador. These locally produced SARS-CoV-2 tests have contributed to overcome supply shortages and reduce diagnosis cost, while maintaining the high quality standards of FDA EUA commercially available kits. This approach could be extended for other diagnostic products to improve infectious diseases surveillance at middle and low income countries beyond COVID-19 pandemic., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The authors of this study are employees of "Universidad de Las Américas". This institution is enrolled in the commercialization "ECUGEN SARS-CoV-2 RT-PCR kit", one of the kits evaluated on this study.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution., Competing Interests: The authors have declared that no competing interests exist.

We conducted a systematic review and meta-analysis of studies assessing HCV infection rates in haemodialysis patients in Brazil (Prospero CRD #42021275068). We included studies on patients under haemodialysis, comprising both convenience samples and exhaustive information from selected services. Patients underwent HCV serological testing with or without confirmation by HCV RNA PCR. Exclusion criteria were the following: absence of primary empirical information and studies without information on their respective settings, study year, accurate infection rates, or full specification of diagnostic tests. Studies with samples ≤ 30 and serial assessments with repeated information were also excluded. Reference databases included PubMed, LILACS, Scopus, and Web of Science for the period 1989-2019. A systematic review was carried out, followed by two independent meta-analyses: (i) studies with data on HCV prevalence and (ii) studies with a confirmatory PCR (i.e., active infection), respectively. A comprehensive set of different methods and procedures were used: forest plots and respective statistics, polynomial regression, meta-regression, subgroup influence, quality assessment, and trim-and-fill analysis. 29 studies and 11,290 individuals were assessed. The average time patients were in haemodialysis varied from 23.5 to 56.3 months. Prevalence of HCV infection was highly heterogeneous, with a pronounced decrease from 1992 to 2001, followed by a plateau and a slight decrease in recent years. The summary measure for HCV prevalence was 34% (95% CI 26-43%) for studies implemented before 2001. For studies implemented after 2001, the corresponding summary measure was 11% (95% CI 8-15%). Estimates for prevalence of active HCV infection were also highly heterogeneous. There was a marked decline from 1996 to 2001, followed by a plateau and a slight increase after 2010. The summary measure for active HCV infection was 19% (95% CI 15-25%) in studies carried out before 2001. For studies implemented after 2001, the corresponding summary measure was 9% (95% CI 6-13%). Heterogeneity was pervasive, but different analyses helped to identify its underlying sources. Besides the year each study was conducted, the findings differed markedly between geographic regions and were heavily influenced by the size of the studies and publication biases. Our systematic review and meta-analysis documented a substantial decline in HCV prevalence among Brazilian haemodialysis patients from 1992 to 2015. CKD should be targeted with specific interventions to prevent HCV infection, and if prevention fails, prompt diagnosis and treatment. Although the goal of HCV elimination by 2030 in Brazil remains elusive, it is necessary to adopt measures to achieve micro-elimination and to launch initiatives towards targeted interventions to curb the spread of HCV in people with CKD, among other high-risk groups. This is of particular concern in the context of a protracted COVID-19 pandemic and a major economic and political crisis., (© 2022. The Author(s).)

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

Hepatitis A virus (HAV) is the major cause of enterically transmitted infectious hepatitis. Between 2016 and 2017, the number of confirmed cases of hepatitis A virus (HAV) increased from 64 to 786 in São Paulo affecting mainly adults aged between 18 and 39 years (80%) and males (88%). To support epidemiological surveillance, the present study monitored the presence of HAV in urban sewage samples collected bimonthly for 1 year (November 2017-November 2018) in the central region of the city, where most of cases were detected. Sewage samples were concentrated by polyethylene glycol precipitation and HAV RNA was quantified by RT-qPCR. Nucleotide sequencing targeting the VP1/2A junction region was carried out to genotype the HAV strains. HAV was detected in 76.9% (40/52) of the samples, with a geometric mean viral load of 5.09 × 10 4 (± SD 4.51 × 10 5 ) genome copies (GC/L) (Mauá Street) and 5.27 × 10 4 (± SD 1.26 × 10 6 ) GC/L (Prestes Maia Avenue). Of the 40 positive samples, 8 were typed as HAV subgenotype IA [100% nucleotide (nt) identity with HAV strain VRD&#95;521&#95;2016]. Highest homology was obtained with sequences from European countries (Italy, Spain) and Israel, all of which had reported recent HAV outbreaks associated with men who have sex with men. Our results highlight that wastewater surveillance is a useful tool to support investigating HAV outbreaks in the community, including circulating genotypes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Since the beginning of the SARS-CoV-2 spread in Brazil, few studies have been published analysing the variability of viral genome. Herein, we described the dynamic of SARS-CoV-2 strains circulating in Brazil from May to September 2020, to better understand viral changes that may affect the ongoing pandemic. Our data demonstrate that some of the mutations identified are currently observed in variants of interest and variants of concern, and emphasize the importance of studying previous periods in order to comprehend the emergence of new variants. From 720 SARS-CoV-2 genome sequences, we found few sites under positive selection pressure, such as the D614G (98.5 %) in the spike, that has replaced the old variant; the V1167F in the spike (41 %), identified in the P.2 variant that emerged from Brazil during the period of analysis; and I292T (39 %) in the N protein. There were a few alterations in the UTRs, which was expected, however, our data suggest that the emergence of new variants was not influenced by mutations in UTR regions, since it maintained its conformational structure in most analysed sequences. In phylogenetic analysis, the spread of SARS-CoV-2 from the large urban centres to the countryside during these months could be explained by the flexibilization of social isolation measures and also could be associated with possible new waves of infection. These results allow a better understanding of SARS-CoV-2 strains that have circulated in Brazil, and thus, with relevant infomation, provide the potential viral changes that may have affected and/or contributed to the current and future scenario of the COVID-19 pandemic.

We quantified the presence of SARS-CoV-2 RNA in the air of different hospital settings and the autopsy room of the largest medical centre in Sao Paulo, Brazil. Real-time reverse-transcription PCR was used to determine the presence of the envelope protein of SARS-CoV-2 and the nucleocapsid protein genes. The E-gene was detected in 5 out of 6 samples at the ICU-COVID-19 ward and in 5 out of 7 samples at the ward-COVID-19. Similarly, in the non-dedicated facilities, the E-gene was detected in 5 out of 6 samples collected in the ICU and 4 out of 7 samples in the ward. In the necropsy room, 6 out of 7 samples were positive for the E-gene. When both wards were compared, the non-COVID ward presented a significantly higher concentration of the E-gene than in the COVID-19 ward (p = 0.003). There was no significant difference in E-gene concentration between the ICU-COVID-19 and the ICU (p = 0.548). Likewise, there was no significant difference among E-gene concentrations found in the autopsy room versus the ICUs and wards (dedicated or not) (p = 0.245). Our results show the widespread presence of aerosol contamination in different hospital units., (© 2021. The Author(s).)

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations., Competing Interests: The authors have declared that no competing interests exist.

SARS-CoV-2, the novel Coronavirus, was first detected in Wuhan, China, in December 2019, and has since spread rapidly, causing millions of deaths worldwide. As in most countries of the world, in Brazil, the consequences of the COVID-19 pandemic have been catastrophic. Several studies have reported the fecal shedding of SARS-CoV-2 RNA titers from infected symptomatic and asymptomatic individuals. Therefore, the quantification of SARS-CoV-2 in wastewater can be used to track the virus spread in a population. In this study, samples of untreated wastewater were collected for 44 weeks at five sampling sites in the ABC Region (São Paulo, Brazil), in order to evaluate the SARS-CoV-2 occurrence in the sewerage system. SARS-CoV-2 RNA titers were detected throughout the period, and the concentration ranged from 2.7 to 7.7 log 10 genome copies.L -1 , with peaks in the last weeks of monitoring. Furthermore, we observed a positive correlation between the viral load in wastewater and the epidemiological/clinical data, with the former preceding the latter by approximately two weeks. The COVID-19 prevalence for each sampling site was estimated via Monte-Carlo simulation using the wastewater viral load. The mean predicted prevalence ranged 0.05 to 0.38%, slightly higher than reported (0.016 ± 0.005%) in the ABC Region for the same period. These results highlight the viability of the wastewater surveillance for COVID-19 infection monitoring in the largest urban agglomeration in South America. This approach can be especially useful for health agencies and public decision-makers in predicting SARS-CoV-2 outbreaks, as well as in local tracing of infection clusters., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 (3.4×10 5 GC/g of stool) and GII (1.9×10 7 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.

Hepatitis B (HBV) and delta (HDV) viruses are endemic in the Amazon region, but vaccine coverage against HBV is still limited. People who use illicit drugs (PWUDs) represent a high-risk group due to common risk behavior and socioeconomic factors that facilitate the acquisition and transmission of pathogens. The present study assessed the presence of HBV and HBV-HDV co-infection, identified viral sub-genotypes, and verified the occurrence of mutations in coding regions for HBsAg and part of the polymerase in HBV-infected PWUDs in municipalities of the Brazilian states of Amapá and Pará, in the Amazon region. In total, 1074 PWUDs provided blood samples and personal data in 30 municipalities of the Brazilian Amazon. HBV and HDV were detected by enzyme-linked immunosorbent assay and polymerase chain reaction. Viral genotypes were identified by nucleotide sequencing followed by phylogenetic analysis, whereas viral mutations were analyzed by specialized software. High rates of serological (32.2%) and molecular (7.2%) markers for HBV were detected, including cases of occult HBV infection (2.5%). Sub-genotypes A1, A2, D4, and F2a were most frequently found. Escape mutations due to vaccine and antiviral resistance were identified. Among PWUDs with HBV DNA, serological (19.5%) and molecular (11.7%) HDV markers were detected, such as HDV genotypes 1 and 3. These are worrying findings, presenting clear implications for urgent prevention and treatment needs for the carriers of these viruses.

The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer's instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

Bovine viral diarrhea virus (BVDV) is a major pathogen in cattle herds. Considering the epidemiological importance of pestiviruses and the process of wild boar invasion in Brazil, this study aimed to investigate the presence of BVDV in free-living boars. Forty-nine free-living wild boars were collected by exotic wildlife controller agents in 2017 and 2018. The presence of BVDV antibodies was evaluated in 42 serum samples using the virus neutralization test, and the detection of BVDV RNA was performed from the 5'UTR genomic region by RT-PCR assay in 49 lung tissue samples followed by sequencing of amplicons. BVDV neutralizing antibodies in serum were not identified in any of the evaluated samples. However, 3/49 (6.12%) lung samples were positive for BVDV RNA and classified one as BVDV-1a and two as 1d subgenotype. This report identified BVDV RNA in free-living wild boars and these results should be considered in BVDV control programs, especially in extensive beef cattle rearing systems.

Most dengue virus (DENV) infections remain asymptomatic. This increases the risk of DENV transfusion transmission (TT-DENV) during outbreaks. We evaluated DENV viremia in 8475 blood donations assembled in minipools for the presence of DENV RNA. The tested samples were obtained between February and May, 2016, during a large DENV outbreak in Ribeirão Preto city, northeast region of the São Paulo State, Brazil. The DENV RNA + samples were serotyped and screened for DENV NS1. We also tested a significant number of plasma samples (n = 372) to estimate the DENV seroprevalence among blood donors in the region. We detected three DENV RNA + samples in the tested blood donations (n = 3/8475, 0.04%). From these, two samples were further serotyped as DENV-1 and one sample as DENV-2. All DENV RNA positive samples were negative for anti-DENV IgG, indicating the presence of primary acute infection. Moreover, two of the DENV RNA + samples were also NS1 antigen positive (antigenemia). The anti-DENV IgG seroprevalence among blood donor population was 50.8% (n = 189/372). Our results are in accordance with the presence of DENV primary infection in blood donors which can lead to transfusion transmission of the infection to recipients. Measures to exclude such donors should be adopted to prevent TT-DENV., (© 2020 Wiley Periodicals LLC.)

Enterovirus (EV) is commonly associated with central nervous system (CNS) syndromes. Recently, gastroenteric viruses, including rotavirus (RVA), human astrovirus (HAstV), and norovirus (NoV), have also been associated with CNS neurological disorders. The aim of the present study was to investigate the presence of EV, RVA, HAst, and NoV associated to CNS infections with undiagnosed etiology in Northwest region of São Paulo State, Brazil, and to conduct the molecular characterization of the positive samples detected. A total of 288 cerebrospinal fluid samples collected from July to December 2017 were tested for EV and NoV by quantitative real-time polymerase chain reaction (RT-qPCR), HAstV by conventional RT-PCR, and RVA by enzyme-linked immunosorbent assay. Positive-EV samples were inoculated in cells lines, amplified by RT-PCR and sequenced. RVA, NoV, and HAstV were not detected. EV infection was detected in 5.5% (16/288), and five samples successful genotyped: echovirus 3 (E3) (1/5), coxsackie virus A6 (CVA6) (1/5), and coxsackie virus B4 (CVB4) (3/5). Meningitis was the main syndrome observed (12/16; 75%). CVA6, CVB4, and E3 were identified associated with aseptic meningitis. Reports of CVA6 associated with aseptic meningitis are rare, E3 had not been previously reported in Brazil, and epidemiological data on CVB4 in the country is virtually unknown. The present investigation illustrates the circulation of diverse EV types in a small regional sample set and in a short period of time, highlighting the importance of an active EV surveillance system in CNS infections. Enhanced understanding of undiagnosed CNS infections will assist in public health and health care planning., (© 2020 Wiley Periodicals LLC.)

Respiratory syncytial virus (RSV) is a relevant cause of acute respiratory infection among children. Viral replication and immune conditions may account for severity. RSV viral load (VL) was assessed in 486 children (290 hospitalized and 196 from primary care) attended at São Paulo Hospital from 2009 to 2013. VL was calculated by real-time reverse transcription-polymerase chain reaction and expressed in Log 10 RNA copies/mL. Coinfection with rhinovirus (RV) and influenza A virus was also tested. Young children (<1 year of age) had a higher mean VL than older children at primary care (6.35 and 4.34 Log 10 RNA copies/mL, respectively; P = .0006). Conversely, hospitalized children ≥2 years of age, presented higher mean VL compared with the same age children of primary care (6.10 and 4.26, respectively; P = .0024). RV was the most codetected virus in RSV positive patients (20% from primary care and 14% in hospitalized), and influenza A virus was found in 11% of primary care and 0.4% in hospitalized children with RSV, without RSV VL association (P = .2903). These findings may guide future therapies and immunization policies considering the role of viral load on clinical presentation among older hospitalized children and also the change of infection transmissions., (© 2020 Wiley Periodicals LLC.)

Flaviviruses as West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilhéus virus (ILHV), and Rocio virus (ROCV) are previously reported in different Brazilian regions, but studies in Southern Brazil are still scarce. To improve the information regarding flaviviruses in Southern Brazil, horse serum samples were analyzed using RT-qPCR and a commercial ELISA-Ab against WNV followed by PRNT 75 . All 1000 samples analyzed by real-time RT-PCR resulted negative. The 465 subsampled samples were analyzed by a commercial ELISA-Ab against WNV, and the 18.5% (86/465) positive samples were further analyzed by PRNT 75 . In the PRNT 75 , 13/86 and 2/86 horses were positive for SLEV and WNV, respectively. It was observed that 5.8% (13/226) of the farms presented at least one positive animal for SLEV in PRNT 75 , whereas 0.9% (2/226) for WNV. Apart from the lower seroprevalences identified when compared to data previously reported in other Brazilian regions, our results suggest that public health professionals must be aware of the presence of these potential zoonotic pathogens.

This study is aimed at detecting Feline paramyxovirus (FPaV) and Feline morbillivirus (FeMV) in 35 urine samples from domestic cats, collected in 2019, with or without clinical signs of uropathies using a reverse transcription-polymerase chain reaction (RT-PCR) followed by semi-nested polymerase chain reaction (SN-PCR) assays to amplify a partial paramyxovirus L gene. Eight (22.9%) out of the 35 urine samples were positive for paramyxoviruses. Sequencing and phylogenetic analyses revealed that three samples were positive for FPaV, four samples were positive for FeMV, and it was not possible to determine which virus was present in one RT-SN-PCR positive urine sample. FPaV strains showed 100% nucleotide (nt) identity with each other and 97% nt identity with a Japanese 163 FPaV strain. The FeMV strains showed 85.9% nt identity with each other; three strains were similar to previously described Brazilian FeMV strains, and one strain clustered in a different branch of the phylogenetic tree together with the first described Chinese FeMV strain. This study provides the first description of FPaV strains in cats from Brazil and provides new information about the molecular characteristics of FPaV and FeMV strains circulating in domestic cats in Brazil.

Background: Respiratory Syncytial Virus (RSV) is the main cause of pediatric morbidity and mortality. The complex evolution of RSV creates a need for worldwide surveillance, which may assist in the understanding of multiple viral aspects., Objectives: This study aimed to investigate RSV features under the Brazilian Influenza Surveillance Program, evaluating the role of viral load and genetic diversity in disease severity and the influence of climatic factors in viral seasonality., Methodology: We have investigated the prevalence of RSV in children up to 3 years of age with severe acute respiratory infection (SARI) in the state of Espirito Santo (ES), Brazil, from 2016 to 2018. RT-qPCR allowed for viral detection and viral load quantification, to evaluate association with clinical features and mapping of local viral seasonality. Gene G sequencing and phylogenetic reconstruction demonstrated local genetic diversity., Results: Of 632 evaluated cases, 56% were caused by RSV, with both subtypes A and B co-circulating throughout the years. A discrete inverse association between average temperature and viral circulation was observed. No correlation between viral load and disease severity was observed, but children infected with RSV-A presented a higher clinical severity score (CSS), stayed longer in the hospital, and required intensive care, and ventilatory support more frequently than those infected by RSV-B. Regarding RSV diversity, some local genetic groups were observed within the main genotypes circulation RSV-A ON1 and RSV-B BA, with strains showing modifications in the G gene amino acid chain., Conclusion: Local RSV studies using the Brazilian Influenza Surveillance Program are relevant as they can bring useful information to the global RSV surveillance. Understanding seasonality, virulence, and genetic diversity can aid in the development and suitability of antiviral drugs, vaccines, and assist in the administration of prophylactic strategies., Competing Interests: The authors have declared that no competing interests exist.

Brazil experienced a large dengue virus (DENV) epidemic in 2019, highlighting a continuous struggle with effective control and public health preparedness. Using Oxford Nanopore sequencing, we led field and classroom initiatives for the monitoring of DENV in Brazil, generating 227 novel genome sequences of DENV1-2 from 85 municipalities (2015-2019). This equated to an over 50% increase in the number of DENV genomes from Brazil available in public databases. Using both phylogenetic and epidemiological models we retrospectively reconstructed the recent transmission history of DENV1-2. Phylogenetic analysis revealed complex patterns of transmission, with both lineage co-circulation and replacement. We identified two lineages within the DENV2 BR-4 clade, for which we estimated the effective reproduction number and pattern of seasonality. Overall, the surveillance outputs and training initiative described here serve as a proof-of-concept for the utility of real-time portable sequencing for research and local capacity building in the genomic surveillance of emerging viruses.

The envelope glycoprotein E2 of pestiviruses is a major target for neutralizing antibodies. In this study, we analyzed the E2 DA domain of 43 pestiviruses from Southern Brazil. The isolates were identified as Bovine viral diarrhea virus (BVDV) subtypes 1a and 1b or BVDV-2b. Compared to reference strains, the BVDV-1 and -2 isolates had four and two mutations in the DA domain, respectively. All BVDV-2 isolates had a deletion of residues 724 and 725. All mutated amino acids in the BVDV isolates had the same aa substitution, and all were in previously identified antibody binding sites. It is possible that an immunity-mediated selection is acting on the pestiviruses circulating in Southern Brazil.

Echoviruses (E) are a diverse group of viruses responsible for various pathological conditions in humans including aseptic meningitis, myocarditis, and acute flaccid paralysis. The detection and identification of echovirus genotypes in clinical samples is challenging due to its high genetic diversity. Here, we report the complete genome sequences of nine echoviruses, obtained by next-generation sequencing of 238 fecal samples from individuals with gastroenteritis in regions of Brazil. Detected viruses were classified into six genotypes: Three E1 sequences (BRA/TO-028, BRA/TO-069 and BRA/TO-236), one E3 (BRA/TO-018), one E11 (BRA/TO-086), one E20 (BRA/TO-016), two E29 (BRA/TO-030 and BRA/TO-193), and one E30 sequence (BRA/TO-032). Phylogenetic analysis indicated that the echoviruses E1 and E29 circulating in Brazil are divergent from strains circulating worldwide. The genotype diversity identified in our study may under-represent the total echovirus diversity in Brazil because of the small sample size and the restricted geographical distribution covered by the survey.

Dengue virus, the etiological agent of dengue fever (DF) occurs in four genetically distinct serotypes (DENV1-4), being transmitted by female Aedes mosquitoes. DF incidence is increasing in Brazil, following vector dispersal, proliferation and DENV serotypes introduction, co-circulation and substitution. Medium- and small-sized cities in Sao Paulo State, such as Marilia (Midwest region), have been affected by huge epidemics. To understand the evolution of DENV epidemics in medium-sized cities, in this study a historical data on DENV incidence (2000-2015) in Marilia, was evaluated. Previous studies disclosed regional and specific DF outcomes associated with 2007 outbreak in that city, when co-circulating DENV1 and DENV3 presented different hematological profiles. In this study, characteristics of 2007 DENV epidemics were compared to the epidemiological, hematological and demographic outlines of the major outbreak of DENV1 in Marilia in 2015. DENV1 genetic diversity was assessed through capsid and pre-membrane junction encoding gene (CprM) sequencing. The results revealed circulation of DENV1 serotype from 2007 to 2015, with epidemics occurring every three-years until 2013 and then, increasing yearly. There were significant differences in hematological profiles of DENV1 patients between 2015 and 2007. CprM showed DENV1 genetic variability in 2015, contrasting with the unique sequence pattern in 2007. These results reinforce the regional and temporal characteristics of DENV epidemics that need local public health research to improve care for people and to limit the spread of new serotypes/genotypes to uninfected areas.