BACKGROUND The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) has changed unevenly over time around the world. Although whole genome sequencing is the gold standard for virus characterisation, the discovery of alpha VOC causing spike gene target failure (SGTF) result, when tested using an reverse transcription real-time polymerase chain reaction (RT-qPCR) assay, has provided a simple tool for tracking the frequencies of variants. OBJECTIVES The aim of this study was to investigate if a multiplex RT-qPCR assay (BioM 4Plex VOC) could be used to detect SARS-CoV-2 and to perform a VOC screening test in a single reaction tube. Here, we present the multicentre study evaluating this assay. METHODS Twelve laboratories have participated in the multicentre study. The BioM 4Plex VOC was distributed to them with detailed instructions of how to perform the test. They were asked to test the BioM 4Plex VOC in parallel with their routine Commercial SARS-CoV-2 diagnostic assay. Additionally, they were requested to select SARS-CoV-2-positive samples with genome sequenced and lineage definition according to PANGO lineage classification. FINDINGS The BioM 4Plex VOC and commercial RT-PCR assay are equally effective in detecting SARS-CoV-2. Results revealed a specificity of 96.5-100% [95% confidence interval (CI)], a sensitivity of 99.8-100% (95% CI), and an accuracy of 99.8-100% (95% CI). A 99% concordance rate was found between results from the BioM 4Plex VOC and that from available genome sequencing data. MAIN CONCLUSIONS The BioM 4Plex VOC provides an effective solution to detect SARS-CoV-2 infections and screening for VOCs in a single reaction. It is a straightforward method to help us monitor the frequency and distribution of VOCs and develop strategies to better cope with the pandemics.

Abstract This study aimed to evaluate the application of thermosonication for the viability of Staphylococcus aureus and the antioxidant activity of HM. The US effect on the S. aureus was measured by counting viable cells in discarded HM immediately before and after different treatments varying time (1, 5 and 10 minutes) and temperature (20, 30, 40, 50 and 60 °C) through surface plating on standard agar. The antioxidant activity evaluation was carried out by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging method. With Regarding microbiological quality, it can be concluded that treatment with US at 60 °C for 10 minutes was the most effective in terms of reducing the viability of S. aureus, in addition to presenting a significant increase in the content of antioxidants when compared to raw and pasteurized milk samples.

BACKGROUND The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has become a major concern contributing to increased morbidity and mortality worldwide. OBJECTIVES Here we describe the replacement of the Gamma variant of concern (VOC) with Delta in the western Brazilian Amazon. METHODS In this study, we analysed 540 SARS-CoV-2 positive samples determined by qualitative real-time RT-PCR selected in the state of Rondônia between June and December 2021. The positive cohort was sequenced through next-generation sequencing (NGS) and each sample was quantified using real-time RT-qPCR, the whole genome sequence was obtained, SARS-CoV-2 lineages were classified using the system Pango and the maximum likelihood (ML) method was used to conduct phylogenetic analyses. FINDINGS A total of 540 high-quality genomes were obtained, where the Delta VOC showed the highest prevalence making up 72%, with strain AY.43 being the most abundant, while the Gamma VOC was present in 28%, where the P.1 strain was the most frequent. In this study population, only 32.96% (178/540) had completed the vaccination schedule. MAIN CONCLUSIONS This study highlighted the presence of Gamma and Delta variants of SARS-CoV-2 in RO. Furthermore, we observed the replacement of the Gamma VOC with the Delta VOC and its lineages.

BACKGROUND The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.33-derived lineage named N.9 was described recently in Brazil and it’s considered a potential variant of interest (VOI) due to the presence of E484K substitution at the receptor-binding domain (RBD) of the Spike (S) protein. OBJECTIVE To describe the first detection of variant N.9 in Rio de Janeiro State. METHODS SARS-CoV-2 N.9 was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), whole-genome sequencing and phylogenetic analysis. FINDINGS Here, we report two SARS-CoV-2 N.9 lineage strains in Rio de Janeiro. One of them had only the E484K substitution of the six N.9 lineage-defining mutations. Other three strains pre-defined as N.9 have the same genomic profile. These four strains are grouped within the B.1.1.33 lineage and basal to the N.9 lineage in our phylogenetic analysis, and we call them “N.9-like/B.1.1.33 + E484K”. MAIN CONCLUSIONS The phylogenetic analysis shows four independent introductions of N.9 in the state of Rio de Janeiro in October and December 2020, January and March 2021. SARS-CoV-2 N.9 dissemination in the Rio de Janeiro could have been limited by the emergence and dominance of other variants, mainly by the lineage P.2 VOI Zeta that emerged in the same period and co-circulated with N.9, as observed in the neighboring State of São Paulo.

BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.

The 2009 pandemic influenza A virus outbreak led to the systematic use of the neuraminidase (NA) inhibitor oseltamivir (OST). Consequently, OST-resistant strains, carrying the mutation H275Y, emerged in the years after the pandemics, with a prevalence of 1-2%. Currently, OST-resistant strains have been found in community settings, in untreated individuals. To spread in community settings, H275Y mutants must contain additional mutations, collectively called permissive mutations. We display the permissive mutations in NA of OST-resistant A(H1N1)pdm09 virus found in Brazilian community settings. The NAs from 2013 are phylogenetically distinct from those of 2012, indicating a tendency of positive selection of NAs with better fitness. Some previously predicted permissive mutations, such as V241I and N369K, found in different countries, were also detected in Brazil. Importantly, the change D344N, also predicted to compensate loss of fitness imposed by H275Y mutation, was found in Brazil, but not in other countries in 2013. Our results reinforce the notion that OST-resistant A(H1N1)pdm09 strains with compensatory mutations may arise in an independent fashion, with samples being identified in different states of Brazil and in different countries. Systematic circulation of these viral strains may jeopardise the use of the first line of anti-influenza drugs in the future.

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.