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.

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.

Coronavirus disease 2019 (COVID-19) surveillance, in Brazil, initiated shortly after its description, in China. Our aim was to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional pathogens in samples from the initial phase of the outbreak in Brazil, from late February to late March. From 707 samples analysed, 29 (4.1%) were SARS-CoV-2 positive. Fever and cough were their most prevalent symptoms. Co-detection of rhinovirus was observed in 2 (6.9%) cases. Additional pathogens were identified in 66.1% of the SARS-CoV-2 negative cases, mainly rhinovirus and influenza A(H1N1)pdm09. Thus, we emphasise the importance of differential diagnosis in COVID-19 suspected cases.

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.

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.

During the influenza pandemic of 2009, the A(H1N1)pdm09, A/H3N2 seasonal and influenza B viruses were observed to be co-circulating with other respiratory viruses. To observe the epidemiological pattern of the influenza virus between May 2009-August 2011, 467 nasopharyngeal aspirates were collected from children less than five years of age in the city of Salvador. In addition, data on weather conditions were obtained. Indirect immunofluorescence, real-time transcription reverse polymerase chain reaction (RT-PCR), and sequencing assays were performed for influenza virus detection. Of all 467 samples, 34 (7%) specimens were positive for influenza A and of these, viral characterisation identified Flu A/H3N2 in 25/34 (74%) and A(H1N1)pdm09 in 9/34 (26%). Influenza B accounted for a small proportion (0.8%) and the other respiratory viruses for 27.2% (127/467). No deaths were registered and no pattern of seasonality or expected climatic conditions could be established. These observations are important for predicting the evolution of epidemics and in implementing future anti-pandemic measures.