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 Yellow Fever (YF) vaccine is produced by the inoculation of embryonated chicken eggs with YF17DD virus on the ninth day of development. Full embryos are collected on the twelfth day of development for vaccine formulation. Skeletal muscle tissue is the main site where biosynthesis of viral particles occurs. OBJECTIVES This study aimed to analyse the experimental infection of skeletal muscle cells of chicken embryos by the 17DD Yellow Fever virus (YFV) in vivo and in vitro. METHODS Chicken embryos infected with YF17DD virus were analysed by immunofluorescence using confocal and super-resolution microscopes. Primary cultures of skeletal muscle cells of non-infected chicken embryos were evaluated for susceptibility and permissiveness to YF17DD virus using different protocols. This evaluation was performed based on morphological, viral titration, molecular biology, and colorimetric techniques. FINDINGS The present work phenotypically characterises embryonic chicken skeletal muscle cells as myogenic precursors expressing the Pax7 transcription factor in some cases. We demonstrated that these cells are susceptible to in vitro infection at different multiplicities of infection (MOIs), reproducing the same infection pattern observed in vivo. Furthermore, myogenic precursors and myoblasts are preferred infection targets, but establishment of infection does not depend on the presence of these cells. The peak of viral production occurred at 48 hpi, with decay occurring 72 hpi, when the cytopathic effect can be observed. MAIN CONCLUSIONS In conclusion, the primary culture of chicken skeletal muscle cells is a good model for studying muscle cells infected with YF17DD virus. This culture system displays satisfactory emulation of the in vitro phenomenon observed, contributing to our understanding of virus infection dynamics and leading to the development of alternative methods of vaccine production.

BACKGROUND In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.