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Background We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), Gamma (P.1), in January 2021. The VOC Gamma, a ramification of the B.1.1.28 lineage first detected in Manaus (northern Brazil), has grown rapidly since December 2020 and was thought to be responsible for the deadly second wave of COVID-19 throughout Brazil. Methods The 333 genomic sequences of SARS-CoV-2 from March 2020 to April 2021 were generated as part of the genomic surveillance carried out by Fiocruz in Brazil Genomahcov Fiocruz. SARS-CoV-2 sequencing was performed using representative samples from all geographic areas of Paraná. Phylogenetic analyses were performed using the 333 genomes also included other SARS-CoV-2 genomes from the state of Paraná and other states in Brazil that were deposited in the GISAID. In addition, the time-scaled phylogenetic tree was constructed with up to 3 random sequences of the Gamma variant from each state in Brazil in each month of 2021. In this analysis we also added the sequences identified as the B.1.1.28 lineage of the Amazonas state and and the Gamma-like-II (P.1-like-II) lineage identified in different regions of Brazil. Results Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC Gamma lineage by WHO/PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage Gamma-like-II. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC Gamma from Paraná in March and April 2021.Finally, we analyzed the correlation between the lineage and the Gamma variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná. Conclusions Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the Gamma strain, as well as a high frequencies of the Gamma-like-II lineage and the S:E661D mutation. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC Gamma in Paraná.

The COVID-19 epidemic in Brazil experienced two major country-wide lineage replacements, the first driven by the lineage P.2, formerly classified as variant of interest (VOI) Zeta in late 2020 and the second by the variant of concern (VOC) Gamma in early 2021. To better understand how these SARS-CoV-2 lineage turnovers occurred in Brazil, we analyzed 11,724 high-quality SARS-CoV-2 whole genomes of samples collected in different country regions between September 2020 and April 2021. Our findings indicate that the spatial dispersion of both variants in Brazil was driven by short and long-distance viral transmission. The lineage P.2 harboring Spike mutation E484K probably emerged around late July 2020 in the Rio de Janeiro (RJ) state, which contributed with most (∼50%) inter-state viral disseminations, and only became locally established in most Brazilian states by October 2020. The VOC Gamma probably arose in November 2020 in the Amazonas (AM) state, which was responsible for 60-70% of the inter-state viral dissemination, and the earliest timing of community transmission of this VOC in many Brazilian states was already traced to December 2020. We estimate that variant Gamma was 1.56-3.06 more transmissible than variant P.2 co-circulating in RJ and that the median effective reproductive number (Re) of Gamma in RJ and SP states (Re = 1.59-1.91) was lower than in AM (Re = 3.55). In summary, although the epicenter of the lineage P.2 dissemination in Brazil was the heavily interconnected Southeastern region, it displayed a slower rate of spatial spread than the VOC Gamma originated in the more isolated Northern Brazilian region. Our findings also support that the VOC Gamma was more transmissible than lineage P.2, although the viral Re of the VOC varied according to the geographic context.

Deemed a variant of concern by the World Health Organization on 15 June, the Lambda variant of SARS-CoV-2 is a growing epidemiological threat in several South American countries, and initial results suggest it exhibits increased infectivity and immune escape qualities. Here, we present evidence of its multiple introductions in Brazil.

The SARS-CoV-2 has infected almost 200 million people worldwide by July 2021 and the pandemic has been characterized by infection waves of viral lineages showing distinct fitness profiles. The simultaneous infection of a single individual by two distinct SARS-CoV-2 lineages provides a window of opportunity for viral recombination and the emergence of new lineages with differential phenotype. Several hundred SARS-CoV-2 lineages are currently well characterized but two main factors have precluded major coinfection/codetection analysis thus far: i) the low diversity of SARS-CoV-2 lineages during the first year of the pandemic which limited the identification of lineage defining mutations necessary to distinguish coinfecting viral lineages; and the ii) limited availability of raw sequencing data where abundance and distribution of intrasample/intrahost variability can be accessed. Here, we have put together a large sequencing dataset from Brazilian samples covering a period of 18 May 2020 to 30 April 2021 and probed it for unexpected patterns of high intrasample/intrahost variability. It enabled us to detect nine cases of SARS-CoV-2 coinfection with well characterized lineage-defining mutations. In addition, we matched these SARS-CoV-2 coinfections with spatio-temporal epidemiological data confirming their plausibility with the co-circulating lineages at the timeframe investigated. These coinfections represent around 0.61% of all samples investigated. Although our data suggests that coinfection with distinct SARS-CoV-2 lineages is a rare phenomenon, it is likely an underestimation and coinfection rates warrants further investigation.DATA SUMMARYThe raw fastq data of codetection cases are deposited on gisaid.org and correlated to gisaid codes: EPI_ISL_1068258, EPI_ISL_2491769, EPI_ISL_2491781, EPI_ISL_2645599, EPI_ISL_2661789, EPI_ISL_2661931, EPI_ISL_2677092, EPI_ISL_2777552, EPI_ISL_3869215. Supplementary data are available on https://doi.org/10.6084/m9.figshare.16570602.v1. The workflow code used in this study is publicly available on: https://github.com/dezordi/IAM_SARSCOV2.

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. Importantly, we detected the community transmission of different P.1 lineages bearing NTD indels ∆69-70 (which can impact several SARS-CoV-2 diagnostic protocols), ∆144 and ins214ANRN, and a new VOI N.10 derived from the B.1.1.33 lineage carrying three NTD deletions (∆141–144, ∆211, and ∆256–258). These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil generates new viral lineages that might be more resistant to antibody neutralization than parental variants of concern.

We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, Southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), P.1, in January 2021. Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC P.1 lineage by PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage P.1-like-II. An extended phylogenetic analysis, including SARS-CoV-2 genomes from all over Brazil, showed that the P.1-like-II lineage appears at a high frequency in the southern region of the country. The P.1-like-II lineage genomes share some, but not all, defining mutations of the VOC P.1. For instance, it has the previously described ORF1a:D2980H and N:P383 L unique mutations and the newly detected ORF1a:P1213 L and ORF1b:K2340N mutations. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC P.1 from Paraná in March and April 2021. We also report the identification of the S:W152C mutation in one genome from Paraná, classified as the N.10 variant. Finally, we analyzed the correlation between the lineage and the P.1 variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná. This analysis does not support an association between the P.1 variant prevalence and COVID-19 severity or age strata. Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the P.1 strain, as well as a high frequencies of the P.1-like-II lineage and the S:E661D mutations. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC P.1 in Paraná.

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies., Structural and functional analysis of the P.1 variant of SARS-CoV-2 from Brazil reveals less resistance to antibodies generated from natural infection or vaccination compared to another similar variant, B.1.351. A monoclonal antibody mAb 222 is able to neutralize all three variants (P.1, B.1.351 and B.1.1.7), with its light chain able to restore neutralization potency to broad group of antibodies.

One of the most remarkable features of the SARS-CoV-2 Variants of Concern (VOC) is the unusually large number of mutations they carry. However, the specific factors that drove the emergence of such variants since the second half of 2020 are not fully resolved. In this study, we described a new SARS-CoV-2 lineage provisionally designated as P.1-like-II that, as well as the previously described lineage P.1-like-I, shares several lineage-defining mutations with the VOC P.1 circulating in Brazil. Reconstructions of P.1 ancestor sequences demonstrate that the entire constellation of mutations that define the VOC P.1 did not accumulate within a single long-term infected individual, but was acquired by sequential addition during interhost transmissions. Our evolutionary analyses further estimate that P.1-ancestors strains carrying half of the P.1-lineage-defining mutations, including those at the receptor-binding domain of the Spike protein, circulated cryptically in the Amazonas state since August 2020. This evolutionary pattern is consistent with the hypothesis that partial human population immunity acquired from natural SARS-CoV-2 infections during the first half of 2020 might have been the major driving force behind natural selection that allowed VOCs' emergence and worldwide spread. These findings also support a long lag-time between the emergence of variants with key mutations of concern and expansion of the VOC P.1 in Brazil.

SARS-CoV-2 has undergone progressive change with variants conferring advantage rapidly becoming dominant lineages e.g. B.1.617. With apparent increased transmissibility variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the UK. Here we study the ability of monoclonal antibodies, convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2 and complement this with structural analyses of Fab/RBD complexes and map the antigenic space of current variants. Neutralization of both viruses is reduced when compared with ancestral Wuhan related strains but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2 suggesting that individuals previously infected by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insight for immunisation policy with future variant vaccines in non-immune populations., The B.1.617 lineage of SARS-CoV-2, especially the delta strain that is B.1.617.2 has contributed to the wave of infection in the Indian subcontinent. Structural and serological analyses show no evidence of antibody escape but individuals previously infected with either the B.1.351 (beta) and P.1 (gamma) variants are likely more susceptible to reinfection by the delta strain. Vaccines based on B.1.1.7 (alpha) are likely to provide the broadest protection against current variants.

First in Manaus in the Brazilian Northern region, the Variant of Concern P.1 traveled 3800 kilometers southeast to endanger Sao Paulo contributing to the collapse of the health system. Here, we show evidence of how fast the VOC P.1 has spread in the most populated city in South America.

OBJECTIVES To investigate the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a vulnerable population of children and their household contacts. METHODS SARS-CoV-2 reverse transcription polymerase chain reaction assays and coronavirus disease 2019 (COVID-19) immunoglobulin G serology tests were performed in children and their household contacts after enrollment during primary health care clinic visits. Participants were followed prospectively with subsequent specimens collected through household visits in Manguinhos, an impoverished urban slum (a favela) in Rio de Janeiro at 1, 2, and 4 weeks and quarterly post study enrollment. RESULTS Six hundred sixty-seven participants from 259 households were enrolled from May to September 2020. This included 323 children (0–13 years), 54 adolescents (14–19 years), and 290 adults. Forty-five (13.9%) children had positive test results for SARS-CoV-2 polymerase chain reaction. SARS-CoV-2 infection was most frequent in children aged CONCLUSIONS In our setting, children do not seem to be the source of SARS-CoV-2 infection and most frequently acquire the virus from adults. Our findings suggest that, in settings such as ours, schools and child care potentially may be reopened safely if adequate COVID-19 mitigation measures are in place and staff are appropriately immunized.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 19 (COVID-19), is a single-stranded positive-sense ribonucleic acid (RNA) virus that typically undergoes one to two single nucleotide mutations per month. COVID-19 continues to spread globally, with case fatality and test positivity rates often linked to locally circulating strains of SARS-CoV-2. Furthermore, mutations in this virus, in particular those occurring in the spike protein (involved in the virus binding to the host epithelial cells) have potential implications in current vaccination efforts. In Rwanda, more than twenty thousand cases have been confirmed as of March 14th 2021, with a case fatality rate of 1.4% and test positivity rate of 2.3% while the recovery rate is at 91.9%. Rwanda started its genomic surveillance efforts, taking advantage of pre-existing research projects and partnerships, to ensure early detection of SARS-CoV-2 variants and to potentially contain the spread of variants of concern (VOC). As a result of this initiative, we here present 203 SARS-CoV-2 whole genome sequences analyzed from strains circulating in the country from May 2020 to February 2021. In particular, we report a shift in variant distribution towards the newly emerging sub-lineage A.23.1 that is currently dominating. Furthermore, we report the detection of the first Rwandan cases of the VOCs, B.1.1.7 and B.1.351, among incoming travelers tested at Kigali International Airport. We also discuss the potential impact of COVID-19 control measures established in the country to control the spread of the virus. To assess the importance of viral introductions from neighboring countries and local transmission, we exploit available individual travel history metadata to inform spatio-temporal phylogeographic inference, enabling us to take into account infections from unsampled locations during the time frame of interest. We uncover an important role of neighboring countries in seeding introductions into Rwanda, including those from which no genomic sequences are currently available or that no longer report positive cases. Our results point to the importance of systematically screening all incoming travelers, regardless of the origin of their travels, as well as regional collaborations for durable response to COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in Brazil was dominated by two lineages designated as B.1.1.28 and B.1.1.33. The two SARS-CoV-2 variants harboring mutations at the receptor-binding domain of the Spike (S) protein, designated as lineages P.1 and P.2, evolved from lineage B.1.1.28 and are rapidly spreading in Brazil. Lineage P.1 is considered a Variant of Concern (VOC) because of the presence of multiple mutations in the S protein (including K417T, E484K, N501Y), while lineage P.2 only harbors mutation S:E484K and is considered a Variant of Interest (VOI). On the other hand, epidemiologically relevant B.1.1.33 deriving lineages have not been described so far. Here we report the identification of a new SARS-CoV-2 VOI within lineage B.1.1.33 that also harbors mutation S:E484K and was detected in Brazil between November 2020 and February 2021. This VOI displayed four non-synonymous lineage-defining mutations (NSP3:A1711V, NSP6:F36L, S:E484K, and NS7b:E33A) and was designated as lineage N.9. The VOI N.9 probably emerged in August 2020 and has spread across different Brazilian states from the Southeast, South, North, and Northeast regions.

SummaryTerminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

The SARS-CoV-2 lineage B.1.1.28 has been evolving in Brazil since February 2020 giving origin to multiple local clades including the new Variant of Concern (VOC) designated P.1 or 501Y.V3. The recent emergence of sub-lineages with convergent mutations in the spike (S) protein raises concern about the potential impact on viral infectivity and immune escape. We describe here the first three confirmed SARS-CoV-2 reinfections cases with the new VOC P.1 in residents of the Amazonas state, Brazil. Three female patients, 29, 40, and 50-year-old, were RT-PCR confirmed for SARS-CoV-2 on two occasions, with at least 92 days apart. Next-generation sequencing and phylogenetic analysis were conducted to precisely access the SARS-CoV-2 lineages of each infection event. SARS-CoV-2 genomic analysis confirmed three cases of reinfections caused by the VOC P.1 in patients that were primo-infected by distinct viral lineages 3–9 months earlier. Case 1 (29-year-old) was positive on March 24, 2020 (lineage B.1.195) and then on December 30, 2020 (lineage P.1); case 2 (50-year-old) was positive on October 19, 2020 (lineage B.1.1.33) and on January 19, 2021 (lineage P.1); case 3 (40-year-old) was positive on April 22, 2020 (lineage B.1.195) and on January 29, 2021 (lineage P.1). The three patients displayed low mean Ct values (

Highlights • Analysis of sewer pipes promoted faster public health actions to control epidemic. • An environmental health indicator was made available to the population. • Environmental data was used in decision making to control the epidemic • SARS-CoV-2 lineages were demonstrated through whole-genome sequencing. • WBE is an excellent surveillance tool to assess the course of the COVID-19 epidemic., Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) wastewater-based epidemiology (WBE) has been applied as a complementary approach for spatial tracking of coronavirus disease 2019 cases (COVID-19) as well as early warning of the occurrence of infected populations. The present study presents the result of the monitoring of sanitary sewerage in the municipality of Niterói, a metropolitan region of Rio de Janeiro (Brazil) and its use as a complementary indicator in the surveillance of COVID-19 cases, thus assisting actions of public health from local authorities. Twelve composite raw sewage samples were weekly collected from two wastewater treatment plants (WWTPs) and alternately from 17 sewer pipes (SP) from surrounding neighbourhoods and slums throughout 20 weeks (April 15th to August 25th, 2020). Two hundred twenty-three samples were concentrated using the ultracentrifugation-based method and SARS-CoV-2 RNA detected and quantified by RT-qPCR using primers and probe targeting the N2 genome. SARS-CoV-2 RNA was detected in 84.3% (188/223) of samples with a positive rate ranging from 42% (5/12) in the first week of monitoring to 100% during the peak of epidemic with viral concentration ranging from 3.1 to 7.1 log10 genome copies /100 mL throughout the studied period. Positive rates were higher in WWTPs when compared to SP, being useful tool for monitoring trends in the evolution of the COVID-19 curve, while SP data were more effective when health public interventions were needed. Whole-genome sequencing using Illumina MiSeq System confirmed the lineage of three genomes as B.1.1.33 (clade G) containing the nucleotide substitutions observed in strains that circulate in the Rio de Janeiro during the period of this study. In addition, geoprocessing tool was used to build heat maps based on SARS-CoV-2 data from sewage samples, which were weekly updated and available online to the general population as an indicator of the ongoing epidemic situation in Niterói city, raising public awareness., Graphical abstract Image, graphical abstract

The Northern Brazilian state of Amazonas is one of the most heavily affected country regions by the COVID-19 epidemic and experienced two exponential growing waves in early and late 2020. Through a genomic epidemiology study based on 250 SARS-CoV-2 genomes from different Amazonas municipalities sampled between March 2020 and January 2021 we revealed that the first exponential growth phase was driven mostly by the dissemination of lineage B.1.195 which was gradually replaced by lineage B.1.1.28. The second wave coincides with the emergence of the variant of concern (VOC) P.1 which evolved from a local B.1.1.28 clade in late November and rapidly replaced the parental lineage in less than two months. Our findings support that successive lineage replacements in Amazonas were driven by a complex combination of variable levels of social distancing measures and the emergence of a more transmissible VOC P.1 virus. These data provide unique insights to understanding the mechanisms that underlie the COVID-19 epidemic waves and the risk of disseminating SARS-CoV-2 VOC P.1 in Brazil and potentially worldwide.

The northern state of Amazonas is among the regions in Brazil most heavily affected by the COVID-19 epidemic and has experienced two exponentially growing waves, in early and late 2020. Through a genomic epidemiology study based on 250 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from different Amazonas municipalities sampled between March 2020 and January 2021, we reveal that the first exponential growth phase was driven mostly by the dissemination of lineage B.1.195, which was gradually replaced by lineage B.1.1.28 between May and June 2020. The second wave coincides with the emergence of the variant of concern (VOC) P.1, which evolved from a local B.1.1.28 clade in late November 2020 and replaced the parental lineage in

On 29 January 2021, we identified a new circulating lineage of Severe acute respiratory syndrome coronavirus 2 in Brazil, located in Sergipe state, Northeast region. The B.1.1.251 lineage was identified in a 32-old-man with headache, sore throat and coryza. Further studies are needed to evaluate the transmissibility and virulence of this emergent coronavirus lineage in Brazil.

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.

SummaryBackgroundUruguay is one of the few countries in the Americas that successfully contained the COVID-19 epidemic during the first half of 2020. Nevertheless, the intensive human mobility across the dry border with Brazil is a major challenge for public health authorities. We aimed to investigate the origin of SARS-CoV-2 strains detected in Uruguayan localities bordering Brazil as well as to measure the viral flux across this ∼1,100 km uninterrupted dry frontier.MethodsUsing complete SARS-CoV-2 genomes from the Uruguayan-Brazilian bordering region and phylogeographic analyses, we inferred the virus dissemination frequency between Brazil and Uruguay and characterized local outbreak dynamics during the first months (May-July) of the pandemic.FindingsPhylogenetic analyses revealed multiple introductions of SARS-CoV-2 Brazilian lineages B.1.1.28 and B.1.1.33 into Uruguayan localities at the bordering region. The most probable sources of viral strains introduced to Uruguay were the Southeast Brazilian region and the state of Rio Grande do Sul. Some of the viral strains introduced in Uruguayan border localities between early May and mid-July were able to locally spread and originated the first outbreaks detected outside the metropolitan region. The viral lineages responsible for Uruguayan suburban outbreaks were defined by a set of between four and 11 mutations (synonymous and non-synonymous) respect to the ancestral B.1.1.28 and B.1.1.33 viruses that arose in Brazil, supporting the notion of a rapid genetic differentiation between SARS-CoV-2 subpopulations spreading in South America.InterpretationAlthough Uruguayan borders have remained essentially closed to non-Uruguayan citizens, the inevitable flow of people across the dry border with Brazil allowed the repeated entry of the virus into Uruguay and the subsequent emergence of local outbreaks in Uruguayan border localities. Implementation of coordinated bi-national surveillance systems are crucial to achieve an efficient control of the SARS-CoV-2 spread across this kind of highly permeable borderland regions around the world.Research in contextEvidence before this studySince the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of coronavirus disease 19 (COVID-19), was first detected in South America on February 26, 2020, it has rapidly spread through the region, causing nearly 350,000 deaths by December, 2020. In contrast to most American countries, Uruguay avoided an early exponential growth of SARS-CoV-2 cases and during the first six months of the pandemic it registered the lowest incidence of SARS-CoV-2 cases and deaths among South American countries. The intensive cross-border human mobility through the ∼1,100 km uninterrupted dry frontier between Uruguay and Brazil, might poses a major challenge for long-term control of the epidemic in Uruguay. Previous genomic studies conducted in Uruguay have analyzed sequences mostly sampled at the capital city, Montevideo, and detected prevalent SARS-CoV-2 lineages different from those described in Brazil, thus finding no evidence of frequent viral exchanges between these countries.Added value of this studyHere we present the first genomic study of SARS-CoV-2 strains detected in different Uruguayan and Brazilian localities along the bordering region. The samples analyzed include 30% (n = 59) of all laboratory confirmed SARS-CoV-2 cases from Uruguayan departments at the Brazilian border between March and July, 2020, as well as 68 SARS-CoV-2 sequences from individuals diagnosed in the southernmost Brazilian state of Rio Grande do Sul between March and August, 2020. We demonstrate that SARS-CoV-2 viral lineages that widely spread in the Southeastern Brazilian region (B.1.1.28 and B.1.1.33) were also responsible for most viral infections in Rio Grande do Sul and neighboring Uruguayan localities. We further uncover that major outbreaks detected in Uruguayan localities bordering Brazil in May and June, 2020, were originated from two independent introduction events of the Brazilian SARS-CoV-2 lineage B.1.1.33, unlike previous outbreaks in the Uruguayan metropolitan region that were seeded by European SARS-CoV-2 lineages.Implications of all the available evidenceOur findings confirm that although Uruguayan borders have remained essentially closed to non-Uruguayan citizens, dissemination of SARS-CoV-2 across the Uruguayan-Brazilian frontier was not fully suppressed and had the potential to ignite local transmission chains in Uruguay. These findings also highlight the relevance of implementing bi-national public health cooperation workforces combining epidemiologic and genomic data to monitor the viral spread throughout this kind of highly permeable dry frontiers around the world.

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), wholegenome 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 Sao Paulo.

Background Infection by SARS-CoV-2 in domestic animals has been related to close contact with humans diagnosed with COVID-19. Objectives: To assess the exposure, infection, and persistence by SARS-CoV-2 of dogs and cats living in the same households of humans that tested positive for SARS-CoV-2, and to investigate clinical and laboratory alterations associated with animal infection. Methods Animals living with COVID-19 patients were longitudinally followed and had nasopharyngeal/oropharyngeal and rectal swabs collected and tested for SARS-CoV-2. Additionally, blood samples were collected for laboratory analysis, and plaque reduction neutralization test (PRNT90) to investigate specific SARS-CoV-2 antibodies. Results Between May and October 2020, 39 pets (29 dogs and 10 cats) of 21 patients were investigated. Nine dogs (31%) and four cats (40%) from 10 (47.6%) households were infected with or seropositive for SARS-CoV-2. Animals tested positive from 11 to 51 days after the human index COVID-19 case onset of symptoms. Three dogs tested positive twice within 14, 30, and 31 days apart. SARS-CoV-2 neutralizing antibodies were detected in one dog (3.4%) and two cats (20%). In this study, six out of thirteen animals either infected with or seropositive for SARS-CoV-2 have developed mild but reversible signs of the disease. Using logistic regression analysis, neutering, and sharing bed with the ill owner were associated with pet infection. Conclusions The presence and persistence of SARS-CoV-2 infection have been identified in dogs and cats from households with human COVID-19 cases in Rio de Janeiro, Brazil. People with COVID-19 should avoid close contact with their pets during the time of their illness.

The COVID-19 epidemic in Brazil experienced two major lineage replacements until mid-2021. The first was driven by lineage P.2, in late 2020, and the second by lineage Gamma, in early 2021. To understand how these SARS-CoV-2 lineages spread in Brazil, we analyzed 11,724 genomes collected throughout the country between September 2020 and April 2021. Our findings indicate that lineage P.2 probably emerged in July 2020 in the Rio de Janeiro state and Gamma in November 2020 in the Amazonas state. Both states were the main hubs of viral disseminations to other Brazilian locations. We estimate that Gamma was 1.56-3.06 times more transmissible than P.2 in Rio de Janeiro and that the median effective reproductive number (Re) of Gamma varied according to the geographic context (Re = 1.59-3.55). In summary, our findings support that lineage Gamma was more transmissible and spread faster than P.2 in Brazil.

Multiple epicenters of the SARS-CoV-2 pandemic have emerged since the first pneumonia cases in Wuhan, China, such as Italy, USA, and Brazil. Brazil is the third-most affected country worldwide, but genomic sequences of SARS-CoV-2 strains are mostly restricted to states from the Southeast region. Pernambuco state, located in the Northeast region, is the sixth most affected Brazilian state, but very few genomic sequences from the strains circulating in this region are available. We sequenced 101 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms that reside in Pernambuco. Phylogenetic reconstructions revealed that all genomes belong to the B lineage and most of the samples (88%) were classified as lineage B.1.1. We detected multiple viral introductions from abroad (likely from Europe) as well as six local B.1.1 clades composed by Pernambuco only strains. Local clades comprise sequences from the capital city (Recife) and other country-side cities, corroborating the community spread between different municipalities of the state. These findings demonstrate that different from Southeastern Brazilian states where the epidemics were majorly driven by one dominant lineage (B.1.1.28 or B.1.1.33), the early epidemic phase at the Pernambuco state was driven by multiple B.1.1 lineages seeded through both national and international traveling.

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 is highly transmissible, and 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. Research groups around the world are strongly focused on this, and the susceptibility of different cell lines to SARS-CoV-2 infection has already been demonstrated by molecular techniques. However, data on the biology of SARS-CoV-2 at the ultrastructural level in these in vitro models is still scarce. In this study, we documented, by transmission electron microscopy and real-time RT-PCR, the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. 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. Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Despite all efforts to control the COVID-19 spread, the SARS-CoV-2 reached South America within three months after its first detection in China, and Brazil became one of the hotspots of COVID-19 in the world. Several SARS-CoV-2 lineages have been identified and some local clusters have been described in this early pandemic phase in Western countries. Here we investigated the genetic diversity of SARS-CoV-2 during the early phase (late February to late April) of the epidemic in Brazil. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 in Brazil and the community transmission of a major B.1.1 lineage defined by two amino acid substitutions in the Nucleocapsid and ORF6. This SARS-CoV-2 Brazilian lineage was probably established during February 2020 and rapidly spread through the country, reaching different Brazilian regions by the middle of March 2020. Our study also supports occasional exportations of this Brazilian B.1.1 lineage to neighboring South American countries and to more distant countries before the implementation of international air travels restrictions in Brazil.

SummaryGenomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.

Neuraminidase inhibitors (NAIs) are the main class of antivirals currently used for the treatment of influenza infections. As influenza viruses are constantly evolving, drug-resistance can emerge resulting in reduced effectiveness of treatment. This study evaluated the presence of molecular markers associated with NAI susceptibility in 724 influenza A(H1N1)pdm09 positive samples from Brazilian surveillance system from the 2014-2016 seasons, including 76 isolates tested for oseltamivir (OST) susceptibility and 23 isolates also tested for zanamivir, peramivir and laninamivir susceptibility. We identified the H275Y (n = 3) and I223K (n = 1) NA substitutions, associated with reduced inhibition (RI) by the NAIs. Noteworthy, no epidemiological links were identified among the patients infected with the mutant viruses. Phylogenetic analysis from NA and hemagglutinin genes showed that mutant viruses were not clustered. All mutant virus strains carried the permissive substitutions V241I and N369K, in addition to the N386K, which has been shown to destabilize the NA structure. Functional NA analysis of one virus containing the H275Y mutation confirmed its highly RI profile to OST and peramivir and demonstrated that it had decreased viral replication and NA thermostability compared to the wild type virus. The remaining tested isolates presented normal inhibition profile to the NAIs tested. In conclusion, the overall frequency of influenza A(H1N1)pdm09 viruses bearing mutations associated with NAI RI was 0.6%, similar to what has been observed in recent global studies.

In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene., In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010–2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10−9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.

The H3N2 subtype of influenza A (H3N2) was the predominant strain during the early months of the 2017 influenza epidemic in Brazil. In Australia, it was responsible for a strong and prolonged 2017 season and reached the Northern hemisphere causing an intense 2017/8 influenza season. Several genetic and antigenic A(H3N2) variants were circulating, which made the decision about which strain to incorporate into the influenza vaccine challenging. For 2018, the WHO selected a new H3N2 strain, A/Singapore/INFIMH-16-0019/2016-like, to replace the strain A/HongKong/4801/2014-like in the Southern Hemisphere trivalent vaccine. The aim of this study was to describe the genetic diversity of influenza A (H3N2) viruses circulating in Brazil between January 2017 and January 2018, checking the match between the vaccines and worldwide circulating strains with the Brazilian influenza strains. Hemagglutinin gene sequencing of the influenza A (H3N2) was performed, followed by a phylogenetic reconstruction using additional database sequences to define genetic groups and compare with other worldwide circulating strains. We observed a large diversity of H3N2 genetic clusters, including 3C.2a, 3C.2a1, 3C.3a, and their subgroups. During the 2016–7, inter-epidemic and 2017 epidemic period the cluster most frequently detected belonged to clade 3C.2a1 (148/185; 80.0%), a distinct group related to the 2017 vaccine strain A/HongKong/4801/2014-like (3C.2a). However, the genetic profile changed during the study period and in the inter-epidemic season 2017–8 the most commonly detected genetic group was the 3C.2a cluster (43/58; 74.1%). Inside this cluster, the majority (34/43; 79.1%) of strains belonged to a single genetic 3C.2a subgroup 2 (3C.2a2), bearing antigenic substitutions T131K and R142K (site A) and R261Q (site E). The dominance of this 3C.2a2 in the 2017–8 inter-epidemic period in Brazil was similar to the 2017–8 season in Europe and Canada according their surveillance data. The new vaccine strain has five to six antigenic changes in comparison to the predominant 3C.2a2 circulating in South America since September 2017 until now. It is possible that the vaccine mismatch will not protect the population against a majority of circulating strains. Surveillance of the vaccine effectiveness supported by antigenic and serological analysis are necessary to prove this hypothesis. However, this highlights the difficulty of vaccine strain selection and highlights the need for of a universal influenza vaccine.

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.

The limited influenza A(H3N2) genetic data available from the Southern Hemisphere (particularly from Africa and Latin America), constrains the accurate reconstruction of viral dissemination dynamics within those regions. Our objective was to describe the spatial dissemination dynamics of influenza A(H3N2) within South America. A total of 469 sequences of the HA1 portion of the hemagglutinin gene (HA) from influenza A(H3N2) viruses sampled in temperate and tropical South American countries between 1999 and 2012 were combined with available contemporary sequences from Australia, Hong Kong, United Kingdom and the United States. Phylogenetic analyses revealed that influenza A(H3N2) sequences from South America were highly intermixed with sequences from other geographical regions, although a clear geographic virus population structure was detected globally. We identified 14 clades mostly (≥80%) composed of influenza sequences from South American countries. Bayesian phylogeographic analyses of those clades support a significant role of both temperate and tropical regions in the introduction and dissemination of new influenza A(H3N2) strains within South America and identify an intensive bidirectional viral exchange between different geographical areas. These findings indicate that seasonal influenza A(H3N2) epidemics in South America are seeded by both the continuous importation of viral variants from other geographic regions and the short-term persistence of local lineages. This study also supports a complex metapopulation model of influenza A(H3N2) dissemination in South America, with no preferential direction in viral movement between temperate and tropical regions.

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.

Analyses of the 2009 H1N1 influenza pandemic and post-pandemic years showed high attack rates and severity among indigenous populations. This study presents the characteristics of the first documented influenza outbreak in indigenous peoples in Brazil, that occurred from 30th March to 14th April 2016 in a Guarani village in Southeast Region. Acute respiratory infections were prospectively investigated. The majority of the 73 cases were influenza-like illness (ILI) (63.0%) or severe acute respiratory infection (SARI) (20.5%). The ILI+SARI attack rate (35.9%) decreased with increasing age. There was a high influenza vaccination rate (86.3%), but no statistically significant difference in vaccination rates between severe and non-severe cases was seen (p = 0.334). Molecular analyses of 19.2% of the cases showed 100% positivity for influenza A(H1N1)pdm09 and/or hRSV. Influenza A(H1N1)pdm09 was included in the 6B.1 genetic group, a distinct cluster with 13 amino acid substitutions of A/California/07/2009-like. The hRSV were clustered in the BA-like genetic group. The early arrival of the influenza season overlapping usual hRSV season, the circulation of a drifted influenza virus not covered by vaccine and the high prevalence of risk factors for infection and severity in the village jointly can explain the high attack rate of ARI, even with a high rate of influenza vaccination. The results reinforce the importance of surveillance of respiratory viruses, timely vaccination and controlling risk factors for infection and severity of in the indigenous populations in order to preventing disease and related deaths, particularly in children.

International audience; Pandemic influenza A H1N1 [A(H1N1)pdm09] was first detected in Brazil in May 2009, and spread extensively throughout the country causing a peak of infection during June to August 2009. Since then, it has continued to circulate with a seasonal pattern, causing high rates of morbidity and mortality. Over this period, the virus has continually evolved with the accumulation of new mutations. In this study we analyze the phylogenetic relationship in a collection of 220 A(H1N1)pdm09 hemagglutinin (HA) gene sequences collected during and after the pandemic period (2009 to 2014) in Brazil. In addition, we have looked for evidence of viral polymorphisms associated with severe disease and compared the range of viral variants with the vaccine strain (A/California/7/2009) used throughout this period. The phylogenetic analyses in this study revealed the circulation of at least eight genetic groups in Brazil. Two (G6-pdm and G7-pdm) co-circulated during the pandemic period, showing an early pattern of viral diversification with a low genetic distance from vaccine strain. Other phylogenetic groups, G5, G6 (including 6B, 6C and 6D subgroups), and G7 were found in the subsequent epidemic seasons from 2011 to 2014. These viruses exhibited more amino acid differences from the vaccine strain with several substitutions at the antigenic sites. This is associated with a theoretical decrease in the vaccine efficacy. Furthermore, we observed that the presence of any polymorphism at residue 222 of the HA gene was significantly associated with severe/fatal cases, reinforcing previous reports that described this residue as a potential virulence marker. This study provides new information about the circulation of some viral variants in Brazil, follows up potential genetic markers associated with virulence and allows infer if the efficacy of the current vaccine against more recent A(H1N1)pdm09 strains may be reduced.

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.

The 2009 pandemic influenza A(H1N1)pdm09 virus emerged and caused considerable morbidity and mortality in the third world, especially in Brazil. Although circulating strains of A(H1N1)pdm09 are A/California/04/2009-like (CA-04-like) viruses, various studies have suggested that some mutations in the viral hemagglutinin (HA) may be associated with enhanced severity and fatality. This phenomenon is particularly challenging for immunocompromised individuals, such as those who have undergone bone marrow transplant (BMT), because they are more likely to display worse clinical outcomes to influenza infection than non-immunocompromised individuals. We studied the clinical and viral aspects of post-BMT patients with confirmed A(H1N1)pdm09 diagnosis in the largest cancer hospital in Brazil. We found a viral strain with K-15E, P83S and Q293H polymorphisms in the HA, which is presumably more virulent, in these individuals. Despite that, these patients showed only mild symptoms of infection. Our findings complement the discovery of mild cases of infection with the A(H1N1)pdm09 virus with the K-15E, P83S and Q293H mutations in Brazil and oppose other studies that have linked these changes with increased disease severity. These results could be important for a better comprehension of the impact of the pandemic influenza in the context of BMT.

Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012. We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other Latin American countries to confirm or rule out the community transmission of OST-resistant strains of A(H1N1)pdm09.

8 páginas, 3 figuras, We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2., We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from GISAID’s EpiCoV Database used in the phylogenetic analysis. We gratefully acknowledge all the staff working with sample collection, sample preparation, sequencing, data analysis and data sharing in all laboratories in the WHO European Region for making this work possible.