Immunoglobulin A as a key immunological molecular signature of post-COVID-19 conditions (preprint)

COVID-19 has infected humans worldwide, causing millions of deaths or prolonged symptoms in survivors. The transient or persistent symptoms after SARS-CoV-2 infection have been defined as post-COVID-19 conditions (PCC). We conducted a study of 151 Brazilian PCC patients to analyze symptoms and immunoglobulin profiles, taking into account gender, vaccination, hospitalization and age. Fatigue and myalgia were the most common symptoms and lack of vaccination, hospitalization, and neuropsychiatric and metabolic comorbidities were relevant for the development of PCC. Analysis of serological immunoglobulins showed that IgA was higher in PCC patients, especially in the adult and elderly groups. Also, non-hospitalized and hospitalized PCC patients produced high and similar levels of IgA. Our results indicated that the detection of IgA antibodies against SARS-CoV-2 during the course of the disease could be associated with the development of PCC and may be an immunological signature to predict prolonged symptoms in COVID-19 patients.

Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance and high Spike-specific neutralizing antibodies (preprint)

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Control of pathogen load (host resistance) and prevention of tissue damage (disease tolerance) are essential for the outcome of infectious diseases. Both low resistance and high disease tolerance might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immune response of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly-symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Interestingly, the plasma of these patients showed a higher spike-specific neutralization capacity. The identification of very early distinct immune responses in long-term carriers adds up to our understanding on essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Evaluation of the Panbio™ COVID-19 Antigen Rapid Diagnostic Test in subjects infected with Omicron using different specimens (preprint)

Community testing is a crucial tool for the early identification of SARS-CoV-2 infection and transmission control. The emergence of the highly mutated Omicron variant (B.1.1.259) raised concerns about its primary site of replication, impacting sample collection, and its detectability by rapid antigens tests. We tested the Antigen Rapid Diagnostic Test (Ag-RDT) performance using nasal, oral, and saliva specimens for COVID-19 diagnosis in 192 symptomatic individuals, using RT-qPCR from nasopharyngeal samples as control. Variant of Concern (VOC) investigation was determined by the 4Plex SARS-CoV-2 screening kit. SARS-CoV-2 positivity rate was 66.2%, with 99% of the positive samples showing an amplification profile consistent with that of the Omicron variant. Nasal Ag-RDT showed higher sensitivity (89%) than oral (12.6%) and saliva (22.1%) Ag-RDTs. Our data showed the good performance of the Ag-RDT in a pandemic scenario dominated by the Omicron VOC. Furthermore, our data also demonstrated that nasal specimens perform better than oral and saliva ones for Omicron Ag-RDT detection.

A SARS-CoV-2 negative antigen rapid diagnostic in RT-qPCR positive samples correlates with a low likelihood of infectious viruses in the nasopharynx. (preprint)

SARS-CoV-2 transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDT) are highly specific, but sensitivity is variable. Discordant RT- qPCR vs Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between a negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that a positive SARS-CoV-2 Ag-RDT result correlates with the presence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was verified in negative Ag-RDT / positive RT-qPCR samples. The data clearly demonstrates the less likelihood of a negative Ag-RDT sample to harbor infectious SARS-CoV-2 and consequently with a lower transmissible potential.

Phylodynamic analysis of SARS-CoV-2 spread in Rio de Janeiro, Brazil, highlights how metropolitan areas act as dispersal hubs for new variants. (preprint)

During the first semester of 2021, all of Brazil has suffered an intense wave of COVID-19 associated with the Gamma variant. In July, the first cases of Delta variant were detected in the state of Rio de Janeiro. In this work, we have employed phylodynamic methods to analyze more than 1,600 genomic sequences of Delta variant collected until September in Rio de Janeiro to reconstruct how this variant has surpassed Gamma and dispersed throughout the state. After the introduction of Delta, it has initially spread mostly in the homonymous city of Rio de Janeiro, the most populous of the state. In a second stage, dispersal occurred to mid- and long-range cities, which acted as new close-range hubs for spread. We observed that the substitution of Gamma by Delta was possibly caused by its higher viral load, a proxy for transmissibility. This variant turnover prompted a new surge in cases, but with lower lethality than was observed during the peak caused by Gamma. We reason that high vaccination rates in the state of Rio de Janeiro were possibly what prevented a higher number of deaths. Impact statementUnderstanding how SARS-CoV-2 spreads is vital to propose efficient containment strategies, especially when under the perspective of new variants emerging in the next year. Still, models of SARS-CoV-2 dispersal are still largely based in large cities from high-income countries, resulting in an incomplete view of the possible scenarios consequent of a new variant introduction. The work improves this discussion by reconstructing the spatio-temporal dispersal of Delta variant since its introduction in Rio de Janeiro, a densely populated region in South America. We also analyzed the epidemiological outcome of this spread, with a decrease in lethality rate uncommon to the observed in other countries. Data summaryFour supplementary figures, one supplementary table and one supplementary file are available with the online version of this article. Raw short reads of the newly sequenced genomes are available at SRA-NCBI database (https://www.ncbi.nlm.nih.gov/sra) under the BioProject PRJNA774631 and the assembled genomes are deposited at GISAID database (https://www.gisaid.org/) under the accession numbers listed in Table S1. Other genomic sequences used in the analyses are listed in Table S2. Epidemiological data for the state of Rio de Janeiro was obtained from https://www.saude.rj.gov.br/informacao-sus/dados-sus/2020/11/covid-19.

Emergence of within-host SARS-CoV-2 recombinant genome after coinfection by Gamma and Delta variants (preprint)

Since the first reports of patients coinfected by two genetically-distinct lineages of SARS-CoV-2, the scientific community raised concerns about the recombination of intra-host viral RNA sequences as a possible mechanism underlying the emergence of novel variants. Indeed, this phenomenon occurs at a relatively high frequency among betacoronaviruses. Nevertheless, the few existing studies about recombination between genetically-distinct lineages of SARS-CoV-2 are restricted to detect the inter-host dissemination of genomes post-recombination events. However, the high genomic similarity between the current co-circulating lineages challenges the identification of these events. Here, we report the first case of intra-host SARS-CoV-2 recombination during a coinfection by the variants of concern (VOC) AY.33 (Delta) and P.1 (Gamma) supported by sequencing reads harboring a mosaic of lineage-defining mutations. By using next-generation sequencing reads intersecting regions that simultaneously overlap lineage-defining mutations from Gamma and Delta, we were able to identify a total of six recombinant regions across the SARS-CoV-2 genome within a sample. Four of them mapped in the spike gene and two in the nucleocapsid gene. We detected mosaic reads harboring a combination of lineage-defining mutations from each VOC. To our knowledge, this is the first report of intra-host RNA-RNA recombination between two lineages of SARS-CoV-2, which can represent a threat to public health management during the COVID-19 pandemic due to the possibility of the emergence of viruses with recombinant phenotypes.

Immunogenicity of trimetric spike protein associated to Poly(I:C) plus Alum (preprint)

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (S Ptn with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

Turnover of SARS-CoV-2 lineages shaped the pandemic and enabled the emergence of new variants in the state of Rio de Janeiro, Brazil (preprint)

In the present study, we provide a retrospective genomic epidemiology analysis of the SARS-CoV-2 pandemic in the state of Rio de Janeiro, Brazil. We gathered publicly available data from GISAD and sequenced more 1,927 new genomes sampled periodically from March 2021 to June 2021 from 91 out of the 92 cities of the state. Our results showed that the pandemic was characterized by three different phases driven by a successive replacement of lineages. All stages occurred in distinct mortality and mobility contexts, with higher evidence of social distancing measures being observed in early pandemic and relaxed in the last two phases. Interestingly, we noticed that viral supercarriers accounted for the overwhelming majority of the circulating virus (> 90%) among symptomatic individuals in the state. Moreover, SARS-CoV-2 genomic surveillance also revealed the emergence and spread of two new variants (P.5 and P.1.2) firstly reported in this study. Altogether, our findings provided important lessons learned from the different epidemiological aspects of the SARS-CoV-2 dynamic in the state of Rio de Janeiro that have a strong potential to shape future decisions aiming to improve public health management and understanding mechanisms underlying virus dispersion.

Development and Large-Scale Validation of A Highly Accurate SARS-COV-2 Serological Test Using Regular Test Strips for Autonomous and Affordable Finger-Prick Sample Collection, Transportation, and Storage (preprint)

Accurate serological tests are essential tools to allow adequate monitoring and control of COVID-19 spread. Production of a low-cost and high-quality recombinant viral antigen can enable the development of reliable and affordable serological assays, which are urgently needed to facilitate epidemiological surveillance studies in low-income economies. Trimeric SARS-COV-2 spike (S) protein was produced in serum-free, suspension-adapted HEK293 cells. Highly purified S protein was used to develop an ELISA, named S-UFRJ test. It was standardized to work with different types of samples: (i) plasma or serum from venous blood samples; (ii) eluates from dried blood spots (DBS) obtained by collecting blood drops from a finger prick. We developed a cost-effective, scalable technology to produce S protein based on its stable expression in HEK293 cells. Using this recombinant antigen, we presented a workflow for test development in the setting of a pandemic, starting from limited amounts of samples up to reaching final validation with hundreds of samples. Test specificity was determined to be 98.6%, whereas sensitivity was 95% for samples collected 11 or more days after symptoms onset. A ROC analysis allowed optimizing the cut-off and confirming the high accuracy of the test. Endpoint titers were shown to correlate with virus neutralization assessed as PRNT90. There was excellent agreement between plasma and DBS samples, significantly simplifying sample collection, storing, and shipping. An overall cost estimate revealed that the final retail price could be in the range of one US dollar. The S-UFRJ assay developed herein meets the quality requirements of high sensitivity and specificity. The low cost and the use of mailable DBS samples allow for serological surveillance and follow-up of SARS-CoV-2 vaccination of populations regardless of geographical and socio-economic aspects. We hope the detailed guidelines for the development of an affordable and accurate anti-spike SARS-COV-2 ELISA, such as S-UFRJ described here, will stimulate governmental and non-governmental health agencies in other countries to engage in much-needed large-scale studies monitoring the spread and immunity to SARS-COV-2 infection.

Epidemiological dynamics of SARS-CoV-2 VOC Gamma in Rio de Janeiro, Brazil (preprint)

The emergence and widespread circulation of SARS-CoV-2 variants of concern (VOC) or interest (VOI) imposes an enhanced threat to global public health. In Brazil, one of the countries most severely impacted throughout the pandemic, a complex dynamics involving variants co-circulation and turnover events has been recorded with the emergence and spread of VOC Gamma in Manaus in late 2020. In this context, we present a genomic epidemiology investigation based on samples collected between December 2020 and May 2021 in the second major Brazilian metropolis, Rio de Janeiro. By sequencing 244 novel genomes through all epidemiological weeks in this period, we were able to document the introduction and rapid dissemination of VOC Gamma in the city, driving the rise of the third local epidemic wave. Molecular clock analysis indicates this variant has circulated locally since the first weeks of 2021 and only seven weeks were necessary for it to achieve a frequency above 70%, consistent with rates of growth observed in Manaus and other states. Moreover, a Bayesian phylogeographic reconstruction indicates VOC Gamma spread throughout Brazil between December 2020 and January 2021, and that it was introduced in Rio de Janeiro through at least 13 events coming from nearly all regions of the country. Comparative analysis of RT-qPCR cycle threshold (Ct) values provide further evidence that VOC Gamma induces higher viral loads (N1 target; mean reduction of Ct: 2.7, 95% CI = 2.0 - 3.4). This analysis corroborates the previously proposed mechanistic basis for this variant enhanced transmissibility and distinguished epidemiological behavior. Our results document the evolution of VOC Gamma and provide independent assessment of scenarios previously studied in Manaus, therefore contributing to the better understanding of the epidemiological dynamics currently being surveyed in other Brazilian regions.

Polyclonal F(ab’) 2 Fragments of Equine Antibodies Raised Against Recombinant SARS-CoV-2 Spike Protein Neutralize the P.1 and P.2 Strains with High Potency (preprint)

We used the trimeric spike (S) glycoprotein (residues 1-1208) in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by anti-spike ELISA were above 1:106, and neutralizing antibody titer against an early isolate of authentic virus (WT) was 1:14,604 (average PRNT90), which is 140-fold higher than the average neutralizing titer of plasma from three convalescent COVID-19 patients analyzed for comparison. Using the same technology routinely used for industrial production of other horse hyperimmune products, plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab’)2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Repeating the hyperimmunization in a second group of horses confirmed the very high neutralizing titers in serum and in a GMP clinical F(ab’)2 lot. Virus-neutralizing activity in samples from mice that received the F(ab’)22 was similar against WT and P.2 but displaced to higher concentrations by 0.39 log units against P.1. The neutralizing activity against P.1 can be explained by the polyclonal nature of the raised antibody against the whole spike protein. These results support the possibility of using equine F(ab’)2 preparation for the clinical treatment of COVID patients.

COVID-19 in the Brazilian Oil Capital: the successful experience of public and private engagement (preprint)

Background: Brazilian strategy to overcome the spread of COVID-19 has been particularly criticized due to the lack of a national coordinating effort and an appropriate testing program. Here, a successful approach to control the spread of COVID-19 transmission is described by the engagement of public (university and governance) and private sectors (hospitals and oil companies) in Macaé, state of Rio de Janeiro, Brazil, a city known as the National Oil Capital. Methods Until the 38th epidemiological week, over two percent of the 206,728 citizens were subjected to symptom analysis and massive RT-qPCR testing by the Federal University of Rio de Janeiro, with positive individuals being notified up to 48 hours after swab collection. Geocodification and spatial cluster analysis were used to limit COVID-19 spreading in Macaé. Findings: Within the first semester after the outbreak of COVID-19 in Brazil, Macaé recorded 1.8% of fatality associated to COVID-19 up to the 38th epidemiological week, which was at least five times lower than the state capital (10.92%). Overall, considering the successful experience of this joint effort of private and public engagement in Macaé, our data suggest that the development of a similar strategy country wise would have saved over 50,000 lives. Interpretation: Quarantine decree by the local government, molecular massive testing coupled to scientific analysis of COVID-19 spreading prevented the catastrophic consequences of the pandemic as seen in other populous cities within the state of Rio de Janeiro and elsewhere in Brazil.

Genomic characterization of a novel SARS-CoV-2 lineage from Rio de Janeiro, Brazil (preprint)

In this study, we report the sequencing of 180 new viral genomes obtained from different municipalities of the state of Rio de Janeiro from April to December 2020. We identified a novel lineage of SARS-CoV-2, originated from B.1.1.28, distinguished by five single-nucleotide variants (SNVs): C100U, C28253U, G28628U, G28975U, and C29754U. The SNV G23012A (E484K), in the receptor-binding domain of Spike protein, was widely spread across the samples. This mutation was previously associated with escape from neutralizing antibodies against SARS-CoV-2. This novel lineage emerged in late July being first detected by us in late October and still mainly restricted to the capital of the state. However, as observed for other strains it can be rapidly spread in the state. The significant increase in the frequency of this lineage raises concerns about public health management and continuous need for genomic surveillance during the second wave of infections.

Intra-host evolution during SARS-CoV-2 persistent infection (preprint)

Prolonged infection of SARS-CoV-2 represents a challenge to the development of effective public health policies to control the COVID-19 pandemic. The reason why some people have persistent infection and how the virus survives for so long are still not fully understood. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during persistent infection. Thirty-three patients who remained RT-PCR positive in the nasopharynx for at least 16 days were included in this study. Complete SARS-CoV-2 sequences were obtained for each patient at two time points. Phylogenetic, populational, and computational analysis of viral sequences confirmed persistent infection with evidence for a transmission cluster in health care professionals that shared the same workplace. A high number of missense variants targeting crucial structural and non-structural proteins such as Spike and Helicase was found. Interestingly, longitudinal acquisition of substitutions in Spike protein mapped many SARS-CoV-2 predicted T cell epitopes. Furthermore, the mutational profiles observed were suggestive of RNA editing enzyme activities, indicating innate immune mechanisms of the host cell. Viral quasispecies analysis corroborates persistent infection mainly by increasing richness and nucleotide diversity over time. Altogether, our findings highlight a dynamic and complex landscape of host and pathogen interaction during persistent infection suggesting that the host's innate immunity shapes the increase of intra-host diversity with possible implications for therapeutic strategies and public health decisions during the COVID-19 pandemic.

An Affordable Anti-SARS-COV-2 Spike Protein Elisa Test for Early Detection of IgG Seroconversion Suited for Large-Scale Surveillance Studies in Low-Income Countries (preprint)

Background: Accurate serological tests are essential tools to allow adequate monitoring and control of COVID-19 spread. Production of a low-cost and high-quality recombinant viral antigen can enable the development of reliable and affordable serological assays, which are urgently needed to facilitate epidemiological surveillance studies in low-income economies.Methods: Trimeric SARS-COV-2 spike (S) protein was produced in serum-free, suspension-adapted HEK293 cells. Highly purified S protein was used to develop an ELISA, named S-UFRJ test. It was standardized to work with different types of samples: (i) plasma or serum from venous blood samples; (ii) dried blood spots (DBS) from blood drops collected by finger prick.Findings: We developed a cost-effective, scalable technology to produce S protein based on its stable expression in HEK293 cells. The S-UFRJ ELISA displayed 98.4% specificity and sensitivity above 90% already 10 days after symptoms onset, allowing early detection of anti-S IgG seroconversion. Endpoint titers were shown to correlate with virus neutralization assessed as PRNT90. There was excellent agreement between plasma and DBS samples, significantly simplifying sample collection, storing and shipping. The overall cost per test was estimated to be approximately one US dollar.Interpretation: The S-UFRJ assay developed herein meets the quality requirements of high sensitivity and specificity. The low cost and the use of mailable DBS samples allow for serological surveillance of populations regardless of geographical and socio-economic aspects, with special relevance for public health policy actions in low-income countries.Funding Statement: This work was supported by Senai CETIQT, Senai DN and CTG, and by the Brazilian research funding agencies Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Instituto Serrapilheira.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: Samples collected at the State Hematology Institute Hemorio followed a protocol approved by the local ethics committee (CEP Hemorio; approval #4008095). Samples collected at UFRJ COVID Screening Center followed a protocol approved by the national ethics committee (CONEP, Brazil; protocol #30161620000005257; approval #3953368).

Equine hyperimmune globulin raised against the SARS-CoV-2 spike glycoprotein has extremely high neutralizing titers (preprint)

COVID-19 pandemic caused approximately 750,000 deaths and over 20 million confirmed cases of infection by SARS-CoV-2 within 8 months since the emergence of the virus. While there are no vaccines approved and considering the difficulty in meeting the large vaccination demand worldwide, the potential use of passive immunization should be considered based on existing successful therapies against many diseases. Here we demonstrate that hyperimmune globulin preparations raised in horses against the recombinant trimeric spike (S) glycoprotein of SARS-CoV-2 in the prefusion conformation provide very high ELISA titers as well as highly potent neutralizing activity against SARS-CoV-2. Five horses were subcutaneously inoculated for 6 weeks with the recombinant S protein (ectodomain, residues 1-1208). Four out of the 5 horses presented a strong immune response. Considering the average of all 5 horses, ELISA titers above 1:1,000,000 and neutralizing titers (PRNT90) reaching 1:14,604 were observed. When compared with the plasma of three convalescent COVID-19 patients, sera of immunized horses displayed approximately 140-fold higher neutralizing titers measured as PRNT90. To prevent eventual side effects caused by horse antiserum, IgG was digested with pepsin and purified by fractional salt precipitation to eliminate Fc fragments, a process that is industrially used for the production of passive immunization F(ab)2 concentrates against rabies, tetanus and snake venoms. The high neutralizing titers against SARS-CoV-2 obtained for the unprocessed sera were confirmed for the F(ab)2 fragments and were 150-fold higher than the PRNT90 neutralizing titers of plasma of three COVID-19 convalescent patients. The great advantage of using the recombinant trimeric S glycoprotein is that it is safe and provides quick adaptive immunity in horses. Our data show the perspective of using hyperimmune anti-SARS-CoV-2 F(ab)2 preparations as a passive immunization therapy in humans, similar to therapies that have been safely used for decades against rabies, tetanus and snake venoms.

AN AFFORDABLE ANTI-SARS-COV-2 SPIKE ELISA TEST FOR EARLY DETECTION OF IgG SEROCONVERSION SUITED FOR LARGE-SCALE SURVEILLANCE STUDIES IN LOW-INCOME COUNTRIES (preprint)

Background: Accurate serological tests are essential tools to allow adequate monitoring and control of COVID-19 spread. Production of a low-cost and high-quality recombinant viral antigen can enable the development of reliable and affordable serological assays, which are urgently needed to facilitate epidemiological surveillance studies in low-income economies. Methods: Trimeric SARS-COV-2 spike (S) protein was produced in serum-free, suspension-adapted HEK293 cells. Highly purified S protein was used to develop an ELISA, named S-UFRJ test. It was standardized to work with different types of samples: (i) plasma or serum from venous blood samples; (ii) dried blood spots (DBS) from blood drops collected by finger prick. Findings: We developed a cost-effective, scalable technology to produce S protein based on its stable expression in HEK293 cells. The S-UFRJ ELISA displayed 98.4% specificity and sensitivity above 90% already 10 days after symptoms onset, allowing early detection of anti-S IgG seroconversion. Endpoint titers were shown to correlate with virus neutralization assessed as PRNT90. There was excellent agreement between plasma and DBS samples, significantly simplifying sample collection, storing and shipping. The overall cost per test was estimated to be approximately one US dollar. Interpretation: The S-UFRJ assay developed herein meets the quality requirements of high sensitivity and specificity. The low cost and the use of mailable DBS samples allow for serological surveillance of populations regardless of geographical and socio-economic aspects, with special relevance for public health policy actions in low-income countries.

Evolution and epidemic spread of SARS-CoV-2 in Brazil (preprint)

Brazil currently has one of the fastest growing SARS-CoV-2 epidemics in the world. Due to limited available data, assessments of the impact of non-pharmaceutical interventions (NPIs) on virus transmission and epidemic spread remain challenging. We investigate the impact of NPIs in Brazil using epidemiological, mobility and genomic data. Mobility-driven transmission models for Sao Paulo and Rio de Janeiro cities show that the reproduction number (Rt) reached below 1 following NPIs but slowly increased to values between 1 to 1.3 (1.0 - -1.6). Genome sequencing of 427 new genomes and analysis of a geographically representative genomic dataset from 21 of the 27 Brazilian states identified >100 international introductions of SARS-CoV-2 in Brazil. We estimate that three clades introduced from Europe emerged between 22 and 27 February 2020, and were already well-established before the implementation of NPIs and travel bans. During this first phase of the epidemic establishment of SARS-CoV-2 in Brazil, we find that the virus spread mostly locally and within-state borders. Despite sharp decreases in national air travel during this period, we detected a 25% increase in the average distance travelled by air passengers during this time period. This coincided with the spread of SARS-CoV-2 from large urban centers to the rest of the country. In conclusion, our results shed light on the role of large and highly connected populated centres in the rapid ignition and establishment of SARS-CoV-2, and provide evidence that current interventions remain insufficient to keep virus transmission under control in Brazil.

Ultrastructural analysis of SARS-CoV-2 interactions with the host cell via high resolution scanning electron microscopy (preprint)

SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 hours after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2. HighlightsWe used high resolution scanning electron microscopy to investigate Vero cells infected with SARS-CoV-2 at 2 and 48 hours post-infection. The central conclusions of this work include: O_LIInfected cells display polarization of their cytosol forming a restricted viroplasm-like zone dedicated to virus production and morphogenesis. C_LIO_LIThis is the first demonstration of SARS-CoV-2 attachment by scanning electron microscopy. C_LIO_LIThis is the first scanning electron microscopy images of the interior of SARS-CoV-2 infected cells and exploration of their vacuole contents. C_LIO_LIPerspective-viewing of bordering vesicles in close association with vacuoles. C_LIO_LIObservation of membrane ruffles and structures suggestive of exocytosis on the surface of infected cells. C_LIO_LIThe first demonstration of viral surfing in cell-to-cell communication on SARS-CoV-2 infection. C_LI