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Viruses
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SARS-CoV-2 Research in Brazil
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SARS-CoV-2 Research in Brazil

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "General Virology".

Deadline for manuscript submissions: closed (28 February 2023) | Viewed by 16841

Special Issue Editor

Dr. Fernando Spilki

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Guest Editor
Laboratório de Microbiologia Molecular, Instituto de Ciências da Saúde, Universidade Feevale, Novo Hamburgo 93525-075, Brazil
Interests: emerging viruses; pandemic preparedness; one-health
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Brazil was one of the countries most impacted by the COVID-19 pandemic. Successive waves with a large volume of SARS-CoV-2 infection cases and deaths occurred in the country, especially between the year 2020 and the first half of 2021. This process fortunately began to be circumvented as vaccination evolved in the country. Despite the difficulties, Brazilian science gave important answers, and Brazilian researchers definitely contributed to tackling the pandemic. Brazilian research groups were involved in diagnosis, epidemiological surveys, genomic surveillance, clinical studies of vaccines, in addition to relevant studies on the biology of infection and pathogenesis in vitro and in vivo. The country still has several initiatives in progress for the development of new vaccines, monitoring vaccine effectiveness, therapeutic strategies, long COVID-19, and continuous monitoring of the virus’ evolution. In this Special Issue, we intend to receive contributions from colleagues in the most diverse aspects of SARS-CoV-2 and COVID-19 in Brazil, seeking to compile some of Brazilian science’s relevant contributions to this topic.

Dr. Fernando Spilki
Guest Editor

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Keywords

  • SARS-CoV-2
  • Brazil
  • emerging viruses
  • COVID-19
  • pandemic

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Published Papers (6 papers)

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16 pages, 3392 KiB  
Article
Virulence Profiles of Wild-Type, P.1 and Delta SARS-CoV-2 Variants in K18-hACE2 Transgenic Mice
by Yasmin da Silva Santos, Thais Helena Martins Gamon, Marcela Santiago Pacheco de Azevedo, Bruna Larotonda Telezynski, Edmarcia Elisa de Souza, Danielle Bruna Leal de Oliveira, Jamille Gregório Dombrowski, Livia Rosa-Fernandes, Giuseppe Palmisano, Leonardo José de Moura Carvalho, Maria Cecília Rui Luvizotto, Carsten Wrenger, Dimas Tadeu Covas, Rui Curi, Claudio Romero Farias Marinho, Edison Luiz Durigon and Sabrina Epiphanio
Viruses 2023, 15(4), 999; https://doi.org/10.3390/v15040999 - 19 Apr 2023
Cited by 5 | Viewed by 2402
Abstract
Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and [...] Read more.
Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and the P.1 (Gamma) and Delta variants using infected K18-hACE2 mice. The clinical manifestations, behavior, virus load, pulmonary capacity, and histopathological alterations were analyzed. The P.1-infected mice showed weight loss and more severe clinical manifestations of COVID-19 than the Wt and Delta-infected mice. The respiratory capacity was reduced in the P.1-infected mice compared to the other groups. Pulmonary histological findings demonstrated that a more aggressive disease was generated by the P.1 and Delta variants compared to the Wt strain of the virus. The quantification of the SARS-CoV-2 viral copies varied greatly among the infected mice although it was higher in P.1-infected mice on the day of death. Our data revealed that K18-hACE2 mice infected with the P.1 variant develop a more severe infectious disease than those infected with the other variants, despite the significant heterogeneity among the mice. Full article
(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)
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Figure 1

Figure 1
<p>The P.1 variant induced a greater weight loss and clinical manifestation than the Delta variant and Wt strain in ACE2-transgenic mice. (<b>A</b>,<b>B</b>) The average of weight loss of K18-hACE2 transgenic mice infected intranasally with 10<sup>5</sup> PFU of the Wt strain and the P.1 and Delta variants. The P.1-infected mice were euthanized on the 5th dpi, and the Wt- and Delta-infected mice on the 7th dpi. Analysis of body weight (<b>B</b>) on the 5th dpi showed significant differences between the P.1 variant and the control. The clinical manifestations of the disease in K18-hACE2 mice were more pronounced in the group infected with the P.1 variant when compared to the Delta variant or the Wt strain (<b>C</b>). The probability of survival was reduced when the mice were infected with the P.1 variant compared to Delta or Wt strain (<b>D</b>). The hypothesis test performed by a one-way ANOVA, with Bonferroni multivariance analysis for the parametric variables, or Brown–Forsythe and Welch ANOVA tests with the Games–Howell multiple comparisons test for values with asymmetric distribution, the survival Log-rank (Mantel–Cox) test was applied, using GraphPad Prism 8.0, <span class="html-italic">p</span> &lt; 0.05 was considered significant (* <span class="html-italic">p</span> ≤ 0.05; ** <span class="html-italic">p</span> ≤ 0.005; **** <span class="html-italic">p</span> ≤ 0.0001).</p> Full article ">Figure 2
<p>The whole-body plethysmography indicates severe lung dysfunction in the P.1-infected mice. The evaluation of lung function for respiratory frequency (<b>A</b>), enhanced pause (Penh) (<b>B</b>), and exhalatory flow curve (Rpef) (<b>C</b>) in the Wt strain and the P.1 and Delta-infected K18-hACE mice. A multiple <span class="html-italic">t</span> test was applied, using GraphPad Prism 8.0; <span class="html-italic">p</span> &lt; 0.05, was considered significant (** <span class="html-italic">p</span> ≤ 0.005 and **** <span class="html-italic">p</span> ≤ 0.0001).</p> Full article ">Figure 3
<p>Histopathological changes in the lungs of K18-hACE2 mice after infection with the Wt strain, or the P.1 or Delta SARS-CoV-2 variants. (<b>A</b>) The sum of all histological parameters analyzed per group. (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>,<b>J</b>,<b>L</b>,<b>N</b>,<b>P</b>,<b>R</b>,<b>T</b>,<b>V</b>) Individual histological parameters were quantified in three groups of infected mice. (<b>C</b>,<b>E</b>,<b>G</b>,<b>I</b>,<b>K</b>,<b>M</b>,<b>O</b>,<b>Q</b>,<b>S</b>,<b>U</b>,<b>W</b>) Representative photomicrographs of SARS-CoV-2-infected mice lungs. (<b>C</b>) Severe interstitial pneumonia with a mixed inflammatory infiltrate vasculitis and peri-vascular infiltrate (asterisks) from a P.1-infected mouse on the 5th dpi (20×). (<b>E</b>) Interstitial pneumonia with thickness of alveolar septa (arrows), from a Wt-infected mouse on the 6th dpi (20×). (<b>G</b>) Interstitial pneumonia showing extensive inflammatory infiltrate and consolidation area (asterisk) from a Wt-infected mouse on the 7th dpi (20×). (<b>I</b>) Vein vasculitis and diapedesis (arrows) of polymorph and mononuclear cells from a Wt-infected mouse on the 7th dpi (40×). (<b>K</b>) The congestion (arrows) and severe bronchopneumonia with extensive inflammatory infiltrate and consolidation, showing exudate inside bronchioles (asterisks) from a P.1-infected mouse on the 5th dpi (20×). (<b>M</b>) Extensive and diffuse hemorrhagic areas (arrows) associated with inflammatory infiltrate from a Wt-infected mouse euthanized on the 5th dpi (20×). (<b>O</b>) Brownish hemosiderin pigments (arrows) visualized in an extensive hemorrhagic area in a Wt-infected mouse on the 5th dpi (40×). (<b>Q</b>) Alveolar oedema, evidenced by light eosinophilic areas (asterisks) and reactive alveolar macrophages (arrows) from a Wt-infected mouse on the 7th dpi (20×). (<b>U</b>) Emphysema alveolar areas (asterisks) in a Wt-infected mouse on the 7th dpi (20×). (<b>S</b>) White thrombus (arrow) surrounded by bleeding (asterisks) and congested areas. The right upper area shows emphysema in a P.1-infected mouse on the 5th dpi (20×). (<b>W</b>) Fibrin (arrow) and congestion areas (asterisks) in a P.1-infected mouse on the 5th dpi. Scale bars represent 40 μm (objective 20×) and 20μm (objective 40×). Wild type = Wt; dpi = days post infection. Data are representative of three independent experiments. Non-parametric variables were compared using Kruskal–Wallis test which was followed by Dunn’s post hoc test using GraphPad Prism 8.0, <span class="html-italic">p</span> &lt; 0.05 was considered significant (* <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.005).</p> Full article ">Figure 4
<p>Quantitative detection and viral load analysis in oral swabs and lungs of K18-hACE2 mice. (<b>A</b>) Viral load detection by RT-qPCR in oral swabs and lungs of K18-hACE2 mice infected with P.1 variant (<b>A</b>), Wt strain (<b>B</b>), and Delta variant (<b>C</b>) on the day of death (timepoint or endpoint). Test performed by Mann–Whittney test using GraphPad Prism 8.0, considering <span class="html-italic">p</span> &lt; 0.05 as significant (** <span class="html-italic">p</span> ≤ 0.005).</p> Full article ">
11 pages, 1111 KiB  
Article
Genomic Surveillance of SARS-CoV-2 in Healthcare Workers: A Critical Sentinel Group for Monitoring the SARS-CoV-2 Variant Shift
by Dayane Azevedo Padilha, Doris Sobral Marques Souza, Eric Kazuo Kawagoe, Vilmar Benetti Filho, Ariane Nicaretta Amorim, Fernando Hartmann Barazzetti, Marcos André Schörner, Sandra Bianchini Fernandes, Bruna Kellet Coelho, Darcita Buerger Rovaris, Marlei Pickler Debiase Dos Anjos, Juliana Righetto Moser, Fernanda Rosene Melo, Bianca Bittencourt De Souza, Dimitri da Costa Bessa, Fernando Henrique de Paula e Silva Mendes, Alexandra Crispim Boing, Antonio Fernando Boing, Josimari Telino de Lacerda, Guilherme Valle Moura, Daniela Carolina De Bastiani, Milene Höehr de Moraes, Luiz Felipe Valter De Oliveira, Renato Simões Moreira, Patricia Hermes Stoco, Maria Luiza Bazzo, Gislaine Fongaro and Glauber Wagneradd Show full author list remove Hide full author list
Viruses 2023, 15(4), 984; https://doi.org/10.3390/v15040984 - 17 Apr 2023
Cited by 1 | Viewed by 1790
Abstract
SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa [...] Read more.
SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa Catarina, southern Brazil, and evaluated the similarity between variants present in the population and healthcare workers (HCW). A total of 5291 sequenced genomes demonstrated the circulation of 55 strains and four variants of concern (Alpha, Delta, Gamma and Omicron—sublineages BA.1 and BA.2). The number of cases was relatively low in May 2021, but the number of deaths was higher with the Gamma variant. There was a significant increase in both numbers between December 2021 and February 2022, peaking in mid-January 2022, when the Omicron variant dominated. After May 2021, two distinct variant groups (Delta and Omicron) were observed, equally distributed among the five Santa Catarina mesoregions. Moreover, from November 2021 to February 2022, similar variant profiles between HCW and the general population were observed, and a quicker shift from Delta to Omicron in HCW than in the general population. This demonstrates the importance of HCW as a sentinel group for monitoring disease trends in the general population. Full article
(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)
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Figure 1

Figure 1
<p>Number of cases/deaths and variant distribution of SARS-CoV-2 from 1 May 2021 to 30 April 2022. (<b>A</b>) Daily cases and (<b>B</b>) daily deaths, according to the Boa Vista platform (<a href="https://dados.sc.gov.br" target="_blank">https://dados.sc.gov.br</a>, accessed on 15 December 2022). SARS-CoV-2 variant profile (<b>C</b>) by lineage and (<b>D</b>) by variant of concern (VOC).</p> Full article ">Figure 2
<p>Comparison of relative frequency for each SARS-CoV-2 lineage in healthcare workers and general population during the variant shift from Delta to Omicron.</p> Full article ">Figure 3
<p>Multidimensional scale (MDS) with Jaccard dissimilarity plot of genomes based on amino acids and presence/absence of mutations, deletions, and insertions—a comparison between healthcare workers and the general population of Santa Catarina, from November 2021 to February 2022. (<b>A</b>) Displaying Delta and Omicron variants (<b>B</b>) Omicron BA.1 lineage and sublineages.</p> Full article ">
16 pages, 2263 KiB  
Article
Identification of B-Cell Linear Epitopes in the Nucleocapsid (N) Protein B-Cell Linear Epitopes Conserved among the Main SARS-CoV-2 Variants
by Rodrigo N. Rodrigues-da-Silva, Fernando P. Conte, Gustavo da Silva, Ana L. Carneiro-Alencar, Paula R. Gomes, Sergio N. Kuriyama, Antonio A. F. Neto and Josué C. Lima-Junior
Viruses 2023, 15(4), 923; https://doi.org/10.3390/v15040923 - 6 Apr 2023
Cited by 4 | Viewed by 2377
Abstract
The Nucleocapsid (N) protein is highlighted as the main target for COVID-19 diagnosis by antigen detection due to its abundance in circulation early during infection. However, the effects of the described mutations in the N protein epitopes and the efficacy of antigen testing [...] Read more.
The Nucleocapsid (N) protein is highlighted as the main target for COVID-19 diagnosis by antigen detection due to its abundance in circulation early during infection. However, the effects of the described mutations in the N protein epitopes and the efficacy of antigen testing across SARS-CoV-2 variants remain controversial and poorly understood. Here, we used immunoinformatics to identify five epitopes in the SARS-CoV-2 N protein (N(34–48), N(89–104), N(185–197), N(277–287), and N(378–390)) and validate their reactivity against samples from COVID-19 convalescent patients. All identified epitopes are fully conserved in the main SARS-CoV-2 variants and highly conserved with SARS-CoV. Moreover, the epitopes N(185–197) and N(277–287) are highly conserved with MERS-CoV, while the epitopes N(34–48), N(89–104), N(277–287), and N(378–390) are lowly conserved with common cold coronaviruses (229E, NL63, OC43, HKU1). These data are in accordance with the observed conservation of amino acids recognized by the antibodies 7R98, 7N0R, and 7CR5, which are conserved in the SARS-CoV-2 variants, SARS-CoV and MERS-CoV but lowly conserved in common cold coronaviruses. Therefore, we support the antigen tests as a scalable solution for the population-level diagnosis of SARS-CoV-2, but we highlight the need to verify the cross-reactivity of these tests against the common cold coronaviruses. Full article
(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)
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Figure 1

Figure 1
<p>Location of predicted epitopes in SARS-CoV-2 N protein 3D structure. The protein chain is indicated by a gray cartoon and transparent surface. The locations of epitopes N<sub>(34–48)</sub>, N<sub>(89–104)</sub>, N<sub>(185–197)</sub>, N<sub>(277–287),</sub> and N<sub>(378–390)</sub> were indicated by colors blue, teal, purple, red, and orange, respectively. In the cartoon, round helices, flat sheets, and smooth loops are applied to allow better visualization of the predicted structure. Different rotations of the protein are shown in (<b>a</b>) and (<b>b</b>).</p> Full article ">Figure 2
<p>Heatmap of IgM and IgG reactivity indexes against synthetic epitopes. Values higher than 1 represent responder individuals and were indicated in the color scale, and non-responders were indicated by a light blue color.</p> Full article ">Figure 3
<p>Evaluation of natural immunogenicity of predicted epitopes. (<b>a</b>) Frequencies of IgM, IgG, and overall responders to N<sub>(34–48)</sub> (blue bar), N<sub>(89–104)</sub> (green bar), N<sub>(185–197)</sub> (gray bar), N<sub>(277–287)</sub> (red bar), and N<sub>(378–390)</sub> (light blue bar). The frequencies of responders to epitopes were compared by Fisher’s exact test, and statistical differences were indicated by asterisks: (*) = <span class="html-italic">p</span> &lt; 0.05; (**) = <span class="html-italic">p</span> &lt; 0.01; (***) = <span class="html-italic">p</span> &lt; 0.001. (<b>b</b>) IgM (white dots) and IgG (black dots) reactivity indexes against predicted epitopes. Responders to peptides were indicated above the traced line.</p> Full article ">Figure 4
<p>B-cell epitopes and key mutations in SARS-CoV-2 N protein—3D structure. The N protein was presented as cartoon (gray), in which the identified epitopes were represented by colored sticks: N<sub>(34–48)</sub> (Blue), N<sub>(89–104)</sub> (teal), N<sub>(185–197)</sub> (magenta), N<sub>(277–287)</sub> (red), and N<sub>(378–390)</sub> (orange). The positions of key mutations (D3, Q9, P13, Del31/33, D63, P80, E136, R203, G204, T205, G215, L230, S235, D377, and S413) were represented by green spheres. In the cartoon, round helices, flat sheets, and smooth loops are applied to allow better visualization of the 3D structure.</p> Full article ">
12 pages, 1186 KiB  
Article
Antibody Response to the SARS-CoV-2 Spike and Nucleocapsid Proteins in Patients with Different COVID-19 Clinical Profiles
by Sinei Ramos Soares, Maria Karoliny da Silva Torres, Sandra Souza Lima, Kevin Matheus Lima de Sarges, Erika Ferreira dos Santos, Mioni Thieli Figueiredo Magalhães de Brito, Andréa Luciana Soares da Silva, Mauro de Meira Leite, Flávia Póvoa da Costa, Marcos Henrique Damasceno Cantanhede, Rosilene da Silva, Adriana de Oliveira Lameira Veríssimo, Izaura Maria Vieira Cayres Vallinoto, Rosimar Neris Martins Feitosa, Juarez Antônio Simões Quaresma, Tânia do Socorro Souza Chaves, Giselle Maria Rachid Viana, Luiz Fábio Magno Falcão, Eduardo José Melo dos Santos, Antonio Carlos Rosário Vallinoto and Andréa Nazaré Monteiro Rangel da Silvaadd Show full author list remove Hide full author list
Viruses 2023, 15(4), 898; https://doi.org/10.3390/v15040898 - 31 Mar 2023
Cited by 4 | Viewed by 2296
Abstract
The first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Brazil was diagnosed on February 26, 2020. Due to the important epidemiological impact of COVID-19, the present study aimed to analyze the specificity of IgG [...] Read more.
The first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Brazil was diagnosed on February 26, 2020. Due to the important epidemiological impact of COVID-19, the present study aimed to analyze the specificity of IgG antibody responses to the S1, S2 and N proteins of SARS-CoV-2 in different COVID-19 clinical profiles. This study enrolled 136 individuals who were diagnosed with or without COVID-19 based on clinical findings and laboratory results and classified as asymptomatic or as having mild, moderate or severe disease. Data collection was performed through a semistructured questionnaire to obtain demographic information and main clinical manifestations. IgG antibody responses to the S1 and S2 subunits of the spike (S) protein and the nucleocapsid (N) protein were evaluated using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The results showed that among the participants, 87.5% (119/136) exhibited IgG responses to the S1 subunit and 88.25% (120/136) to N. Conversely, only 14.44% of the subjects (21/136) displayed S2 subunit responses. When analyzing the IgG antibody response while considering the different proteins of the virus, patients with severe disease had significantly higher antibody responses to N and S1 than asymptomatic individuals (p ≤ 0.0001), whereas most of the participants had low antibody titers against the S2 subunit. In addition, individuals with long COVID-19 showed a greater IgG response profile than those with symptomatology of a short duration. Based on the results of this study, it is concluded that levels of IgG antibodies may be related to the clinical evolution of COVID-19, with high levels of IgG antibodies against S1 and N in severe cases and in individuals with long COVID-19. Full article
(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)
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Figure 1

Figure 1
<p>Analysis of the total IgG response to SARS-CoV-2 S and N proteins in individuals with different clinical profiles of COVID-19. (<b>A</b>) Analysis of the total IgG response to the S1 subunit of S. (<b>B</b>) Analysis of the total IgG response to the S2 subunit of S. (<b>C</b>) Analysis of the total IgG response to N. Pos: Positive ELISA result. Neg: Negative ELISA result. IND: Indeterminate ELISA result. ns: Non-significant.</p> Full article ">Figure 2
<p>IgG antibody response to the (<b>A</b>) S1 and (<b>B</b>) S2 subunits and <b>(C)</b> N protein in individuals with different clinical profiles of COVID-19. Significance value <span class="html-italic">p</span> &lt; 0.002 ** and <span class="html-italic">p</span> &lt; 0.0001 ***. ns: Non-significant.</p> Full article ">Figure 3
<p>IgG antibody response to (<b>A</b>) (N = 56 Acute COVID; N = 30 Long COVID) the S1 and (<b>B</b>) (N = 13 Acute COVID; N = 8 Long COVID) S2 subunits and (<b>C</b>) (N = 59 Acute COVID; N = 31 Long COVID) the N protein in subjects with acute COVID-19 and long COVID-19. Significance value <span class="html-italic">p</span> &lt; 0.0001 ***. ns: Non-significant.</p> Full article ">
25 pages, 3453 KiB  
Article
Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling
by Carolline Soares Motta, Silvia Torices, Barbara Gomes da Rosa, Anne Caroline Marcos, Liandra Alvarez-Rosa, Michele Siqueira, Thaidy Moreno-Rodriguez, Aline da Rocha Matos, Braulia Costa Caetano, Jessica Santa Cruz de Carvalho Martins, Luis Gladulich, Erick Loiola, Olivia R. M. Bagshaw, Jeffrey A. Stuart, Marilda M. Siqueira, Joice Stipursky, Michal Toborek and Daniel Adesse
Viruses 2023, 15(3), 745; https://doi.org/10.3390/v15030745 - 14 Mar 2023
Cited by 17 | Viewed by 3700
Abstract
Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure [...] Read more.
Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood–brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19. Full article
(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)
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Figure 1
<p>Characterization of infectivity profile of HBMECs and Vero cells by SARS-CoV-2. (<b>A</b>) Cells were exposed to different MOIs of SARS-CoV-2 (variant D614G) and viral production, and release to supernatant was analyzed by RT-qPCR for Envelope (E) gene from 0 to 72 h post-infection (hpi). As compared to Vero cells, HBMECs showed a non-productive infection. (<b>B</b>) At desired time points (6 and 24 hpi), total RNA from HBMEC cultures and expression of Spike1 and E genes were analyzed by RT-qPCR. HBMECs exposed to MOI 0.1 showed an increase in the expression of these two transcripts at 24 hpi (<span class="html-italic">p</span> &gt; 0.05). (<b>C</b>) Evaluation of SARS-CoV-2 receptors expression in HBMECs after SARS-CoV-2 challenge. ACE2 mRNA had a significant decrease at 24 hpi with the MOI 0.1, which did not translate to protein levels (right panel). TMPRSS2 had a slight increase in protein content at 24 hpi. (<b>D</b>) Exposure to SARS-CoV-2 increased immunoreactivity for cleaved caspase-3 in Vero cells and HBMECs at 24 hpi. *: <span class="html-italic">p</span> &lt; 0.05; ****: <span class="html-italic">p</span> &lt; 0.0001, Two-Way ANOVA with Bonferroni post-test of at least five independent experiments.</p> Full article ">Figure 2
<p>Effects of SARS-CoV-2 on tight junctional proteins in Vero and HBMECs. (<b>A</b>)<b>:</b> Cells were stained for tight junction adaptor protein ZO-1 (red) and SARS-CoV-2 Spike1 (in green). ZO-1 was affected in infected cultures at 24 hpi, as shown in higher magnification in the insets. (<b>B</b>): Morphometrical analyses of ZO-1 fluorescence intensity and TiJOR in HBMECs (<b>B</b>) showed increased ZO-1 signal and TiJOR index 6 h after exposure to the MOI 0.01. Levels of mRNA encoding for ZO-1 and claudin-5 TJ genes remained unaffected by SARS-CoV-2 challenge (<b>C</b>), but a significant increase 6 h after exposure to MOI 0.1 was observed at the protein level (<b>D</b>). *: <span class="html-italic">p</span> &lt; 0.05 one-way ANOVA with Bonferroni post-test (in (<b>D</b>)) or two-way ANOVA with Bonferroni post-test (in (<b>B</b>,<b>C</b>)); ***: <span class="html-italic">p</span> &lt; 0.001; ****: <span class="html-italic">p</span> &lt; 0.0001, Two-Way ANOVA with Bonferroni post-test. Each symbol in (<b>C</b>,<b>D</b>) corresponds to independent cultures, and in (<b>B</b>) corresponds to microscopic field from four independent cultures. Representative blots in (<b>D</b>) from 3–4 independent experiments.</p> Full article ">Figure 3
<p>Transcriptomic profiling of SARS-CoV-2 challenge on HBMECs. Cells were exposed to MOIs 0.01 and 0.1 and analyzed by RNA-Seq. (<b>A</b>) Volcano plot depicting the overall profile of differentially expressed genes in cultures after 24 h exposure to the MOI 0.1, with up-regulated genes shown in purple and down-regulated—in green. (<b>B</b>) Heatmap diagram depicting expression levels of the most significantly altered genes by MOI 0.1 (3 right columns), as compared to uninfected controls (3 left columns). (<b>C</b>) Cnetplot visualization of functional enrichment results with up-regulated genes, depicting the functional correlation of genes with the most significant GO terms. (<b>D</b>) Enrichment functional analysis of GO terms most affected by SARS-CoV-2 challenge in HBMECs indicates inflammatory endothelial activation, as well as mitochondrial dysfunction and ribosomal-related gene expression. (<b>E</b>) RT-qPCR validation of most significantly altered genes detected in the RNA-Seq indicates activation of non-canonical NF-κB pathway, with massive increase in TNF-α, lymphotoxin B (LTB, or TNF-C), and downstream target genes, such as IL-6, CXCL1, -2, and -8. NFKB1 (p105/p50) and NFKB2 (p100/p52), as well as JUNB, showed no significant alteration in SARS-CoV-2-exposed cultures. *: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01; ****: <span class="html-italic">p</span> &lt; 0.0001, two-way ANOVA with Bonferroni post-test of at least 5 independent experiments. MOI: multiplicity of infection; GO: gene ontology.</p> Full article ">Figure 4
<p>Production of angiogenic-related molecules is modulated by SARS-CoV-2 in HBMECs. (<b>A</b>) Conditioned medium from Mock and SARS-CoV-2-exposed HBMEC cultures (both with MOI 0.01 and 0.1) were analyzed via Proteome Profiler Human Angiogenic Antibody Array and detected by chemoluminescence, each protein detected in duplicated spots. (<b>B</b>) Densitometric analysis of membranes in (<b>A</b>) revealed the analytes with the strongest signal and which were affected by the SARS-CoV-2 challenge. Spots labelled 1-15 in (<b>A</b>) correspond to the analytes depicted in (<b>B</b>). (<b>C</b>) RT-qPCR analysis of angiogenesis-related genes in HBMECs revealed that PTX3 and HIF-1α were increased following SARS-CoV-2 exposure. *: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01, two-way ANOVA with Bonferroni post-test of at least five independent experiments.</p> Full article ">Figure 5
<p>SARS-CoV-2-induced mitochondrial remodeling in HBMECs. Mitochondrial networks were detected by TOMM20 immunostaining (<b>A</b>) and TEM (<b>B</b>). MiNA analysis of TOMM20 revealed that exposure to SARS-CoV-2 induced an increase in mitochondrial footprint, branch length mean, and summed branch length (<b>C</b>). Mitochondrial density was calculated by TEM images (<b>D</b>), which also revealed increased fusion and association with multivesicular bodies (<b>B</b>). (<b>E</b>): RT-qPCR (<b>upper panel</b>) and western blotting (<b>lower panel</b>) analyses revealed that although fission-related genes (Fis1 and Drp1) were up-regulated in MOI 0.01-exposed cultures, only Mfn2 protein levels were increased in MOI 0.1-exposed cultures. TOMM20 protein levels also remained unaltered. *: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01; ****: <span class="html-italic">p</span> &lt; 0.0001, two-way ANOVA with Bonferroni post-test. Each symbol in graphs represents one cell (<b>C</b>), one mitochondrion (<b>D</b>), or one independent experiment (<b>E</b>). Bottom right panels depict blots from (<b>E</b>). Scale bars: 50 µm for (<b>A</b>) and 500 nm for (<b>B</b>).</p> Full article ">

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Case Report
Persistence of SARS-CoV-2 Antigens in the Nasal Mucosa of Eight Patients with Inflammatory Rhinopathy for over 80 Days following Mild COVID-19 Diagnosis
by Juliana Costa dos Santos, Marjory Ximenes Rabelo, Luana Mattana Sebben, Matheus Vinicius de Souza Carneiro, João Bosco Lopes Botelho, José Cardoso Neto, Anderson Nogueira Barbosa, Diego Monteiro de Carvalho and Gemilson Soares Pontes
Viruses 2023, 15(4), 899; https://doi.org/10.3390/v15040899 - 31 Mar 2023
Cited by 4 | Viewed by 2762
Abstract
The nasal mucosa is the main gateway for entry, replication and elimination of the SARS-CoV-2 virus, the pathogen that causes severe acute respiratory syndrome (COVID-19). The presence of the virus in the epithelium causes damage to the nasal mucosa and compromises mucociliary clearance. [...] Read more.
The nasal mucosa is the main gateway for entry, replication and elimination of the SARS-CoV-2 virus, the pathogen that causes severe acute respiratory syndrome (COVID-19). The presence of the virus in the epithelium causes damage to the nasal mucosa and compromises mucociliary clearance. The aim of this study was to investigate the presence of SARS-CoV-2 viral antigens in the nasal mucociliary mucosa of patients with a history of mild COVID-19 and persistent inflammatory rhinopathy. We evaluated eight adults without previous nasal diseases and with a history of COVID-19 and persistent olfactory dysfunction for more than 80 days after diagnosis of SARS-CoV-2 infection. Samples of the nasal mucosa were collected via brushing of the middle nasal concha. The detection of viral antigens was performed using immunofluorescence through confocal microscopy. Viral antigens were detected in the nasal mucosa of all patients. Persistent anosmia was observed in four patients. Our findings suggest that persistent SARS-CoV-2 antigens in the nasal mucosa of mild COVID-19 patients may lead to inflammatory rhinopathy and prolonged or relapsing anosmia. This study sheds light on the potential mechanisms underlying persistent symptoms of COVID-19 and highlights the importance of monitoring patients with persistent anosmia and nasal-related symptoms. Full article
(This article belongs to the Special Issue SARS-CoV-2 Research in Brazil)
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Figure 1

Figure 1
<p>Flowchart illustrating the study’s design and the total number of individuals included.</p> Full article ">Figure 2
<p>Clinical characteristics of patients complaining of mild post-COVID-19 olfactory disorders. (<b>A</b>) absolute frequency of clinical symptoms observed during application of the clinical-epidemiological questionnaire (A, B, C, D, E, F, G) and otorhinolaryngological physical examination (H, I). The letters inside the circle represent the <span class="html-italic">X</span>-axis and indicate which group each variable belongs to. The lines between the groups represent the <span class="html-italic">Y</span>-axis and indicate the absolute frequency of each variable. (<b>B</b>) Absolute frequency of surgery and previous nasal trauma. * patients’ perception of symptoms.</p> Full article ">Figure 3
<p>Nasal mucociliary mucosa of patients complaining of mild post-COVID-19 olfactory dysfunction. Negative control (<b>A</b>); positive control (<b>B</b>); patients (<b>C</b>–<b>E</b>). SARS-CoV-2 S/N proteins, in green; nucleic acid labeling (DAPI), in blue; ciliary structures-tubulin, in red. The “Merge” column shows all overlapping markups. Images (<b>C</b>–<b>E</b>) are representative of samples from 8 patients. The images were selected among a total of 24 images through three replicates. Scale bars represent 25 µm. Arrows indicate viral antigen (nucleocapsid and spike proteins) staining.</p> Full article ">
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