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Article ; Online: SARS-CoV-2 egress from Vero cells: a morphological approach.

Caldas, Lucio Ayres / Carneiro, Fabiana Avila / Augusto, Ingrid / Corrêa, Isadora Alonso / da Costa, Luciana Jesus / Miranda, Kildare / Tanuri, Amilcar / de Souza, Wanderley

Histochemistry and cell biology

2023 Volume 161, Issue 1, Page(s) 59–67

Abstract: Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is ... ...

Abstract	Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.
MeSH term(s)	Animals ; Chlorocebus aethiops ; Vero Cells ; SARS-CoV-2 ; Cell Line ; COVID-19 ; Microscopy, Electron, Scanning ; Mammals
Language	English
Publishing date	2023-09-22
Publishing country	Germany
Document type	Journal Article
ZDB-ID	1222930-1
ISSN	1432-119X ; 0301-5564 ; 0948-6143
ISSN (online)	1432-119X
ISSN	0301-5564 ; 0948-6143
DOI	10.1007/s00418-023-02239-9
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Article ; Online: Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

Miranda, Guilherme A S C / Corrêa, Isadora Alonso / Amorim, Érica Almeida / Caldas, Lucio Ayres / Carneiro, Fabiana Ávila / da Costa, Luciana Jesus / Granjeiro, José Mauro / Tanuri, Amilcar / de Souza, Wanderley / Baptista, Leandra Santos

Artificial organs

2024

Abstract: Background: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects ... ...

Abstract	Background: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. Methods: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. Results: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. Conclusion: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.
Language	English
Publishing date	2024-02-22
Publishing country	United States
Document type	Journal Article
ZDB-ID	441812-8
ISSN	1525-1594 ; 0160-564X
ISSN (online)	1525-1594
ISSN	0160-564X
DOI	10.1111/aor.14729
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Article: SARS-CoV-2 Variant Determination Through SNP Assays in Samples From Industry Workers From Rio de Janeiro, Brazil.

Henriques-Santos, Bianca Monteiro / Farjun, Bruna / Corrêa, Isadora Alonso / Figueiredo, Janaina de Barros / Fidalgo-Neto, Antonio Augusto / Kuriyama, Sergio Noboru

Frontiers in microbiology

2022 Volume 12, Page(s) 757783

Abstract: Since the first reported case in December 2019, SARS-CoV-2 infections have become a major public health worldwide. Even with the increasing vaccination in several countries and relaxing of social distancing measures, the pandemic remains a threat ... ...

Abstract	Since the first reported case in December 2019, SARS-CoV-2 infections have become a major public health worldwide. Even with the increasing vaccination in several countries and relaxing of social distancing measures, the pandemic remains a threat especially due to the emergence of new SARS-CoV-2 variants. Despite the presence of an enzyme capable of proofreading its genome, high rates of replication provide a source of accumulation of mutations within the viral genome. In this retrospective study, samples from a cohort of industry workers tested by the SESI's COVID-19 mass testing program from September 2020 to May 2021 were analyzed using a mutation panel in order to describe the circulation of currently identified SARS-CoV-2 variants within the samples obtained in Rio de Janeiro State. Our results demonstrated that the variant of interest (VOI) Zeta has been in circulation since October 2020 and reached 87% of prevalence in February 2021 followed by a decrease due to the emergence of Gamma variant of concern (VOC). Gamma was detected in January 2021 in our studied population, and its prevalence increased during the following months, reaching absolute prevalence within positive samples in May. The Alpha variant was detected only in 4-7% of samples during March and April while Beta VOC was not detected in our study. Our data agree with sequencing genomic surveillance databases and highlight the importance of continuous mass testing programs and variant detection in order to control viral spread and guide public health measures.
Language	English
Publishing date	2022-02-09
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2021.757783
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Article ; Online: Boosting SARS-CoV-2 detection combining pooling and multiplex strategies.

Correa, Isadora Alonso / de Souza Rodrigues, Tamires / Queiroz, Alex / de França Nascimento, Leon / Wolff, Thiago / Akamine, Rubens Nobumoto / Kuriyama, Sergio Noboru / da Costa, Luciana Jesus / Fidalgo-Neto, Antonio Augusto

Scientific reports

2022 Volume 12, Issue 1, Page(s) 8684

Abstract: RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for ... ...

Abstract	RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario.
MeSH term(s)	Brazil/epidemiology ; COVID-19/diagnosis ; COVID-19/epidemiology ; COVID-19 Testing ; Humans ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; Specimen Handling/methods
Chemical Substances	RNA, Viral
Language	English
Publishing date	2022-05-23
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-12747-8
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Article ; Online: Hydroxypropyl-beta-cyclodextrin (HP-BCD) inhibits SARS-CoV-2 replication and virus-induced inflammatory cytokines.

Bezerra, Bruno Braz / Silva, Gustavo Peixoto Duarte da / Coelho, Sharton Vinicius Antunes / Correa, Isadora Alonso / Souza, Marcos Romario Matos de / Macedo, Keylla Vitória Gomes / Matos, Bruna Machado / Tanuri, Amilcar / Matassoli, Flavio Lemos / Costa, Luciana Jesus da / Hildreth, James E K / Arruda, Luciana Barros de

Antiviral research

2022 Volume 205, Page(s) 105373

Abstract: COVID-19 is marked by extensive damage to the respiratory system, often accompanied by systemic manifestations, due to both viral cytopathic effects and hyperinflammatory syndrome. Therefore, the development of new therapeutic strategies or drug ... ...

Abstract	COVID-19 is marked by extensive damage to the respiratory system, often accompanied by systemic manifestations, due to both viral cytopathic effects and hyperinflammatory syndrome. Therefore, the development of new therapeutic strategies or drug repurposing aiming to control virus replication and inflammation are required to mitigate the impact of the disease. Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol-sequestering agent with antiviral activity that has been demonstrated against enveloped viruses in in vitro and in vivo experimental models. We also demonstrated that HP-BCD has an immunomodulatory effect, inhibiting the production of selected proinflammatory cytokines induced by microbial products. Importantly, this drug has been used in humans for decades as an excipient in drug delivery systems and as a therapeutic agent in the treatment of Niemann pick C disease. The safety profile for this compound is well established. Here, we investigated whether HP-BCD would affect SARS-CoV-2 replication and virus-induced inflammatory response, using established cell lines and primary human cells. Treating virus or cells with HP-BCD significantly inhibited SARS-CoV-2 replication with a high selective index. A broad activity against distinct SARS-CoV-2 variants was evidenced by a remarkable reduction in the release of infectious particles. The drug did not alter ACE2 surface expression, but affected cholesterol accumulation into intracellular replication complexes, lowering virus RNA and protein levels, and reducing virus-induced cytopathic effects. Virus replication was also impaired by HP-BCD in Calu-3 pulmonary cell line and human primary monocytes, in which not only the virus, but also the production of proinflammatory cytokines were significantly inhibited. Given the pathophysiology of COVID-19 disease, these data indicate that the use HP-BCD, which inhibits both SARS-CoV2 replication and production of proinflammatory cytokines, as a potential COVID-19 therapeutic warrants further investigation.
MeSH term(s)	2-Hydroxypropyl-beta-cyclodextrin/pharmacology ; Cholesterol/metabolism ; Cytokines/metabolism ; Humans ; RNA, Viral ; SARS-CoV-2 ; Virus Replication ; COVID-19 Drug Treatment
Chemical Substances	Cytokines ; RNA, Viral ; 2-Hydroxypropyl-beta-cyclodextrin (1I96OHX6EK) ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2022-07-04
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	306628-9
ISSN	1872-9096 ; 0166-3542
ISSN (online)	1872-9096
ISSN	0166-3542
DOI	10.1016/j.antiviral.2022.105373
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Article ; Online: Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

Simas, Maria Clara da Costa / Costa, Sara Mesquita / Gomes, Priscila da Silva Figueiredo Celestino / Cruz, Nádia Vaez Gonçalves da / Corrêa, Isadora Alonso / de Souza, Marcos Romário Matos / Dornelas-Ribeiro, Marcos / Nogueira, Tatiana Lucia Santos / Santos, Caleb Guedes Miranda Dos / Hoffmann, Luísa / Tanuri, Amilcar / Moura-Neto, Rodrigo Soares de / Damaso, Clarissa R / Costa, Luciana Jesus da / Silva, Rosane

Viruses

2023 Volume 15, Issue 3

Abstract: The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported ... ...

Abstract	The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.
MeSH term(s)	Humans ; Cell Culture Techniques ; COVID-19 ; SARS-CoV-2/genetics ; Sequence Analysis ; Sequence Deletion ; Viral Proteins/genetics ; Subgenomic RNA/genetics
Chemical Substances	ORF7a protein, SARS-CoV-2 ; Viral Proteins ; Subgenomic RNA
Language	English
Publishing date	2023-03-21
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v15030801
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Article: Chikungunya Virus: An Emergent Arbovirus to the South American Continent and a Continuous Threat to the World.

Cunha, Marcela S / Costa, Pedro A G / Correa, Isadora Alonso / de Souza, Marcos R M / Calil, Pedro Teles / da Silva, Gustavo P Duarte / Costa, Sara Mesquita / Fonseca, Vinícius Wakoff P / da Costa, Luciana J

Frontiers in microbiology

2020 Volume 11, Page(s) 1297

Abstract: Chikungunya virus (CHIKV) is an arthropod-borne virus (arbovirus) of epidemic concern, transmitted ... ...

Abstract	Chikungunya virus (CHIKV) is an arthropod-borne virus (arbovirus) of epidemic concern, transmitted by
Language	English
Publishing date	2020-06-26
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2020.01297
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Article ; Online: HIV-1 genetic diversity and resistance to antiretroviral drugs among pregnant women in Ribeirão Preto (SP), Brazil. Cross-sectional study.

Pimenta, Ana Teresa Mancini / Correa, Isadora Alonso / Melli, Patricia Pereira Dos Santos / Abduch, Renata / Duarte, Geraldo / Couto-Fernandez, José Carlos / Quintana, Silvana Maria

Sao Paulo medical journal = Revista paulista de medicina

2017 Volume 136, Issue 2, Page(s) 129–135

Abstract: Background: Increasing genetic diversity of HIV-1 and emergence of drug-resistant mutations may reduce the efficacy of antiretroviral therapy and prophylaxis that are used to prevent mother-to-child transmission. The aim of this study was to assess the ... ...

Abstract	Background: Increasing genetic diversity of HIV-1 and emergence of drug-resistant mutations may reduce the efficacy of antiretroviral therapy and prophylaxis that are used to prevent mother-to-child transmission. The aim of this study was to assess the genetic diversity and prevalence of drug-resistant mutations among HIV-infected pregnant women. Design and setting: Cross-sectional study at an outpatient clinic for infectious diseases within gynecology and obstetrics. Methods: This study evaluated the dynamics of HIV-1 subtypes and the prevalence of transmitted and acquired drug-resistant mutations among 38 HIV-infected pregnant women (20 previously exposed to antiretroviral therapy and 18 naive), in Ribeirão Preto (SP), Brazil, between 2010 and 2011. Genotyping was performed by means of molecular sequencing of the protease and reverse transcriptase regions of the HIV-1 pol gene. Results: Subtype B was identified in 84.2% of the samples, recombinant forms between B and F in 7.9%, subtype F1 in 5.3% and the recombinant form K/F in 2.6%. No mutation associated with transmitted drug resistance was detected in the samples from the naive pregnant women, whereas mutations associated with acquired drug resistance were found in 35.0% of the pregnant women previously exposed to antiretroviral therapy. Conclusion: The results showed that subtype B predominated, while there was low prevalence of sequences with transmitted drug resistance.
MeSH term(s)	Anti-HIV Agents/therapeutic use ; Cross-Sectional Studies ; Drug Resistance, Viral/genetics ; Female ; Genetic Variation ; Genotype ; HIV Infections/drug therapy ; HIV Infections/virology ; HIV-1/drug effects ; HIV-1/genetics ; Humans ; Mutation/genetics ; Phylogeny ; Pregnancy ; Pregnancy Complications, Infectious/drug therapy ; Pregnancy Complications, Infectious/virology ; Prevalence ; RNA, Viral/genetics ; Socioeconomic Factors
Chemical Substances	Anti-HIV Agents ; RNA, Viral
Language	English
Publishing date	2017-01-22
Publishing country	Brazil
Document type	Journal Article
ZDB-ID	1203171-9
ISSN	1806-9460 ; 1516-3180 ; 0035-0362
ISSN (online)	1806-9460
ISSN	1516-3180 ; 0035-0362
DOI	10.1590/1516-3180.2017.0233011017
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Article ; Online: Unique RNA replication characteristics and nucleocapsid protein expression may explain differences in the replication capacity of SARS-COV-2 lineages.

Correa, Isadora Alonso / de Souza, Marcos Romario Matos / da Silva, Gustavo Peixoto Duarte / Pimentel, Anna Beatriz Sampaio Vianna Macedo / Calil, Pedro Telles / Cunha, Marcela Sabino / Mariani, Diana / Brindeiro, Rodrigode Moares / Costa, Sara Mesquita / Simas, Maria Clara da Costa / Ota, Victor Akira / Pereira, Elisa Cavalcante / Siqueira, Marilda Mendonca / Resende, Paola Cristina / Galliez, Rafael Mello / Faffe, Debora Souza / Silva, Rosane Jesus / Castineiras, Terezinha Marta Pereira Pinto / Tanuri, Amilcar Jesus /

da Costa, Luciana Jesus

bioRxiv

Abstract: COVID-19 pandemic in Brazil was characterized by the sequential circulation of the SARS-CoV-2 lineages B.1.1.33, and variants Zeta (P.2), Gamma (P.1/P.1.*), Delta (B.1.617.2/AY.*), and Omicron (BA.*). Our research aimed to compare the biological traits ... ...

Abstract	COVID-19 pandemic in Brazil was characterized by the sequential circulation of the SARS-CoV-2 lineages B.1.1.33, and variants Zeta (P.2), Gamma (P.1/P.1.), Delta (B.1.617.2/AY.), and Omicron (BA.*). Our research aimed to compare the biological traits of these lineages and variants by analyzing aspects of viral replication including binding, entry, RNA replication, and viral protein production. We demonstrated that the replication capacity of these variants varies depending on the cell type, with Omicron BA.1 exhibiting the lowest replication in the human pulmonary cells. Additionally, the nucleocapsid proteoforms generated during infection exhibit distinct patterns across variants. Our findings suggest that factors beyond the initial stages of virus entry influence the efficiency of viral replication among different SARS-CoV-2 variants. Thus, our study underscores the significance of RNA replication and the role of nucleocapsid proteins in shaping the replicative characteristics of SARS-CoV-2 variants.
Keywords	covid19
Language	English
Publishing date	2024-05-14
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2024.05.14.594070
Database	COVID19

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Article ; Online: Boosting SARS-CoV-2 qRT-PCR detection combining pool sample strategy and mathematical modeling

Correa, Isadora Alonso / Rodrigues, Tamires de Souza / Queiroz, Alex / Nascimento, Leon de Franca / Wolff, Thiago / Akamine, Rubens Nobumoto / Kuriyama, Sergio Noboru / Costa, Luciana / Fidalgo-Neto, Antonio Augusto

medRxiv

Abstract: qRT-PCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in the actual pandemic scenario. Due to high testing demand, pooling ... ...

Abstract	qRT-PCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in the actual pandemic scenario. Due to high testing demand, pooling sample strategy is an interesting approach to allow cost savings. We aim to evaluate pooling tests in experimental procedures, as well as perform in silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals was observed, the high-test sensitivity is maintained even when 16 and 8 samples were pooled. The in silico analysis showed high-cost savings in populations with positive rates lower than 15.0% according to the pool size. This data was validated with the results obtained in our mass testing program: statistical modeling predicted a cost saving of 48.0%, which in practice, was 51.5%, already considering the expenditures with pool sampling that were analyzed individually. Our data confirmed that mathematical modeling is a powerful strategy to improve the pooling approach for SARS-CoV-2 mass testing around the world while maintaining high sensitivity and robustness.
Keywords	covid19
Language	English
Publishing date	2020-08-19
Publisher	Cold Spring Harbor Laboratory Press
Document type	Article ; Online
DOI	10.1101/2020.08.16.20167536
Database	COVID19

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