Análise da replicação viral das variantes de SARS-CoV-2 mediante tratamento com oligonucleotídeos específicos para transcription regulatory sequences (TRS)
Data
2023-12-13
Autores
Silva, Gabrielly Alves Rodrigues da 
Orientadores
Janini, Luiz Mário Ramos
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Em 2019, houve o surgimento de uma pandemia de origem zoonótica, cujo agente causador é
um coronavírus altamente patogênico e transmissível, o SARS-CoV-2, que se alastrou pelo
mundo e gerou impactos na saúde pública e na economia global. Ao longo da pandemia,
eclodiram diversas variações genéticas do vírus original, o que é preocupante pois pode afetar
a transmissibilidade, virulência e taxa de reinfecção, escapando da imunidade natural e da
induzida por vacinas. Segundo a Organização Mundial da Saúde (OMS), até então em 2023,
foram notificados cerca de 772 milhões de casos confirmados de COVID-19 e 6,9 milhões de
mortes. Em 05 de maio de 2023, devido às perspectivas positivas em relação à doença, a OMS
declarou o fim da Emergência de Saúde Pública de Importância Internacional (ESPII)
concernente à COVID-19. Esse fato, por sua vez, não exime a necessidade de estudos na área
haja vista que a doença continua sendo uma ameaça à saúde e sua propagação segue classificada
como uma pandemia. Atualmente, sabe-se que o genoma do SARS-CoV-2 é composto por um
RNA de polaridade positiva, organizado em duas regiões UTR, nas extremidades 5’ e 3’, com
14 quadros de leitura abertos (ORFs) antecedidos por transcription regulatory sequences
(TRSs). O ciclo de replicação viral, por sua vez, ocorre por meio da síntese contínua e
descontínua de RNA, sendo a última o foco do presente projeto. Na síntese descontínua do
RNA há a produção de RNAs subgenômicos (RNAsgs), que codificam proteínas estruturais.
Durante esse processo, a interação entre o complexo de transcrição e as regiões TRSs promove
a regulação da produção dos RNAs subgenômicos. Desse modo, o silenciamento gênico de
regiões específicas das TRSs visa a interferência na transcrição dos RNAsgs, com consequente
redução ou ausência da síntese das proteínas estruturais e acessórias necessárias para replicação
viral. Nesse contexto, o presente projeto teve como objetivo interferir no ciclo viral por meio
do tratamento com oligonucleotídeos para regiões TRSs de SARS-CoV-2. Para isso, foram
conduzidos ensaios de viabilidade celular, além dos experimentos de infecção viral e tratamento
com posterior quantificação da carga viral via titulação viral e técnica de RT-qPCR. Foram
utilizados 3 oligonucleotídeos e 1 scramble, todos possuindo mesma região alvo diferindo
apenas em tamanho, com exceção do último que configura um controle. A oligoterapia foi
efetuada após infecções de células Vero E6, pelos isolados virais de Wuhan, Delta e Ômicron
num MOI de 0,05 nas concentrações de 250 nM e 500 nM e em 2 horas pós-inoculação. Após
estes tempos houve as coletas do sobrenadante para análise (hpi 24h e 48h). Os
oligonucleotídeos testados não diminuíram a viabilidade celular do modelo experimental
utilizado nas mais diversas concentrações (desde 15,62 nM até 1.000 nM). Os ensaios
realizados evidenciaram atividade antiviral de todos os oligonucleotídeos, com reduções tanto
dos títulos virais quanto do número de cópias de genoma livre. O oligonucleotídeo OAS1 na
concentração de 250 nM após 24h foi a condição de tratamento que apresentou maior redução
do número de partículas virais viáveis. Em relação à resposta das variantes, tivemos atividade
antiviral dos oligonucleotídeos para Wuhan, Delta e Ômicron, destacando o OAS1 na
concentração de 250 nM, porém para Ômicron o tratamento a 500 nM foi mais efetivo. Nos
ensaios moleculares, todos os oligonucleotídeos utilizados evidenciaram reduções no número
de cópias de genoma livre viral. Para ambas as técnicas de quantificação aqui utilizadas, a
oligoterapia após 24 horas mostrou-se mais efetiva na redução de carga viral quando comparada
à de 48 horas. Quando compilados, consideramos que nossos resultados são importantes e
indicam que as TRSs podem ser objetos de estudos valiosos, não só em relação a possíveis
tratamentos, mas também em relação à biologia viral.
In 2019, a zoonotic-origin pandemic emerged, caused by a highly pathogenic and transmissible coronavirus, SARS-CoV-2, which spread worldwide and had impacts on public health and the global economy. Throughout the pandemic, various genetic variations of the original virus emerged, which is concerning as it can affect transmissibility, virulence, and reinfection rates, evading both natural and vaccine-induced immunity. According to the World Health Organization (WHO), as of 2023, approximately 772 million confirmed cases of COVID-19 and 6.9 million deaths had been reported. On May 5, 2023, due to positive developments regarding the disease, the WHO declared the end of the Public Health Emergency of International Concern (PHEIC) related to COVID-19. However, this does not exempt the need for continued studies as the disease remains a health threat, and its spread is still classified as a pandemic. Currently, it is known that the genome of SARS-CoV-2 consists of positive-sense RNA, organized into two UTR regions at the 5' and 3' ends, with 14 open reading frames (ORFs) preceded by transcription regulatory sequences (TRSs). The viral replication cycle involves both continuous and discontinuous RNA synthesis, with a focus on the latter in this project. Discontinuous RNA synthesis leads to the production of subgenomic RNAs (sgRNAs), which encode structural proteins. During this process, the interaction between the transcription complex and TRSs regulates the production of sgRNAs. Therefore, gene silencing of specific TRS regions aims to interfere with sgRNA transcription, resulting in a reduction or absence of structural and accessory protein synthesis necessary for viral replication. In this context, the present project aimed to interfere with the viral cycle through treatment with oligonucleotides targeting SARS-CoV-2 TRS regions. For this purpose, cell viability assays were conducted, along with viral infection experiments and treatment, followed by quantification of the viral load via viral titration and RT-qPCR techniques. Three oligonucleotides and one scramble were used, all targeting the same region but differing only in size, with the last serving as a control. Oligotherapy was performed after Vero E6 cell infections with Wuhan, Delta, and Omicron viral isolates at an MOI of 0.05, at concentrations of 250 nM and 500 nM, and 2 hours post inoculation. Supernatant samples were collected for analysis at 24h and 48h post-inoculation. The tested oligonucleotides did not decrease cell viability at various concentrations (from 15.62 nM to 1,000 nM). The experiments showed antiviral activity of all oligonucleotides, with reductions in viral titers and the number of free genome copies. Oligonucleotide OAS1 at a concentration of 250 nM after 24h exhibited the highest reduction in viable viral particles. Regarding variant responses, antiviral activity was observed for Wuhan, Delta, and Omicron with the oligonucleotides, highlighting OAS1 at 250 nM, but for Omicron, 500 nM treatment was more effective. Molecular assays demonstrated reductions in the number of free viral genome copies for all used oligonucleotides. In both quantification techniques used, oligotherapy after 24 hours proved more effective in reducing viral load compared to 48 hours. In summary, these results suggest that TRSs could be valuable subjects of study, not only for potential treatments but also for viral biology.
In 2019, a zoonotic-origin pandemic emerged, caused by a highly pathogenic and transmissible coronavirus, SARS-CoV-2, which spread worldwide and had impacts on public health and the global economy. Throughout the pandemic, various genetic variations of the original virus emerged, which is concerning as it can affect transmissibility, virulence, and reinfection rates, evading both natural and vaccine-induced immunity. According to the World Health Organization (WHO), as of 2023, approximately 772 million confirmed cases of COVID-19 and 6.9 million deaths had been reported. On May 5, 2023, due to positive developments regarding the disease, the WHO declared the end of the Public Health Emergency of International Concern (PHEIC) related to COVID-19. However, this does not exempt the need for continued studies as the disease remains a health threat, and its spread is still classified as a pandemic. Currently, it is known that the genome of SARS-CoV-2 consists of positive-sense RNA, organized into two UTR regions at the 5' and 3' ends, with 14 open reading frames (ORFs) preceded by transcription regulatory sequences (TRSs). The viral replication cycle involves both continuous and discontinuous RNA synthesis, with a focus on the latter in this project. Discontinuous RNA synthesis leads to the production of subgenomic RNAs (sgRNAs), which encode structural proteins. During this process, the interaction between the transcription complex and TRSs regulates the production of sgRNAs. Therefore, gene silencing of specific TRS regions aims to interfere with sgRNA transcription, resulting in a reduction or absence of structural and accessory protein synthesis necessary for viral replication. In this context, the present project aimed to interfere with the viral cycle through treatment with oligonucleotides targeting SARS-CoV-2 TRS regions. For this purpose, cell viability assays were conducted, along with viral infection experiments and treatment, followed by quantification of the viral load via viral titration and RT-qPCR techniques. Three oligonucleotides and one scramble were used, all targeting the same region but differing only in size, with the last serving as a control. Oligotherapy was performed after Vero E6 cell infections with Wuhan, Delta, and Omicron viral isolates at an MOI of 0.05, at concentrations of 250 nM and 500 nM, and 2 hours post inoculation. Supernatant samples were collected for analysis at 24h and 48h post-inoculation. The tested oligonucleotides did not decrease cell viability at various concentrations (from 15.62 nM to 1,000 nM). The experiments showed antiviral activity of all oligonucleotides, with reductions in viral titers and the number of free genome copies. Oligonucleotide OAS1 at a concentration of 250 nM after 24h exhibited the highest reduction in viable viral particles. Regarding variant responses, antiviral activity was observed for Wuhan, Delta, and Omicron with the oligonucleotides, highlighting OAS1 at 250 nM, but for Omicron, 500 nM treatment was more effective. Molecular assays demonstrated reductions in the number of free viral genome copies for all used oligonucleotides. In both quantification techniques used, oligotherapy after 24 hours proved more effective in reducing viral load compared to 48 hours. In summary, these results suggest that TRSs could be valuable subjects of study, not only for potential treatments but also for viral biology.