Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

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.

Hydroxypropyl-beta-cyclodextrin (HP-BCD) inhibits SARS-CoV-2 replication and virus-induced inflammatory cytokines.

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.

Recombinaçäo e seleçäo durante a infecçäo pelo HIV-1: analises de resultados "in vivo" e "in vitro" / Recombination and selection in HIV-1 infections: analyzes of \"in vivo\" and \"in vitro\" results

A ocorrência de múltiplas infecçoes por HIV-1 em um único indivíduo sao freqüentemente descritas e representam um cenário ideal para estudos de evoluçao das diferentes populaçoes virais e a contribuiçao de cada um dos eventos genéticos; seleçao e recombinaçao, direcionando esta evoluçao. Neste trabalho foram realizados três estudos distintos que procuravam identificar os eventos de competiçao e recombinaçao entre diferentes subtipos de HIV-1 ou populaçoes distintas de um mesmo subtipo que estavam presentes em um único hospedeiro. No primeiro estudo verificamos que a co-infecçao de uma criança que recebeu dois diferentes estoques de sangue contaminado por HIV1 distintos pertencentes ao subtipo B levou a seleçao de uma populaçao viral que apresentou um padrao complexo de recombinaçao na regiao C2V3C3 do gene do envelope viral. Enquanto a regiao C4-V5 foi contribuída por apenas uma das populaçoes. A comparaçao dos regimes seletivos atuando em cada uma destas regioes desta populaçao viral após dois anos de infecçao, em relaçao a populaçao viral ocorrendo nos dois doadores após este mesmo período de tempo, demonstrou que a evoluçao da populaçao viral na receptora dupla foi direcionada por pressao negativa em ambas as regioes. No segundo estudo, amostras de diferentes tempos de três indivíduos de uma mesma família, epidemiologicamente relacionados, infectados com pelo menos dois subtipos distintos de HIV-1 foram analisadas. Pudemos observar um constante processo de competiçao entre as distintas populaçoes virais, com a flutuaçao da representatividade de cada um dos subtipos ao longo da infecçao...(au)