Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2

Influenza A virus’s (IAV’s) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV’s genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem–loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term ‘programmable antivirals’, with implications for antiviral prophylaxis and post-exposure therapy

Avaliação da função de macrófagos alveolares em cavalos clinicamente sadios Evaluation of alveolar macrophage function in healthy horses

Devido à importância dos macrófagos alveolares (MA) nos mecanismos de defesa pulmonar, foram realizados estudos para avaliar a atividade desses fagócitos em cavalos hígidos. Foram realizados lavados broncoalveolares (LBA) em cinco cavalos clinicamente sadios. A citologia foi realizada pela citocentrifugação das amostras e posterior confecção de lâminas coradas pelo método de Rosenfeld. Todas as amostras do LBA foram centrifugadas e a concentração celular foi ajustada para 2×10(6) células/ml, para a mensuração da atividade macrofágica (testes de espraiamento, fagocitose e liberação de peróxido de hidrogênio). A contagem diferencial das células presentes no LBA demonstrou a predominância de macrófagos (59,0&plusmn; 6,9%). Os resultados obtidos nos testes de mensuração da atividade macrofágica foram: índice de espraiamento 25,1&plusmn; 19,7%, índice de fagocitose 89,4&plusmn; 6,2% e liberação de peróxido de hidrogênio 1,6&plusmn; 0,3nmoles/2×10(5) células (sem PMA - phorbol 12-myristate 13-acetate) e 1,8&plusmn; 0,4nmoles/2×10(5) células (com PMA). Os resultados demonstraram um padrão de atividade para MA de cavalos hígidos, os quais apresentaram índices de ativação mesmo sem elicitação prévia, indicando que as técnicas utilizadas foram adequadas para tal propósito.<br>Due to the importance of alveolar macrophages (AM) in pulmonary defense mechanisms, studies were performed in order to evaluate the activity of these cells. Bronchoalveolar lavages (BAL) were obtained from five healthy horses, and cytology was performed on glass slides after cytocentrifugation of the samples. Slides were stained by Rosenfeld. All BAL samples were centrifuged and cell concentration was adjusted to 2×10(6) cells/ml, for the measurement of AM activity (spreading, phagocytosis and hydrogen peroxide release tests). Differential counting of the BAL cells demonstrated that macrophages were the predominant type of cell (59.0&plusmn; 6.9%). Measurement of AM activity presented the following results: spreading rate, 25.1&plusmn; 19.7%, phagocytosis rate, 89.4&plusmn; 6.2% and hydrogen peroxide release, 1.6&plusmn; 0.3nmoles/2×10(5) cells (without PMA- phorbol 12-myristate 13-acetate) and 1.8&plusmn; 0.4nmoles/2×10(5) cells (with PMA). Results presented a pattern for the activity of AM in healthy horses enabling the demonstration of an activation rate even without known previous eliciting factors. These results indicate that the tests of macrophage activity measurement are adequate for evaluation of phagocytic activity of AMs