Total Osteopontin and Its Isoform OPN4 Are Differently Expressed in Respiratory Samples during Influenza A(H1N1)pdm09 Infection and Progression.

Influenza A virus (IAV) infection affects the human respiratory tract, causing an acute and highly contagious disease. Individuals with comorbidities and in the extremes of age are classified as risk groups for serious clinical outcomes. However, part of the severe infections and fatalities are observed among young healthy individuals. Noteworthy, influenza infections lack specific prognostic biomarkers that would predict the disease severity. Osteopontin (OPN) has been proposed as a biomarker in a few human malignancies and its differential modulation has been observed during viral infections. However, OPN expression levels in the primary site of IAV infection have not been previously investigated. Therefore, we evaluated the transcriptional expression patterns of total OPN (tOPN) and its splicing isoforms (OPNa, OPNb, OPNc, OPN4, and OPN5) in 176 respiratory secretion samples collected from human influenza A(H1N1)pdm09 cases and a group of 65 IAV-negative controls. IAV samples were differentially classified according to their disease severity. tOPN was more frequently detected in IAV samples (34.1%) when compared with the negative controls (18.5%) (p < 0.05), as well as in fatal (59.1%) versus non-fatal IAV samples (30.5%) (p < 0.01). OPN4 splice variant transcript was more prevalent in IAV cases (78.4%) than in the negative controls (66.1%) (p = 0.05) and in severe cases (85.7%) in relation to the non-severe ones (69.2%) (p < 0.01). OPN4 detection was also associated with severity symptoms such as dyspnea (p < 0.05), respiratory failure (p < 0.05), and oxygen saturation < 95% (p < 0.05). In addition, the OPN4 expression level was increased in the fatal cases of respiratory samples. Our data indicated that tOPN and OPN4 had a more pronounced expression pattern in IAV respiratory samples, pointing to the potential use of these molecules as biomarkers to evaluate disease outcomes.

Identification of Hypericin as a Candidate Repurposed Therapeutic Agent for COVID-19 and Its Potential Anti-SARS-CoV-2 Activity.

The COVID-19 pandemic has had an unprecedented impact on the global economy and public health. Its etiologic agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible, pathogenic and has a rapid global spread. Currently, the increase in the number of new confirmed cases has been slowed down due to the increase of vaccination in some regions of the world. Still, the rise of new variants has influenced the detection of additional waves of rising cases that some countries have experienced. Since the virus replication cycle is composed of many distinct stages, some viral proteins related to them, as the main-protease (Mpro) and RNA dependent RNA polymerase (RdRp), constitute individual potential antiviral targets. In this study, we challenged the mentioned enzymes against compounds pre-approved by health regulatory agencies in a virtual screening and later in Molecular Mechanics/Poisson-Bolzmann Surface Area (MM/PBSA) analysis. Our results showed that, among the identified potential drugs with anti-SARS-CoV-2 properties, Hypericin, an important component of the Hypericum perforatum that presents antiviral and antitumoral properties, binds with high affinity to viral Mpro and RdRp. Furthermore, we evaluated the activity of Hypericin anti-SARS-CoV-2 replication in an in vitro model of Vero-E6 infected cells. Therefore, we show that Hypericin inhibited viral replication in a dose dependent manner. Moreover, the cytotoxicity of the compound, in cultured cells, was evaluated, but no significant activity was found. Thus, the results observed in this study indicate that Hypericin is an excellent candidate for repurposing for the treatment of COVID-19, with possible inhibition of two important phases of virus maturation.

Low prevalence of influenza A strains with resistance markers in Brazil during 2017-2019 seasons.

The influenza A virus (IAV) is of a major public health concern as it causes annual epidemics and has the potential to cause pandemics. At present, the neuraminidase inhibitors (NAIs) are the most widely used anti-influenza drugs, but, more recently, the drug baloxavir marboxil (BXM), a polymerase inhibitor, has also been licensed in some countries. Mutations in the viral genes that encode the antiviral targets can lead to treatment resistance. Worldwide, a low prevalence of antiviral resistant strains has been reported. Despite that, this situation can change rapidly, and resistant strain surveillance is a priority. Thus, the aim of this was to evaluate Brazilian IAVs antiviral resistance from 2017 to 2019 through the identification of viral mutations associated with reduced inhibition of the drugs and by testing the susceptibility of IAV isolates to oseltamivir (OST), the most widely used NAI drug in the country. Initially, we analyzed 282 influenza A(H1N1)pdm09 and 455 A(H3N2) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S247N was detected in one A(H1N1)pdm09 strain. We also identified NA:I222V (n = 6) and NA:N329K (n = 1) in A(H3N2) strains. In addition, we performed a molecular screening for NA:H275Y in 437 A(H1N1)pdm09 samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC50 values for 222 A(H1N1)pdm09 and 83 A(H3N2) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H3N2) virus presenting with PA:E119D AAS. Moreover, the majority of the IAV sequences had the M2:S31N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis.

Neutralizing antibodies for SARS-CoV-2 in stray animals from Rio de Janeiro, Brazil.

The epidemic of coronavirus disease 2019 (COVID-19), caused by a novel Betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a public health emergency worldwide. Few reports indicate that owned pets from households with at least one human resident that was diagnosed with COVID-19 can be infected by SARS-CoV-2. However, the exposure to SARS-CoV-2 of pets from households with no COVID-19 cases or stray animals remains less assessed. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and plaque reduction neutralization test (PRNT90), we investigated the infection and previous exposure of dogs and cats to SARS-CoV-2 during the ongoing COVID-19 epidemic in Rio de Janeiro, Brazil. From June to August 2020, 96 animals were sampled, including 49 cats (40 owned and 9 stray) and 47 dogs (42 owned and 5 stray). Regarding owned pets, 75.6% (62/82) belonged to households with no COVID-19 cases. Samples included serum, and rectal and oropharyngeal swabs. All swabs were negative for SARS-CoV-2 RNA, but serum samples of a stray cat and a stray dog presented neutralizing antibodies for SARS-CoV-2, with PRNT90 titer of 80 and 40, respectively. Serological data presented here suggest that not only owned pets from households with COVID19 cases, but also stray animals are being exposed to SARS-CoV-2 during the COVID-19 pandemic.

Avaliação da susceptibilidade dos vírus influenza A, circulantes no Brasil, aos antivirais

Infecções pelo vírus influenza A (FluAV) têm grande importância para a saúde pública mundial, principalmente pelos subtipos A(H1N1)pdm09 e o A(H3N2), que causam epidemias anuais e pandemias esporádicas. Além da vacinação, o uso de fármacos antivirais é essencial para o controle destes patógenos. Atualmente, a classe dos inibidores de neuraminidase (NAIs) é a mais utilizada, sendo o fármaco oseltamivir (OST) o mais relevante. Mais recentemente, o fármaco baloxavir marboxil (BXM), da classe de inibidores de polimerase, foi licenciado em alguns países. Porém, devido à constante evolução dos genomas dos FluAVs, mutações nos genes que codificam as proteínas alvos dos antivirais podem levar à resistência ao tratamento. Este fenômeno foi previamente observado para a classe dos fármacos adamantanos, com disseminação de cepas com o marcador S31N no gene M2. Sendo assim, a vigilância de cepas carreando mutações associadas à redução da susceptibilidade (RS) aos NAIs deve ser realizada regularmente. Com relação ao BXM, também já foram relatadas mutações associadas à RS no gene da polimerase ácida (PA). Desta forma, esse estudo tem o objetivo de avaliar os FluAVs circulantes no Brasil, no período de 2017 a 2020, com relação a sua susceptibilidade aos principais antivirais, através de metodologias de sequenciamento genético e análises funcionais dos isolados virais com o OST. Para tal, foram analisadas 1020 amostras positivas para FluAV, oriundas de 9 estados do Brasil. Destas, 699 foram positivas para A(H1N1)pdm09 e 321 para A(H3N2). Analisamos 65,9% das amostras de A(H1N1)pdmp09 através de triagem do alvo mais frequente (H275Y na NA), por pirossequenciamento, detectando-se uma amostra com esta substituição Em seguida, através do sequenciamento completo dos genes alvo dos fármacos (NA, PA e M2), geramos 390 sequências do gene NA, 209 do gene M2 e 182 do gene PA. Assim, detectaram-se as seguintes substituições associadas à RS ao antivirais no gene NA: I222V (n=6), V149A (n=3), N329K (n=1), Q136k (n=1), Y155H (n=1) e Y155H/V149A (n=4). No gene M2, detectamos a alteração S31N (n=208) e, no gene PA, observamos a substituição E199D (n=1). Adicionalmente, foram obtidas sequências genéticas de FluAVs no banco de sequências GISAID, oriundas de 14 estados do Brasil. Destas, 323 sequências eram do gene NA, 248 do gene M2 e 245 do gene PA. Nestas, foram detectados os seguintes marcadores: Y155H (n=3), S247N (n=1), V149A (n=1) e V149A (n=15), na NA. Todas as sequências de M2 apresentavam o marcador S31N. Paralelamente, foram realizadas análises funcionais de susceptibilidade ao OST, em 314 isolados virais, sendo 231 A(H1N1)pdm09 e 83 de A(H3N2). Observou-se que todos os isolados apresentavam perfil normal de susceptibilidade ao OST. Desta forma, conclui-se que a frequência de cepas de FluAV estudadas, carreando mutações associadas à RS aos NAIs e BXM ainda é baixa (<1%) nas amostras circulantes no Brasil e que tais vírus, em sua maioria, continuam apresentando o marcador de resistência aos adamantanos. Esses dados podem auxiliar condutas clínicas e políticas públicas de compra, estocagem e licenciamento de fármacos contra influenza no Brasil