Influenza virus isolation in cell cultures can be a foremost activity in the surveillance of the epidemiological characteristics involved in the vaccination strategies. Here, the virus isolation effectiveness from double positive throat swab to influenza A virus (H1N1pdm09 or H3N2) and SARS-CoV-2 compared to those obtained from single positive throat swab samples was investigated by serial passages of throat swabs samples in cell cultures. Viral replication and IFN-α, IL-1α, and IL-6 cytokine expression at three passages were measured by RT-PCR amplification. Virus isolation using a throat swab samples resulted double positive to influenza A virus (H1N1pdm09 or H3N2) and SARS-CoV-2 showed a different effectiveness compared to a throat samples single positive to each of these viruses. The virus isolation efficacy was independent from an active replication of the two viruses in the inoculum and showed a virus strain-specific reduction in cytokines profile compared to a single infection. Collectively the results point out to take into account the presence of co-circulating respiratory viruses in human samples for virus isolation effectiveness.

BACKGROUND:SARS-CoV-2 fusion inhibitory peptides represent promising antiviral candidates. Recently, a 19-mer peptide (PN19)-designed in our laboratory to mimic the internal fusion peptide of the SARS-CoV-2 spike S2 subunit-demonstrated potent antiviral activity and stable conformational features. OBJECTIVES:To investigate how this antiviral activity depends on membrane interactions, we designed synthetic PN19 lipopeptide derivatives and evaluated their efficacy against SARS-CoV-2 replication. METHODS:Lipopeptides were synthesized by conjugating cholesterol to either the N- or C-terminus of the PN19 peptide, utilizing a Gly/Ser pentapeptide (GSGSG) and/or various polyethylene glycol (PEG) spacers. Antiviral activity against SARS-CoV-2 variants was evaluated by plaque reduction assays, and cytotoxicity was assessed in Vero E6 cells. RESULTS:The lipopeptides exhibited potent inhibitory activity at sub-micromolar concentrations. Compared to the unmodified PN19 peptide, antiviral efficacy was significantly enhanced by cholesterol conjugation at either terminus. Evaluation of six PN19 lipopeptides bearing the GSGSG sequence and different PEG spacers revealed that C-terminal cholesterol conjugation yielded higher antiviral activity than N-terminal derivatives. Furthermore, thirteen shorter PN19 lipopeptide derivatives (8-13-mers) confirmed this robust efficacy, which was most pronounced with C-terminal cholesterol conjugation and further enhanced by the spacers. Noteworthy, all tested PN19 lipopeptides displayed broad activity against multiple SARS-CoV-2 variants in the absence of cytotoxicity. CONCLUSIONS:Collectively, peptides conjugated with cholesterol at the C-terminus emerged as highly potent inhibitors of SARS-CoV-2, likely driven by enhanced peptide-membrane interactions. These findings warrant further investigation to fully elucidate the role of lipidation in the inhibitory mechanism, supporting the development of novel antiviral lipopeptides for SARS-CoV-2 therapy.

Membrane fusion between SARS-CoV-2 and host cells is mediated by the spike protein and involves the membrane-proximal external region (MPER), a tryptophan-rich sequence implicated in viral entry. Here, we investigated the feasibility of MPER-derived peptides as potential antiviral agents and examined the structural determinants underlying their activity. A series of truncated MPER peptides was synthesized and evaluated for antiviral activity against SARS-CoV-2 in cellular assays, revealing a potent inhibitory activity with nanomolar IC50 values associated to sequences rich in aromatic residues. Using NMR spectroscopy, we demonstrate that these peptides adopt a stable a-helical conformation in solution and membrane-mimetic environments that is stabilized by intramolecular aromatic π-π stacking interactions among tryptophan and tyrosine side chains. Structure–activity analysis indicates that this aromatic network promotes a-helix stabilization mimicking MPER structure of the spike post-fusion and correlates with enhanced antiviral potency. Our findings reveal a structural mechanism by which aromatic stacking stabilizes MPER helicity and drives antiviral activity, providing insights into peptide-based inhibition of viral membrane fusion and offering a framework for the rational design of SARS-CoV-2 entry inhibitors.

Background: West Nile virus (WNV) is a mosquito-borne RNA virus, with birds as reservoirs and humans as incidental hosts. WNV often causes asymptomatic infections, but severe neuroinvasive disease occurs in fewer than 1% of human cases. Recent climatic changes and occupational exposure have increased its spread, particularly in Southern Italy. This study aimed to assess WNV seroprevalence and occupational risks among outdoor workers to guide targeted public health interventions. Methods: This cross-sectional study was conducted in the Apulia region, southeastern Italy, from November 2023 to April 2024. Participants completed a detailed questionnaire on socio-demographics, occupational exposure, travel history, and health symptoms. Blood samples were analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization assays to detect WNV-specific antibodies. Results: 250 outdoor workers in southeastern Italy were recruited, including agricultural workers, veterinarians, forestry workers, and livestock breeders. The latter showed the highest WNV prevalence at 6.5%. Protective measures such as repellent use (β = −0.145, OR = 0.95, p = 0.019) and personal protective equipment (PPE) usage (β = −0.12, OR = 0.94, p = 0.04) significantly reduced the likelihood of WNV infection. Conclusions: The study highlights the significant occupational risk posed by WNV to outdoor workers involved in livestock breeding in Southern Italy, likely due to their frequent exposure to mosquito-prone environments. Tailored public health strategies and education programs are needed to protect high-risk outdoor workers from WNV, amidst the backdrop of changing climatic conditions that favor increased transmission.

The mechanisms underlying post-acute sequelae of SARS-CoV-2 infection (PASC) are a topic of debate. This study examined the presence of SARS-CoV-2 microRNA (miRNA)-like small RNAs in extracellular fluids and their potential link to PASC by using a quantitative stem-loop RT-PCR MiRNA assay. Initially, it was demonstrated that three previously identified SARS-CoV-2 miRNA-like small RNAs, specifically svRNA 1 and 2 and miR-07a, were significantly expressed in infected cells in vitro and released into the supernatant following infection by different SARS-CoV-2 variants. Then, the expression of three SARS-CoV-2 small RNAs was studied in both nasopharyngeal swabs (NPS) and sera from 24 patients at their initial COVID-19 diagnosis (T0) and in sera collected 91 to 193 days post-diagnosis (T1). Notably, 11 out of 24 patients (46%) reported PASC consequences. All NPS samples showed SARS-CoV-2 small RNA expression with an altered cytokine network during acute infection, but it did not correlate with PASC outcomes. Serum samples had similar small RNA statuses, though PASC patients, notably at T1, but not at T0, displayed reduced overall positivity compared to those without PASC. The host target expression of SARS-CoV-2 small RNAs was not significantly different between groups. This suggests a need for further research into SARS-CoV-2 small RNA and its role in viral behavior and PASC consequences.

The I38T substitution in the influenza virus polymerase-acidic (PA) subunit is a resistance marker of concern for treatment with the antiviral baloxavir marboxil (BXM). Thus, monitoring PA/I38T mutations is of clinical importance. Here, we developed three rapid and sensitive assays for the detection and monitoring of the PA/I38T mutation. In addition, we updated our previously developed methods to monitor the D197N mutation in the neuraminidase (NA) of influenza B virus, which is associated with resistance to oseltamivir. Real-time PCR high-resolution melting analysis (HRMA) was developed for the rapid detection of the PA/I38T and NA/D197N mutations using oligonucleotides with substitutions of interest and influenza viruses isolated in our laboratory. HRMA was subsequently performed on 94 clinical samples that were positive for A/H1N1pdm09, A/H3N2, and type-B influenza viruses and on viruses that were selected in vitro to grow in the presence of BXA (baloxavir acid, BXM active compound). The HRMAs were able to discriminate PA/I38 from the PA/I38T mutation and NA substitutions in synthetic oligonucleotides. However, the I38T mutation and NA mutations were not detected in any of our clinical samples, indicating the absence of these resistance markers in the circulating viruses examined. Only one out of 43 A/H3N2 clinical samples analyzed contained a virus with mutations associated with resistance to oseltamivir. All the HRMA results were confirmed by sequencing. Finally, HRMA was performed on A/H1N1pdm09 and A/H3N2 influenza viruses following BXA selection in vitro. The presence of the I38T mutation in the BXA-selected A/H3N2 variant, but not in the A/H1N1pdm09 variant, was identified by HRMA after 12 passages. Overall, these findings indicate that HRMA could be a powerful tool for rapidly monitoring BXM resistance in influenza viruses during seasonal circulation.

Background. Understanding the interference patterns of respiratory viruses could be important for shedding light on potential strategies to combat these human infectious agents. Objective. To investigate the possible interactions between adenovirus type 2 (AdV2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/H1N1 pandemic (H1N1pdm09) using the A549 cell line. Methods. Single infections, co-infections, and superinfections (at 3 and 24 h after the first virus infection) were performed by varying the multiplicity of infection (MOI). Virus replication kinetics and the mRNA expression of IFN-α, IL-1α and IL-6 were assessed by real-time qPCR. Results. Co-infection experiments showed different growth dynamics, depending on the presence of the specific virus and time. AdV2 replication remained stable or possibly enhanced in the presence of co-infection with each of the two H1N1pdm09 and SARS-CoV-2 viruses used. In contrast, SARS-CoV-2 replication was facilitated by H1N1pdm09 but hindered by AdV2, indicating possible different interactions. Finally, H1N1pdm09 replication exhibited variably effectiveness in the presence of AdV2 and SARS-CoV-2. Superinfection experiments showed that the replication of all viruses was affected by time and MOI. The mRNA expression of IFN-α, IL-1α and IL-6 showed divergent results depending on the virus used and the time of infection. Conclusions. Further investigation of co-infection or superinfection may be helpful in understanding the potential relationship involved in the outcome of viral respiratory infection in the human population.

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors.

Saliva has emerged as a promising diagnostic fluid for viral infections, enabling the direct analysis of viral genetic material and the detection of infection markers such as proteins, metabolites, microRNAs, and immunoglobulins. This comprehensive review aimed to explore the use of saliva as a diagnostic tool for viral infections, emphasizing its advantages and limitations. Saliva stands out due to its simplicity and safety in collection, along with the convenience of self-collection without the need for healthcare supervision, while potentially being comparable to urine and blood in terms of effectiveness. Herein, we highlighted the significant potential of saliva in assessing viral loads and diagnosing viral infections, such as herpesviruses, HPV, PyV, TTV, SARS-CoV-2, and MPXV. The detection of viral shedding in saliva underscores its utility in early diagnosis, the monitoring of infection progression, and evaluating treatment responses. The non-invasive nature of saliva collection makes it an appealing alternative to more invasive methods, promoting better patient compliance and facilitating large-scale screening and surveillance. As such, we further highlight current evidence on the use of saliva as a prognostic tool. Although a significant amount of data is already available, further investigations are warranted to more comprehensively assess the added benefit from the utilization of salivary biomarkers in the clinics. Salivary biomarkers show great promise for the early detection and prevention of viral infection complications, potentially improving disease management and control at the population level. Integrating these non-invasive tools into routine clinical practice could enhance personalized healthcare strategies and patient outcomes. Future studies should focus on establishing standardization protocols, validating the accuracy of salivary diagnostics, and expanding clinical research to enhance the diagnostic and monitoring capabilities of salivary biomarkers.

Background. Torquetenovirus (TTV) is a small DNA virus constituting the human virome. High levels of TTV-DNA have been shown to be associated with immunosuppression and inflammatory chronic disorders. Aim. To assess the possible association between the salivary viral load of TTV-DNA in patients hospitalised due to COVID-19 and disease severity. Methods. Saliva samples collected from 176 patients infected with SARS-CoV-2 were used to investigate the presence of SARS-CoV-2 and TTV-DNA by use of real-time RT-PCR. Results. The majority of patients were male with severe COVID-19. Presence of SARS-CoV-2 was observed in the saliva of 64.77% of patients, showing TTV-DNA in 55.68% of them. Patients with impaired clinical conditions (p < 0.001), which evolved to death (p = 0.003), showed a higher prevalence of TTV-DNA. The median viral load in patients with severe condition was 4.99 log10 copies/mL, in which those who were discharged and those evolving to death had values of 3.96 log10 copies/mL and 6.27 log10 copies/mL, respectively. A statistically significant association was found between the distribution of TTV-DNA viral load in saliva samples and severity of COVID-19 (p = 0.004) and disease outcomes (p < 0.001). Conclusions. These results indicate that TTV-DNA in saliva could be a useful biomarker of COVID-19 severity and prognosis.

Introduction: Influenza is characterized by an acute viral infection, which can lead to severe conditions and death, especially in vulnerable populations, such as older adults. Therefore, we sought to analyze cases of severe acute respiratory syndrome (SARS) due to influenza in older adults registered in Brazil and investigate the factors related to death due to this disease. Methodology: This is a cross-sectional, population-based study that used secondary data from the Influenza Epidemiological Surveillance Information System (IESIS-Influenza). Older adults aged 60 years and above with laboratory diagnosis of influenza were included. Results: A total of 3,547 older adults with SARS due to influenza were included, out of which 1,185 cases with death as the outcome were identified. Among older adults with death as the outcome, 87.4% were not vaccinated against influenza. The main risk factors for death were invasive ventilatory support use, intensive care unit admission, brown skin color and dyspnea (p < 0.001). Conclusions: This study described the profile of older adults with SARS due to influenza in Brazil. Factors associated with death in this population were identified. Moreover, the need to encourage compliance with vaccination among older adults is evident in order to prevent severe cases and unfavorable outcomes related to influenza.

The aim of this manuscript is to describe a hybrid care model to paediatric patients on a virtual basis for dental treatment before conducting an in-person surveillance by using combined nasal/oral swabbing (NOS). This longitudinal study used a convenience sample of paediatric patients and members of the dental team from an undergraduate paediatric dentistry clinic at the University of São Paulo School of Dentistry during the COVID-19 pandemic. Firstly, parents were contacted and teledentistry was used for screening children who need dental treatment. Appointments were scheduled once a week for two months, in which a pre-COVID-19 screening was performed. Dental team and children’s parents completed a questionnaire addressing COVID-19-related symptoms. Members of the dental team and children were tested for COVID-19 before entering the dental clinic, by NOS and RT-PCR screening. Ninety-three individuals were enrolled and all of them completed the electronic questionnaire on symptoms and had NOS collected weekly, totalising 241 pairs of swabs. No participant reported COVID-19 symptoms before entering the clinic for treatment. Only one child tested positive in the third week of sampling. The hybrid care model associated with molecular testing for asymptomatics provided a safe clinical environment regarding the transmission of SARS-CoV-2.

COPYRIGHT © 2023 Tozetto-Mendoza, Braz-Silva, Giannecchini and Witkin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Editorial: Torquetenovirus: predictive biomarker or innocent bystander in pathogenesis

Despite the availability of vaccines, COVID-19 continues to be aggressive, especially in immunocompromised individuals. Therefore, the development of a specific therapeutic agent with antiviral activity against SARS-CoV-2 is necessary. The infection pathway starts when the receptor binding domain of the viral spike protein interacts with the angiotensin converting enzyme 2 (ACE2), which acts as a host receptor for the RBD expressed on the host cell surface. In this scenario, ACE2 analogs binding to the RBD and preventing the cell entry can be promising antiviral agents. Most of the ACE2 residues involved in the interaction belong to the α1 helix, more specifically to the minimal fragment ACE2(24-42). In order to increase the stability of the secondary structure and thus antiviral activity, we designed different triazole-stapled analogs, changing the position and the number of bridges. The peptide called P3, which has the triazole-containing bridge in the positions 36-40, showed promising antiviral activity at micromolar concentrations assessed by plaque reduction assay. On the other hand, the double-stapled peptide P4 lost the activity, showing that excessive rigidity disfavors the interaction with the RBD.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a great concern in human population. To fight coronavirus emergence, we have dissected the conserved amino acid region of the internal fusion peptide in the S2 subunit of Spike glycoprotein of SARS-CoV-2 to design new inhibitory peptides. Among the 11 overlapping peptides (9-23-mer), PN19, a 19-mer peptide, exhibited a powerful inhibitory activity against different SARS-CoV-2 clinical isolate variants in absence of cytotoxicity. The PN19 inhibitory activity was found to be dependent on conservation of the central Phe and C-terminal Tyr residues in the peptide sequence. Circular dichroism spectra of the active peptide exhibited an alpha-helix propensity, confirmed by secondary structure prediction analysis. The PN19 inhibitory activity, exerted in the first step of virus infection, was reduced after peptide adsorption treatment with virus-cell substrate during fusion interaction. Additionally, PN19 inhibitory activity was reduced by adding S2 membrane-proximal region derived peptides. PN19 showed binding ability to the S2 membrane proximal region derived peptides, confirmed by molecular modelling, playing a role in the mechanism of action. Collectively, these results confirm that the internal fusion peptide region is a good candidate on which develop peptidomimetic anti SARS-CoV-2 antivirals.

Respiratory viruses infection is a worldwide human concern annually. The main viral respiratory diseases are caused by a variety of viruses sharing similar threats and affecting the respiratory system. Among all, influenza viruses, respiratory syncytial virus, parainfluenza viruses, respiratory adenoviruses, rhinoviruses, human bocaviruses, human metapneumovirus and coronaviruses are the main common respiratory viruses affecting human population. The recent coronavirus disease 19 pandemic has revealed critical knowledge gaps required to update in the transmission and pathological induced pathways for respiratory viruses. To date, several evidences suggest that human viruses can hijack extracellular vesicles (EVs) to deliver proteins, mRNAs, microRNAs and whole viral particles during viral life cycle in the host. Thus, several investigations have reported that also respiratory viruses use EVs to deliver viral nucleic acid and proteins, even including the potentiality of carrying whole viral particle. This evidence demonstrates the ability of the EVs produced in infected cells to deliver respiratory viral components to uninfected cells, positively or negatively counteracting new viral infection. Additionally, EVs derived from biological fluids of clinical samples may increase the risk to induce severe respiratory viruses-associated diseases in site far from the respiratory tract and for prolonged time. Here, it has been reviewed the advantages of the respiratory viruses EVs interaction regarding their ability to enhance viral infection, to evade antiviral response, to regulate virus-immune response and to mediate diseases. All these data confirm a potential role of the association between EVs and respiratory viruses infection. This suggests that further studies to define the implication of this interaction in viral life cycle in human population are needed.

INTRODUCTION Influenza is characterized by an acute viral infection, which can lead to severe conditions and death, especially in vulnerable populations, such as older adults. Therefore, we sought to analyze cases of severe acute respiratory syndrome (SARS) due to influenza in older adults registered in Brazil and investigate the factors related to death due to this disease. METHODOLOGY This is a cross-sectional, population-based study that used secondary data from the Influenza Epidemiological Surveillance Information System (IESIS-Influenza). Older adults aged 60 years and above with laboratory diagnosis of influenza were included. RESULTS A total of 3,547 older adults with SARS due to influenza were included, out of which 1,185 cases with death as the outcome were identified. Among older adults with death as the outcome, 87.4% were not vaccinated against influenza. The main risk factors for death were invasive ventilatory support use, intensive care unit admission, brown skin color and dyspnea (p < 0.001). CONCLUSIONS This study described the profile of older adults with SARS due to influenza in Brazil. Factors associated with death in this population were identified. Moreover, the need to encourage compliance with vaccination among older adults is evident in order to prevent severe cases and unfavorable outcomes related to influenza.