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The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses. Here Zhao et al. report a promising broad-spectrum antiviral alkaline peptide—P9R—that is active against several respiratory, pH-dependent viruses, including Influenza and SARS-CoV-2. P9R interferes with virus internalization by binding to the virus and subsequent inhibition of endosomal acidification.

Infection with either influenza A H5N1 virus in 1997 or avian influenza A H7N9 virus in 2013 caused severe pneumonia that did not respond to typical or atypical antimicrobial treatment, and resulted in high mortality. Both viruses are reassortants with internal genes derived from avian influenza A H9N2 viruses that circulate in Asian poultry. Both viruses have genetic markers of mammalian adaptation in their haemagglutinin and polymerase PB2 subunits, which enhanced binding to human-type receptors and improved replication in mammals, respectively. Hong Kong (affected by H5N1 in 1997) and Shanghai (affected by H7N9 in 2013) are two rapidly flourishing cosmopolitan megacities that were increasing in human population and poultry consumption before the outbreaks. Both cities are located along the avian migratory route at the Pearl River delta and Yangtze River delta. Whether the widespread use of the H5N1 vaccine in east Asia—with suboptimum biosecurity measures in live poultry markets and farms—predisposed to the emergence of H7N9 or other virus subtypes needs further investigation. Why H7N9 seems to be more readily transmitted from poultry to people than H5N1 is still unclear.

Up to date, effective antivirals have not been widely available for treating COVID-19. In this study, we identify a dual-functional cross-linking peptide 8P9R which can inhibit the two entry pathways (endocytic pathway and TMPRSS2-mediated surface pathway) of SARS-CoV-2 in cells. The endosomal acidification inhibitors (8P9R and chloroquine) can synergistically enhance the activity of arbidol, a spike-ACE2 fusion inhibitor, against SARS-CoV-2 and SARS-CoV in cells. In vivo studies indicate that 8P9R or the combination of repurposed drugs (umifenovir also known as arbidol, chloroquine and camostat which is a TMPRSS2 inhibitor), simultaneously interfering with the two entry pathways of coronaviruses, can significantly suppress SARS-CoV-2 replication in hamsters and SARS-CoV in mice. Here, we use drug combination (arbidol, chloroquine, and camostat) and a dual-functional 8P9R to demonstrate that blocking the two entry pathways of coronavirus can be a promising and achievable approach for inhibiting SARS-CoV-2 replication in vivo. Cocktail therapy of these drug combinations should be considered in treatment trials for COVID-19. Until today effective antivirals for COVID-19 treatment are not widely available. Here, Zhao et al. characterize a dual-functional cross-linking peptide, 8P9R, that can inhibit SARS-CoV-2 virus entry in vitro and suppresses viral replication in vivo in golden Syrian hamster.

Background. Infections caused by the pandemic H1N1 2009 influenza virus range from mild upper respiratory tract syndromes to fatal diseases. However, studies comparing virological and immunological profile of different clinical severity are lacking. Methods. We conducted a retrospective cohort study of 74 patients with pandemic H1N1 infection, including 23 patients who either developed acute respiratory distress syndrome (ARDS) or died (ARDS-death group), 14 patients with desaturation requiring oxygen supplementation and who survived without ARDS (survived-without-ARDS group), and 37 patients with mild disease without desaturation (mild-disease group). We compared their pattern of clinical disease, viral load, and immunological profile. Results. Patients with severe disease were older, more likely to be obese or having underlying diseases, and had lower respiratory tract symptoms, especially dyspnea at presentation. The ARDS-death group had a slower decline in nasopharyngeal viral loads, had higher plasma levels of proinflammatory cytokines and chemokines, and were more likely to have bacterial coinfections (30.4%), myocarditis (21.7%), or viremia (13.0%) than patients in the survived-without-ARDS or the mild-disease groups. Reactive hemophagocytosis, thrombotic phenomena, lymphoid atrophy, diffuse alveolar damage, and multiorgan dysfunction similar to fatal avian influenza A H5N1 infection were found at postmortem examinations. Conclusions. The slower control of viral load and immunodysregulation in severe cases mandate the search for more effective antiviral and immunomodulatory regimens to stop the excessive cytokine activation resulting in ARDS and death.

The D225G (aspartic acid to glycine) substitution in the hemagglutinin of H1N1 influenza virus may alter its receptorbinding specificity. Direct analysis of polymorphisms in 126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N1 2009 virus from 117 clinical specimens in Hong Kong found the D225G substitution for 7 (12.5%) of 57 patients with severe disease and for 0 (0%) of 60 patients with mild disease. D225G quasispecies were identified mainly in endotracheal aspirate samples and were identified less frequently in nasopharyngeal aspirate samples from patients with severe disease. Continuous monitoring of the prevalence and tissue tropism of this variant during its circulation among humans is important.

Human infection caused by the avian influenza A H7N9 virus has a case-fatality rate of over 30%. Systematic study of the pathogenesis of avian H7N9 isolate and effective therapeutic strategies are needed. BALB/c mice were inoculated intranasally with an H7N9 virus isolated from a chicken in a wet market epidemiologically linked to a fatal human case, (A/chicken/Zhejiang/DTID-ZJU01/2013 [CK1]), and with an H7N9 virus isolated from a human (A/Anhui/01/2013 [AH1]). The pulmonary viral loads, cytokine/chemokine profiles and histopathological changes of the infected mice were compared. The therapeutic efficacy of a non-steroidal anti-inflammatory drug (NSAID), celecoxib, was assessed. Without prior adaptation, intranasal inoculation of 106 plaque forming units (PFUs) of CK1 caused a mortality rate of 82% (14/17) in mice. Viral nucleoprotein and RNA expression were limited to the respiratory system and no viral RNA could be detected from brain, liver and kidney tissues. CK1 caused heavy alveolar inflammatory exudation and pulmonary hemorrhage, associated with high pulmonary levels of proinflammatory cytokines. In the mouse lung cell line LA-4, CK1 also induced high levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA. Administration of the antiviral zanamivir did not significantly improve survival in mice infected with CK1, but co-administration of the non-steroidal anti-inflammatory drug (NSAID) celecoxib in combination with zanamivir improved survival and lung pathology. Our findings suggested that H7N9 viruses isolated from chicken without preceding trans-species adaptation can cause lethal mammalian pulmonary infection. The severe proinflammatory responses might be a factor contributing to the mortality. Treatment with combination of antiviral and NSAID could ameliorate pulmonary inflammation and may improve survival.

Positive detection of viral RNA in blood and other non-respiratory specimens occurs in severe human influenza A/H5N1 viral infection but is not known to occur commonly in seasonal human influenza infection. Recently, viral RNA was detected in the blood of patients suffering from severe pandemic influenza A/H1N1/2009 viral infection, although the significance of viremia had not been previously studied. Our study aims to explore the clinical and virological factors associated with pandemic influenza A/H1N1/2009 viremia and to determine its clinical significance. Clinical data of patients admitted to hospitals in Hong Kong between May 2009 and April 2010 and tested positive for pandemic influenza A/H1N1/2009 was collected. Viral RNA was detected by reverse-transcription polymerase chain reactions (RT-PCR) targeting the matrix (M) and HA genes of pandemic influenza A/H1N1/2009 virus from the following specimens: nasopharyngeal aspirate (NPA), endotracheal aspirate (ETA), blood, stool and rectal swab. Stool and/ or rectal swab was obtained only if the patient complained of any gastrointestinal symptoms. A total of 139 patients were included in the study, with viral RNA being detected in the blood of 14 patients by RT-PCR. The occurrence of viremia was strongly associated with a severe clinical presentation and a higher mortality rate, although the latter association was not statistically significant. D222G/N quasispecies were observed in 90% of the blood samples. Presence of pandemic influenza A/H1N1/2009 viremia is an indicator of disease severity and strongly associated with D222G/N mutation in the viral hemagglutinin protein.

Background/Objectives: We have previously reported the engineering of a point-of-care (POC) system that fully automates the procedures for nucleic acid extraction and multiplexed real-time RT-PCR, with a major advantage of high-level multiplexing. In this study, we applied and validated the system in a respiratory tract infection setting. Methods: An automatic nested real-time RT-PCR assay was developed (POCm). It was a 40-plex assay that simultaneously detected 39 epidemiologically important respiratory pathogens in 1.5 h in the POC system. The analytical and clinical performance was evaluated. Results: The analytical sensitivities of the POCm assay were comparable to those of its single-plex counterparts performed manually on a bench-top. The minimum detectable concentrations ranged from 53 copies/mL to 5.3 × 103 copies/mL for all pathogen targets except hCoV-NL63 (5.3 × 104 copies/mL). The quantitative performance was demonstrated by the linear correlations between Ct values and input concentrations for all pathogen targets, with 24 of them demonstrating coefficients of correlation (r) greater than 0.9. The POCm assay was subsequently evaluated in 283 clinical samples. A high level of agreement (98.2–100%) was achieved for pathogen detection results between POCm and standard diagnostic methods. The POCm result was also fully concordant with the result of another commercial POC multiplex platform. For positive clinical samples, pairwise Ct values measured by POCm closely correlated with those of the bench-top reference method (r = 0.70). The feasibility of mutation genotyping of the viral subtype was further demonstrated. Conclusions: This study demonstrated the practicality of POCm for routine testing in clinical laboratories. Further clinical trials are being conducted to evaluate the clinical performance of the system.

The COVID-19 (caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) epidemic started in Wuhan (Hubei Province, China) in mid-December 2019 and quickly spread across the world as a pandemic. As a key to tracing the disease and to implement strategies aimed at breaking the chain of disease transmission, extensive testing for SARS-CoV-2 was suggested. Although nasopharyngeal/oropharyngeal swabs are the most commonly used biological samples for SARS-CoV-2 diagnosis, they have a number of limitations related to sample collection and healthcare personnel safety. In this context, saliva is emerging as a promising alternative to nasopharyngeal/oropharyngeal swabs for COVID-19 diagnosis and monitoring. Saliva collection, being a non-invasive approach with possibility for self-collection, circumvents to a great extent the limitations associated with the use of nasopharyngeal/oropharyngeal swabs. In addition, various salivary biomarkers including the salivary metabolomics offer a high promise to be useful for better understanding of COVID-19 and possibly in the identification of patients with various degrees of severity, including asymptomatic carriers. This review summarises the clinical and scientific basis for the potential use of saliva for COVID-19 diagnosis and disease monitoring. Additionally, we discuss saliva-based biomarkers and their potential clinical and research applications related to COVID-19.

Pregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear. We showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes. The adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation.