4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

Epidemiological features and trends in the mortality rates of 10 notifiable respiratory infectious diseases in China from 2004 to 2020: Based on national surveillance.

Objectives: The aim of this study is to describe, visualize, and compare the trends and epidemiological features of the mortality rates of 10 notifiable respiratory infectious diseases in China from 2004 to 2020. Setting: Data were obtained from the database of the National Infectious Disease Surveillance System (NIDSS) and reports released by the National and local Health Commissions from 2004 to 2020. Spearman correlations and Joinpoint regression models were used to quantify the temporal trends of RIDs by calculating annual percentage changes (APCs) in the rates of mortality. Results: The overall mortality rate of RIDs was stable across China from 2004 to 2020 (R = -0.38, P = 0.13), with an APC per year of -2.2% (95% CI: -4.6 to 0.3; P = 0.1000). However, the overall mortality rate of 10 RIDs in 2020 decreased by 31.80% (P = 0.006) compared to the previous 5 years before the COVID-19 pandemic. The highest mortality occurred in northwestern, western, and northern China. Tuberculosis was the leading cause of RID mortality, and mortality from tuberculosis was relatively stable throughout the 17 years (R = -0.36, P = 0.16), with an APC of -1.9% (95% CI -4.1 to 0.4, P = 0.1000). Seasonal influenza was the only disease for which mortality significantly increased (R = 0.73, P = 0.00089), with an APC of 29.70% (95% CI 16.60-44.40%; P = 0.0000). The highest yearly case fatality ratios (CFR) belong to avian influenza A H5N1 [687.5 per 1,000 (33/48)] and epidemic cerebrospinal meningitis [90.5748 per 1,000 (1,010/11,151)]. The age-specific CFR of 10 RIDs was highest among people over 85 years old [13.6551 per 1,000 (2,353/172,316)] and was lowest among children younger than 10 years, particularly in 5-year-old children [0.0552 per 1,000 (58/1,051,178)]. Conclusions: The mortality rates of 10 RIDs were relatively stable from 2004 to 2020 with significant differences among Chinese provinces and age groups. There was an increased mortality trend for seasonal influenza and concerted efforts are needed to reduce the mortality rate of seasonal influenza in the future.

Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

SERS-based immunomagnetic bead for rapid detection of H5N1 influenza virus.

The surface-enhanced Raman scattering (SERS) has recently drawn attention in the detection of respiratory viruses, but there have been few reports of the direct detection of viruses. In this study, a sandwich immunomagnetic bead SERS was established for the rapid diagnosis of the H5N1 influenza virus. The detection limit was estimated to be 5.0 × 10-6 TCID50/ml. The method showed excellent specificity with no cross-reaction with H1N1, H5N6 or H9N2. The H5N1 influenza virus detection accuracy of the SERS method was 100% in chicken embryos. The results hold great promise for the utilization of SERS as an innovative approach in the diagnosis of influenza virus.

The health of influenza surveillance and pandemic preparedness in the wake of the COVID-19 pandemic.

The COVID-19 pandemic is the first to have emerged when Next Generation Sequencing was readily available and it has played the major role in following evolution of the causative agent, Severe Acute Respiratory Syndrome Coronavirus 2. Response to the pandemic was greatly facilitated though use of existing influenza surveillance networks: World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS), focussing largely on human influenza, and the OFFLU network of expertise on avian influenza established by the Food and Agricultural Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH). Data collection/deposition platforms associated with these networks, notably WHO's FluNet and the Global Initiative on Sharing All Influenza Data (GISAID) were/are being used intensely. Measures introduced to combat COVID-19 resulted in greatly decreased circulation of human seasonal influenza viruses for approximately 2 years, but circulation continued in the animal sector with an upsurge in the spread of highly pathogenic avian influenza subtype H5N1 with large numbers of wild bird deaths, culling of many poultry flocks and sporadic spill over into mammalian species, including humans, thereby increasing pandemic risk potential. While there are proposals/implementations to extend use of GISRS and GISAID to other infectious disease agents (e.g. Respiratory Syncytial Virus and Monkeypox), there is need to ensure that influenza surveillance is maintained and improved in both human and animal sectors in a sustainable manner to be truly prepared (early detection) for the next influenza pandemic.

Omicron-induced interferon signaling prevents influenza A H1N1 and H5N1 virus infection.

Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface cultures of primary human bronchial epithelial cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active types I (α/ß) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signaling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knockout prevented it. The JAK/STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/STAT signaling. In conclusion, our study (1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, (2) shows that Omicron infection protects cells from influenza A virus super-infection, and (3) indicates that BA.1 and BA.5 induce comparable antiviral states.

Bird flu spread between mink is a 'warning bell'.

Big outbreak at a Spanish farm reignites fears of an H5N1 influenza pandemic.

Vaccine models predict rules for updating vaccines against evolving pathogens such as SARS-CoV-2 and influenza in the context of pre-existing immunity.

Currently, vaccines for SARS-CoV-2 and influenza viruses are updated if the new vaccine induces higher antibody-titers to circulating variants than current vaccines. This approach does not account for complex dynamics of how prior immunity skews recall responses to the updated vaccine. We: (i) use computational models to mechanistically dissect how prior immunity influences recall responses; (ii) explore how this affects the rules for evaluating and deploying updated vaccines; and (iii) apply this to SARS-CoV-2. Our analysis of existing data suggests that there is a strong benefit to updating the current SARS-CoV-2 vaccines to match the currently circulating variants. We propose a general two-dose strategy for determining if vaccines need updating as well as for vaccinating high-risk individuals. Finally, we directly validate our model by reanalysis of earlier human H5N1 influenza vaccine studies.

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins.

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

Broad-spectrum antiviral activity of Spatholobus suberectus Dunn against SARS-CoV-2, SARS-CoV-1, H5N1, and other enveloped viruses.

The current COVID-19 pandemic caused by SARS-Cov-2 is responsible for more than 6 million deaths globally. The development of broad-spectrum and cost-effective antivirals is urgently needed. Medicinal plants are renowned as a complementary approach in which antiviral natural products have been established as safe and effective drugs. Here, we report that the percolation extract of Spatholobus suberectus Dunn (SSP) is a broad-spectrum viral entry inhibitor against SARS-CoV-1/2 and other enveloped viruses. The viral inhibitory activities of the SSP were evaluated by using pseudotyped SARS-CoV-1 and 2, HIV-1ADA and HXB2 , and H5N1. SSP effectively inhibited viral entry and with EC50 values ranging from 3.6 to 5.1 µg/ml. Pre-treatment of pseudovirus or target cells with SSP showed consistent inhibitory activities with the respective EC50 value of 2.3 or 2.1 µg/ml. SSP blocked both SARS-CoV-2 spike glycoprotein and the host ACE2 receptor. In vivo studies indicated that there was no abnormal toxicity and behavior in long-term SSP treatment. Based on these findings, we concluded that SSP has the potential to be developed as a drug candidate for preventing and treating COVID-19 and other emerging enveloped viruses.

Influenza Activity and Composition of the 2022-23 Influenza Vaccine - United States, 2021-22 Season.

Before the emergence of SARS-CoV-2, the virus that causes COVID-19, influenza activity in the United States typically began to increase in the fall and peaked in February. During the 2021-22 season, influenza activity began to increase in November and remained elevated until mid-June, featuring two distinct waves, with A(H3N2) viruses predominating for the entire season. This report summarizes influenza activity during October 3, 2021-June 11, 2022, in the United States and describes the composition of the Northern Hemisphere 2022-23 influenza vaccine. Although influenza activity is decreasing and circulation during summer is typically low, remaining vigilant for influenza infections, performing testing for seasonal influenza viruses, and monitoring for novel influenza A virus infections are important. An outbreak of highly pathogenic avian influenza A(H5N1) is ongoing; health care providers and persons with exposure to sick or infected birds should remain vigilant for onset of symptoms consistent with influenza. Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences.

Identification of Human Case of Avian Influenza A(H5N1) Infection, India.

A 11-year-old boy with acute myeloid leukemia was brought for treatment of severe acute respiratory infection in the National Capital Region, New Delhi, India. Avian influenza A(H5N1) infection was laboratory confirmed. Complete genome analysis indicated hemagglutinin gene clade 2.3.2.1a. We found the strain to be susceptible to amantadine and neuraminidase inhibitors.

Synthesis of novel pyridine and pyrimidine thioglycoside phosphoramidates for the treatment of COVID-19 and influenza A viruses.

A novel series of pyridine, cytosine, and uracil thioglycoside analogs (4a-i, 9a,b, and 13a,b, respectively) and their corresponding phosphoramidates (6a-I, 10a,b, and 14a,b, respectively) were synthesized and assessed for their antiviral inhibitory activities in a dual-pathogen screening protocol against SARS-CoV-2 and influenza A virus (IAV). MTT cytotoxicity (TC50) and plaque reduction assays were used to explore inhibition and cytotoxicity percentage values for H5N1 influenza virus strain and the half-maximal cytotoxic concentration (CC50) and inhibitory concentration (IC50) for SARS-CoV-2 virus. Most of the tested compounds demonstrated dose-dependent inhibition behavior. Both cytosine thioglycoside phosphoramidates 10a and 10b exhibited the most potent profiles with 83% and 86% inhibition at 0.25 µM concentration against H5N1 and IC50 values of 12.16 µM, 14.9 µM against SARS-CoV-2, respectively. Moreover, compounds 10a and 10b have been shown to have the highest selectivity index (SI) among all the tested compounds against SARS-CoV-2 with 28.2 and 26.9 values, respectively.

Emerging Respiratory Viruses of Cats.

In recent years, advances in diagnostics and deep sequencing technologies have led to the identification and characterization of novel viruses in cats as protoparviruses and chaphamaparvoviruses, unveiling the diversity of the feline virome in the respiratory tract. Observational, epidemiological and experimental data are necessary to demonstrate firmly if some viruses are able to cause disease, as this information may be confounded by virus- or host-related factors. Also, in recent years, researchers were able to monitor multiple examples of transmission to felids of viruses with high pathogenic potential, such as the influenza virus strains H5N1, H1N1, H7N2, H5N6 and H3N2, and in the late 2019, the human hypervirulent coronavirus SARS-CoV-2. These findings suggest that the study of viral infections always requires a multi-disciplinary approach inspired by the One Health vision. By reviewing the literature, we provide herewith an update on the emerging viruses identified in cats and their potential association with respiratory disease.

Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1ß and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.