H1N1 Influenza Virus Research Articles

Janus Fe3O4-Au@Pt nanozymes based lateral flow assay for enhanced-sensitive colorimetric detection of influenza A virus

The traditional colloidal gold-based lateral flow assay (LFA) is widely applied in point-of-care testing (POCT) for pathogens. However, its disadvantage of low sensitivity greatly limits its effectiveness in early detection. In this work, we developed an enhanced-sensitive colorimetric nucleic acid lateral flow assay (NALFA) based on Janus Fe3O4-Au@Pt nanoparticles (Fe3O4-Au@Pt NPs) for H1N1 influenza virus detection. Dumbbell-like heterodimer Fe3O4-Au@Pt NPs were prepared by a seed-mediated method, and their unique Janus structure endowed them with both good plasmonic property and high peroxidase-like activity. With the Fe3O4-Au@Pt NPs as NALFA signal labels, qualitative detection was achieved by observing the color change with the naked eyes, and quantitative results were obtained by measuring the gray values on test strips. Due to the strong peroxidase-like activity of Fe3O4-Au@Pt NPs, they could quickly catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidize to the dark blue chelate (oxTMB), thus resulting in greatly enhanced color on the strips. The limit of detection (LOD) of NALFA reached as low as 50 pM, which was 20-fold lower than that of before catalysis. Therefore, the constructed Janus Fe3O4-Au@Pt NPs could effectively improve the performance of traditional colloidal gold LFA and provide a potential tool for the development of sensitive sensing platforms.

Protection against the H1N1 influenza virus using self-assembled nanoparticles formed by lumazine synthase and bearing the M2e peptide

There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform. The primary objective of this study was to determine the protective potential of M2e proteins expressed on Lumazine synthase (LS) nanoparticles. M2e-LS proteins, produced through the E. coli system, spontaneously assemble into nanoparticles. The study investigated the efficacy of the M2e-LS nanoparticle vaccine in mice. Mice immunized with M2e-LS nanoparticles exhibited significantly higher levels of intracellular cytokines than those receiving soluble M2e proteins. The M2e-LS protein exhibited robust immunogenicity and provided 100% protection against cross-clade influenza.

The recombinant vaccine of Lactobacillus plantarum elicits immune protection against H1N1 and H9N2 influenza virus infection

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, resulting in significant economic losses and numerous fatalities. Current vaccines, typically administered through injection, provide limited protection due to the frequent antigenic shift and drift of IAV strains. Therefore, the development of alternative broad-spectrum vaccine strategies is imperative. Lactic acid bacteria (LAB) represent promising candidates for vaccine engineering due to their low cost, high safety profile, and suitability for oral administration. In this study, we identified a strain of Lactobacillus plantarum (Lp) that is resistant to acid and bile salts and capable of colonizing the intestines of mice. Subsequently, we employed the RecE/T gene editing system to integrate headless hemagglutinins (mini-HA) into the genome of Lp, generating Lp-mini-HA-SP. Remarkably, immunization with Lp-mini-HA-SP elicited serum IgG antibody responses and conferred immune protection against H9N2 and H1N1 influenza virus challenges. Collectively, our findings offer a novel approach for the development of orally administered IAV vaccines and hold significant potential for future drug development endeavors.

Inhibitory effect of 5-hydroxy-6,7-dimethoxyflavone on H1N1 influenza virus-induced ferroptosis and inflammation in A549 cells and its possible mechanisms

To investigate the protective effect of 5-hydroxy-6,7-dimethoxyflavone (5-HDF), a compound extracted from Elsholtzia blanda Benth., against lung injury induced by H1N1 influenza virus and explore its possible mechanism of action. 5-HDF was extracted from Elsholtzia blanda Benth. using ethanol reflux extraction and silica gel chromatography and characterized using NMR and MS analyses. In an A549 cell model of H1N1 influenza virus infection (MOI=0.1), the cytotoxicity of 5-HDF was assessed using MTT assay, and its effect on TRAIL and IL-8 expressions was examined using flow cytometry; Western blotting was used to detect the expression levels of inflammatory, apoptosis, and ferroptosis-related proteins. In a mouse model of H1N1 influenza virus infection established by nasal instillation of 50 μL H1N1 virus at the median lethal dose, the effects of 30 and 60 mg/kg 5-HDF by gavage on body weight, lung index, gross lung anatomy and lung histopathology were observed. 5-HDF exhibited no significant cytotoxicity in A549 cells within the concentration range of 0-200 μg/mL. In H1N1-infected A549 cells, treatment with 5-HDF effectively inhibited the activation of phospho-p38 MAPK and phospho-NF-κB p65, lowered the expressions of IL-8, enhanced the expression of anti-ferroptosis proteins (SLC7A11 and GPX4), and inhibited the expressions of apoptosis markers PARP and caspase-3 and the apoptotic factor TRAIL. In H1N1-infected mice, treatment with 5-HDF for 7 days significantly suppressed body weight loss and increment of lung index and obviously alleviated lung tissue pathologies. 5-HDF offers protection against H1N1 influenza virus infection in mice possibly by suppressing H1N1-induced ferroptosis, inflammatory responses, and apoptosis via upregulating SLC7A11 and GPX4, inhibiting the activation of phospho-NF-κB p65 and phospho-p38 MAPK, and decreasing the expression of cleaved caspase3 and cleaved PARP.

Vaccination reduces central nervous system IL-1β and memory deficits after COVID-19 in mice.

Up to 25% of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit postacute cognitive sequelae. Although millions of cases of coronavirus disease 2019 (COVID-19)-mediated memory dysfunction are accumulating worldwide, the underlying mechanisms and how vaccination lowers risk are unknown. Interleukin-1 (IL-1), a key component of innate immune defense against SARS-CoV-2 infection, is elevated in the hippocampi of individuals with COVID-19. Here we show that intranasal infection of C57BL/6J mice with SARS-CoV-2 Beta variant leads to central nervous system infiltration of Ly6Chi monocytes and microglial activation. Accordingly, SARS-CoV-2, but not H1N1 influenza virus, increases levels of brain IL-1β and induces persistent IL-1R1-mediated loss of hippocampal neurogenesis, which promotes postacute cognitive deficits. Vaccination with a low dose of adenoviral-vectored spike protein prevents hippocampal production of IL-1β during breakthrough SARS-CoV-2 infection, loss of neurogenesis and subsequent memory deficits. Our study identifies IL-1β as one potential mechanism driving SARS-CoV-2-induced cognitive impairment in a new mouse model that is prevented by vaccination.

Mastoparan-7 adjuvanted COBRA H1 and H3 hemagglutinin influenza vaccines

Adjuvants enhance, prolong, and modulate immune responses by vaccine antigens to maximize protective immunity and enable more effective immunization in the young and elderly. Most adjuvants are formulated with injectable vaccines. However, an intranasal route of vaccination may induce mucosal and systemic immune responses for enhancing protective immunity in individuals and be easier to administer compared to injectable vaccines. In this study, a next generation of broadly-reactive influenza hemagglutinin (HA) vaccines were developed using the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology. These HA vaccines were formulated with Mastoparan 7 (M7-NH2) mast cell degranulating peptide adjuvant and administered intranasally to determine vaccine-induced seroconversion of antibodies against a panel of influenza viruses and protection following infection with H1N1 and H3N2 viruses in mice. Mice vaccinated intranasally with M7-NH2-adjuvanted COBRA HA vaccines had high HAIs against a panel of H1N1 and H3N2 influenza viruses and were protected against both morbidity and mortality, with reduced viral lung titers, following challenge with an H1N1 influenza virus. Additionally, M7-NH2 adjuvanted COBRA HA vaccines induced Th2 skewed immune responses with robust IgG and isotype antibodies in the serum and mucosal lung lavages. Overall, this intranasally delivered M7-NH2 -adjuvanted COBRA HA vaccine provides effective protection against drifted H1N1 and H3N2 viruses.

Constructing a Ready-to-Use mRNA Vaccine Delivery System for the Prevention of Influenza A virus, Utilizing FDA-Approved Raw Materials.

Messenger ribonucleic acid (mRNA) vaccines, serving as a rapid and easily scalable emergency preventive measure, have played a pivotal role in preventing infectious diseases. The effectiveness of mRNA vaccines heavily relies on the delivery carrier, but the current market options are predominantly lipid nanoparticles. Their intricate preparation process and high transportation costs pose challenges for widespread use in remote areas. In this study, we harnessed FDA-approved polymer PLGA and lipid components widely employed in clinical experiments to craft a ready-to-use mRNA vaccine delivery system known as lipid-polymer hybrid nanoparticles (LPP). Following formulation optimization, the PDCD nanoparticles emerged as the most effective, showcasing exceptional mRNA delivery capabilities both in vitro and in vivo. Loading PDCD nanoparticles with mRNA encoding the H1N1 influenza virus HA antigen-fused M2e peptide enabled the successful induction of M2e-specific antibodies and T cell immune responses in immunized mice. After three rounds of vaccine immunization, the mice demonstrated weight recovery to normal levels and maintained a survival rate exceeding 80% following an encounter with the H1N1 influenza virus. The innovative mRNA delivery system that we designed demonstrates outstanding effectiveness in preventing infectious diseases, with the potential to play an even more significant role in future clinical applications.

Dynamic Covalent Hydrogel as a Single-Dose Vaccine Adjuvant for Sustained Antigen Release and Significantly Elevated Humoral Immunity.

Vaccine is the most important way for fighting against infection diseases. However, multiple injections and unsatisfied immune responses are the main obstacles for current vaccine application. Herein, a dynamic covalent hydrogel (DCH) is used as a single-dose vaccine adjuvant for eliciting robust and sustained humoral immunity. By adjusting the mass ratio of the DCH gel, 10-30 d constant release of the loaded recombinant protein antigens is successfully realized, and it is proved that sustained release of antigens can significantly improve the vaccine efficacy. When loading SARS-CoV-2 RBD (Wuhan and Omicron BA.1 strains) antigens into this DCH gel, an over 32000 times and 8000 times improvement is observed in antigen-specific antibody titers compared to conventional Aluminum adjuvanted vaccines. The universality of this DCH gel adjuvant is confirmed in a Nipah G antigen test as well as a H1N1 influenza virus antigen test, with much improved protection of C57BL/6 mice against H1N1 virus infection than conventional Aluminum adjuvanted vaccines. This sustainably released, single-dose DCH gel adjuvant provides a new promising option for designing next-generation infection vaccines.

A Self-Fulfilling Prophecy Pandemic: The 1977 "Russian Flu".

Surprisingly, the 1977 "Russian flu" H1N1 pandemic influenza virus was genetically indistinguishable from strains that had circulated decades earlier but had gone extinct in 1957. This essay puts forward the most plausible chronology to explain the reemergence of the 1977 H1N1 pandemic virus: (1) in January-February 1976, a self-limited small outbreak of a swine H1N1 influenza virus occurred among Army personnel at Fort Dix, New Jersey; (2) in March 1976, the US launched a nationwide H1N1 swine influenza vaccine program; (3) other countries then also launched their own H1N1 R&D efforts; (4) a new H1N1 outbreak, genetically unrelated to the Fort Dix swine virus but indistinguishable from previously extinct H1N1 viruses, was detected early in 1977 in China; (5) the leading Chinese influenza virologist later disclosed that the Chinese military had conducted large H1N1 vaccine R&D studies in 1976. It is likely that the resurrected H1N1 influenza viruses were laboratory-stored strains that were unfrozen and studied as part of the emergency response to a perceived epidemic threat, and that accidentally escaped. The fear of a possible H1N1 pandemic was the critical factor that gave rise to the actual H1N1 pandemic, resulting in an avoidable "self-fulfilling prophecy pandemic."

Shufeng Jiedu capsule alleviates influenza A (H1N1) virus induced acute lung injury by regulating the lung inflammatory microenvironment

BackgroundDeath caused by respiratory tract infection is one of the leading causes of death in the world today. Shufeng Jiedu Capsule (SFJDC) is a traditional Chinese medicine that has been widely used clinically for coronavirus disease 2019 (COVID-19), H1N1 influenza virus pneumonia and other diseases. Its pharmacological effect is to inhibit inflammation and improve the body's ability to clear viruses. However, the mechanism of SFJDC in the treatment of viral pneumonia, especially its effect on the inflammatory-immune microenvironment of lung tissue remains unclear. MethodsMice with H1N1 influenza virus pneumonia were used as a model to verify the efficacy of SFJDC through death protection, lung index, viral load, and HE staining of lung tissue. The levels of inflammatory cytokines and chemokines in lung tissue were investigated by multi-analyte immunoassay. The number and proportion of cells in peripheral blood were detected by blood routine. The percentage of infiltrating immune cells in lung tissue was detected by flow cytometry and immunofluorescence. ResultsSFJDC (2.2 g/kg·d−1 and 1.1 g/kg·d−1) increased survival rate (P＜0.01, P＜0.05), prolonged the survival period of mice, and alleviated the histopathological damage in lung (P＜0.01). SFJDC (2.2 g/kg·d−1, 1.1 g/kg·d−1 and 0.055 g/kg·d−1) increased body weight(P＜0.01, P＜0.05), improved activity status, reduced the lung index (P＜0.01, P＜0.05) and viral load (P＜0.01). SFJDC (2.2 g/kg·d−1 and 1.1 g/kg·d−1) reduced interleukin-1β (IL-1β), interleukin-18(IL-18), tumour necrosis factor α (TNF-α), monocyte chemoattractant protein (MCP), chemokine (C-X-C motif) ligand 1 (CXCL1) (P＜0.01, P＜0.05), and SFJDC (2.2 g/kg·d−1) increased IL-10 levels (P＜0.05) to regulate inflammation. SFJDC (2.2 g/kg·d−1) increased the percentages of CD4+ T cells (P＜0.01), CD8+ T cells (P＜0.05), and B cells(P＜0.05), and decreased F4/80+ macrophages (P＜0.05). ConclusionOur findings indicated that SFJDC could inhibit inflammation and lung injury while maintaining the function of the adaptive immune response mediated by T and B cells, and promote the clearance of the virus, thereby treating influenza A (H1N1) virus-induced pneumonia.

A combination influenza mRNA vaccine candidate provided broad protection against diverse influenza virus challenge

Influenza viruses present a significant threat to global health. The production of a universal vaccine is considered essential due to the ineffectiveness of current seasonal influenza vaccines against mutant strains. mRNA technology offers new prospects in vaccinology, with various candidates for different infectious diseases currently in development and testing phases. In this study, we encapsulated a universal influenza mRNA vaccine. The vaccine encoded influenza hemagglutinin (HA), nucleoprotein (NP), and three tandem repeats of matrix protein 2 (3M2e). Twice-vaccinated mice exhibited strong humoral and cell-mediated immune responses in vivo. Notably, these immune responses led to a significant reduction in viral load of the lungs in challenged mice, and also conferred protection against future wild-type H1N1, H3N2, or H5N1 influenza virus challenges. Our findings suggest that this mRNA-universal vaccine strategy for influenza virus may be instrumental in mitigating the impact of future influenza pandemics.

Preparation and characterization of mouse-derived monoclonal antibodies against the hemagglutinin of the H1N1 influenza virus

H1N1 influenza virus is a significant global public health concern. Monoclonal antibodies (mAbs) targeting specific viral proteins such as hemagglutinin (HA) have become an important therapeutic strategy, offering highly specific targeting to block viral transmission and infection. This study focused on the development of mAbs targeting HA of the A/Victoria/2570/2019 (H1N1pdm09, VIC-19) strain by utilizing hybridoma technology to produce two mAbs with high binding capacity. Notably, mAb 2B2 has demonstrated a strong affinity for HA proteins in recent H1N1 influenza vaccine strains. In vitro assessments showed that both mAbs exhibited broad-spectrum hemagglutination inhibition and potent neutralizing effects against various vaccine strains of H1N1pdm09 viruses. 2B2 was also effective in animal models, offering both preventive and therapeutic protection against infections caused by recent H1N1 strains, highlighting its potential for clinical application. By individually co-cultivating each of the aforementioned mAbs with the virus in chicken embryos, four amino acid substitution sites in HA (H138Q, G140R, A141E/V, and D187E) were identified in escape mutants, three in the antigenic site Ca2, and one in Sb. The identification of such mutations is pivotal, as it compels further investigation into how these alterations could undermine the binding efficacy and neutralization capacity of antibodies, thereby impacting the design and optimization of mAb therapies and influenza vaccines. This research highlights the necessity for continuous exploration into the dynamic interaction between viral evolution and antibody response, which is vital for the formulation of robust therapeutic and preventive strategies against influenza.

Pathogenicity and escape to pre-existing immunity of a new genotype of swine influenza H1N2 virus that emerged in France in 2020

In 2020, a new genotype of swine H1N2 influenza virus (H1avN2–HA 1C.2.4) was identified in France. It rapidly spread within the pig population and supplanted the previously predominant H1avN1-HA 1C.2.1 virus. To characterize this new genotype which is genetically and antigenically distant from the other H1avNx viruses detected in France, an experimental study was conducted to compare the outcomes of H1avN2 and H1avN1 infections in pigs and evaluate the protection conferred by the only inactivated vaccine currently licensed in Europe containing an HA 1C (clade 1C.2.2) antigen. Infection with H1avN2 induced stronger clinical signs and earlier shedding than H1avN1. The neutralizing antibodies produced following H1avN2 infection were unable to neutralize H1avN1, and vice versa, whereas the cellular-mediated immunity cross-reacted. Vaccination slightly altered the impact of H1avN2 infection at the clinical level, but did not prevent shedding of infectious virus particles. It induced a cellular-mediated immune response towards H1avN2, but did not produce neutralizing antibodies against this virus. As in vaccinated animals, animals previously infected by H1avN1 developed a cross-reacting cellular immune response but no neutralizing antibodies against H1avN2. However, H1avN1 pre-infection induced a better protection against the H1avN2 infection than vaccination, probably due to higher levels of non-neutralizing antibodies and a mucosal immunity. Altogether, these results showed that the new H1avN2 genotype induced a severe respiratory infection and that the actual vaccine was less effective against this H1avN2-HA 1C.2.4 than against H1avN1-HA 1C.2.1, which may have contributed to the H1avN2 epizootic and dissemination in pig farms in France.

Risk assessment of a highly pathogenic H5N1 influenza virus from mink

Outbreaks of highly pathogenic H5N1 clade 2.3.4.4b viruses in farmed mink and seals combined with isolated human infections suggest these viruses pose a pandemic threat. To assess this threat, using the ferret model, we show an H5N1 isolate derived from mink transmits by direct contact to 75% of exposed ferrets and, in airborne transmission studies, the virus transmits to 37.5% of contacts. Sequence analyses show no mutations were associated with transmission. The H5N1 virus also has a low infectious dose and remains virulent at low doses. This isolate carries the adaptive mutation, PB2 T271A, and reversing this mutation reduces mortality and airborne transmission. This is the first report of a H5N1 clade 2.3.4.4b virus exhibiting direct contact and airborne transmissibility in ferrets. These data indicate heightened pandemic potential of the panzootic H5N1 viruses and emphasize the need for continued efforts to control outbreaks and monitor viral evolution.

Detection of clade 2.3.4.4b highly pathogenic H5N1 influenza virus in New York City.

Highly pathogenic avian influenza viruses of the H5N1 clade 2.3.4.4b were detected in North America in the winter of 2021/2022. These viruses have spread across the Americas, causing morbidity and mortality in both wild and domestic birds as well as some mammalian species, including cattle. Many surveillance programs for wildlife as well as commercial poultry operations have detected these viruses. In this study, we conducted surveillance of avian species in the urban environment in New York City. We detected highly pathogenic H5N1 viruses in six samples from four different bird species and performed whole-genome sequencing. Sequencing analysis showed the presence of multiple different genotypes. Our work highlights that the interface between animals and humans that may give rise to zoonotic infections or even pandemics is not limited to rural environments and commercial poultry operations but extends into the heart of our urban centers.IMPORTANCEWhile surveillance programs for avian influenza viruses are often focused on migratory routes and their associated stop-over locations or commercial poultry operations, many bird species-including migratory birds-frequent or live in urban green spaces and wetlands. This brings them into contact with a highly dense population of humans and pets, providing an extensive urban animal-human interface in which the general public may have little awareness of circulating infectious diseases. This study focuses on virus surveillance of this interface, combined with culturally responsive science education and community outreach.

Very Broadly Effective Hemagglutinin-Directed Influenza Vaccines with Anti-Herpetic Activity.

A universal vaccine that generally prevents influenza virus infection and/or illness remains elusive. We have been exploring a novel approach to vaccination involving replication-competent controlled herpesviruses (RCCVs) that can be deliberately activated to replicate efficiently but only transiently in an administration site in the skin of a subject. The RCCVs are derived from a virulent wild-type herpesvirus strain that has been engineered to contain a heat shock promoter-based gene switch that controls the expression of, typically, two replication-essential viral genes. Additional safety against inadvertent replication is provided by an appropriate secondary mechanism. Our first-generation RCCVs can be activated at the administration site by a mild local heat treatment in the presence of an antiprogestin. Here, we report that epidermal vaccination with such RCCVs expressing a hemagglutinin or neuraminidase of an H1N1 influenza virus strain protected mice against lethal challenges by H1N1 virus strains representing 75 years of evolution. Moreover, immunization with an RCCV expressing a subtype H1 hemagglutinin afforded full protection against a lethal challenge by an H3N2 influenza strain, and an RCCV expressing a subtype H3 hemagglutinin protected against a lethal challenge by an H1N1 strain. Vaccinated animals continued to gain weight normally after the challenge. Protective effects were even observed in a lethal influenza B virus challenge. The RCCV-based vaccines induced robust titers of in-group, cross-group and even cross-type neutralizing antibodies. Passive immunization suggested that observed vaccine effects were at least partially antibody-mediated. In summary, RCCVs expressing a hemagglutinin induce robust and very broad cross-protective immunity against influenza.

Effect of human H3N2 influenza virus reassortment on influenza incidence and severity during the 2017–18 influenza season in the USA: a retrospective observational genomic analysis

During the 2017-18 influenza season in the USA, there was a high incidence of influenza illness and mortality. However, no apparent antigenic change was identified in the dominant H3N2 viruses, and the severity of the season could not be solely attributed to a vaccine mismatch. We aimed to investigate whether the altered virus properties resulting from gene reassortment were underlying causes of the increased case number and disease severity associated with the 2017-18 influenza season. Samples included were collected from patients with influenza who were prospectively recruited during the 2016-17 and 2017-18 influenza seasons at the Johns Hopkins Hospital Emergency Departments in Baltimore, MD, USA, as well as from archived samples from Johns Hopkins Health System sites. Among 647 recruited patients with influenza A virus infection, 411 patients with whole-genome sequences were available in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance network during the 2016-17 and 2017-18 seasons. Phylogenetic trees were constructed based on viral whole-genome sequences. Representative viral isolates of the two seasons were characterised in immortalised cell lines and human nasal epithelial cell cultures, and patients' demographic data and clinical outcomes were analysed. Unique H3N2 reassortment events were observed, resulting in two predominant strains in the 2017-18 season: HA clade 3C.2a2 and clade 3C.3a, which had novel gene segment constellations containing gene segments from HA clade 3C.2a1 viruses. The reassortant re3C.2a2 viruses replicated with faster kinetics and to a higher peak titre compared with the parental 3C.2a2 and 3C.2a1 viruses (48 h vs 72 h). Furthermore, patients infected with reassortant 3C.2a2 viruses had higher Influenza Severity Scores than patients infected with the parental 3C.2a2 viruses (median 3·00 [IQR 1·00-4·00] vs 1·50 [1·00-2·00]; p=0·018). Our findings suggest that the increased severity of the 2017-18 influenza season was due in part to twointrasubtypes, cocirculating H3N2 reassortant viruses with fitness advantages over the parental viruses. This information could help inform future vaccine development and public health policies. The Center of Excellence for Influenza Research and Response in the US, National Science and Technology Council, and Chang Gung Memorial Hospital in Taiwan.

Evaluating the impact of influenza infection on pig lungs by single-cell RNA sequencing (scRNA-seq)

Abstract Despite being the most widely consumed meat worldwide, the pig industry faces economic challenges from respiratory viruses including influenza. Pigs are also used as a reliable translational model for human respiratory disorders due to their similar respiratory tracts. However, limited pig-specific reagents have hindered understanding the pig’s immune system. Hence, here we used single cell RNA sequencing (scRNAseq) for unbiased transcriptional profiling of pig immune responses during influenza infection and oseltamivir antiviral therapy. Fourteen six-week-old mixed-breed pigs were intratracheally infected with pandemic 2009 H1N1 influenza virus. Seven of these pigs were treated for 5 days with oseltamivir. A control group was mock-infected. Lung samples were taken 5 days post-infection for scRNAseq, histopathology, and virus titers. We identified 24 pig leukocytes populations which were integrated with previously published human and mouse lung cells. Influenza virus infection/oseltamivir treatment altered the cellular composition, transcriptional networks, and cell-cell interactions in the lung. γδ T cells that are unique to pigs and abundant in the lung responded strongly to infection. Oseltamivir reduced antiviral and inflammatory responses. Our data provide a resource to better understand the pulmonary immune responses of pigs and advances using swine as a model for studying human pulmonary immune responses.

Guanylate-binding protein 1 inhibits inflammatory factors produced by H5N1 virus through Its GTPase activity

The combination of inflammatory factors resulting from an influenza A virus infection is one of the main causes of death in host animals. Studies have shown that guinea pig guanosine monophosphate binding protein 1 (guanylate-binding protein 1, gGBP1) can downregulate cytokine production induced by the influenza virus. Therefore, exploring the innate immune defense mechanism of GBP1 in the process of H5N1 influenza virus infection has important implications for understanding the pathogenic mechanism, disease prevention, and the control of influenza A virus infections. We found that, in addition to inhibiting the early replication of influenza virus, gGBP1 also inhibited the production of CCL2 and CXCL10 cytokines induced by the influenza virus as well as the proliferation of mononuclear macrophages induced by these cytokines. These findings further confirmed that gGBP1 inhibited the production of cytokines through its GTPase activity and cell proliferation through its C-terminal α-helix structure. This study revealed the effect of gGBP1 on the production of cellular inflammatory factors during influenza virus infection and determined the key amino acid residues that assist in the inhibitory processes mediated by gGBP1.

Therapeutic effect of Yinhuapinggan granules mediated through the intestinal flora in mice infected with the H1N1 influenza virus.

In this study, we examined the therapeutic effects of Yinhuapinggan granules (YHPGs) in influenza-infected mice. We also examined how YHPGs affect the composition of the intestinal flora and associated metabolites. We used the nasal drip method to administer the influenza A virus (IAV) H1N1 to ICR mice. Following successful model construction, the mice were injected with 0.9% sterile saline and low (5.5 g/kg), medium (11 g/kg), and high (22 g/kg) doses of YHPGs. The pathological changes in the lungs and intestines were evaluated by gavage for 5 consecutive days. Detection of sIgA, IL-6, TNF-α, INF-γ, and TGF-β cytokine levels in serum by enzyme-linked immunosorbent assay. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to measure the mRNA and protein expression of the tight junction proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) in the colon. To assess the influence of YHPGs on the intestinal microbiota, feces were obtained from the mice for 16s rRNA sequencing, and short-chain fatty acids (SCFAs) were measured in the feces. By reducing the production of pro-inflammatory cytokines and increasing the relative expression of claudin-1, occludin, and ZO-1 in colon tissues, YHPGs had a protective effect in tissues from the lungs and colon. When YHPGs were administered to mice with IAV infection, the relative abundance of Lactobacillus, Coprobacillus, Akkermansia, Prevotella, Oscillospira, and Ruminococcus increased, whereas the relative abundance of Desulfovibrio decreased. The therapeutic mechanism of YHPGs against IAV infection in mice may be underpinned by modulation of the structural composition of colonic bacteria and regulation of SCFA production.