ABSTRACT
Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.
Subject(s)
Active Transport, Cell Nucleus , Protein Biosynthesis , RNA, Messenger , RNA, Viral , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Animals , Influenza A virus/genetics , Influenza A virus/physiology , Influenza A virus/metabolism , Virus Replication , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H1N1 Subtype/physiology , Cell Nucleus/metabolism , Cell Nucleus/virology , 5' Untranslated Regions/genetics , Nucleocapsid Proteins/metabolism , Nucleocapsid Proteins/genetics , Madin Darby Canine Kidney Cells , HEK293 Cells , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Dogs , Influenza, Human/virology , Influenza, Human/metabolism , Influenza, Human/genetics , Mutation , Host-Pathogen Interactions/genetics , Viral Core Proteins/metabolism , Viral Core Proteins/geneticsABSTRACT
Upregulation of ADAMTS-4 has been reported to have an important role in lung injury, and ADAMTS-4 expression is regulated by miR-126a-5p in abdominal aortic aneurysms. The aim of this study was to investigate whether miR-126a-5p/ADAMTS-4 plays a role in influenza-virus-induced lung injury. Lung fibroblasts were infected with H1N1 influenza virus to detect changes in miR-126a-5p and ADAMTS-4 expression, and cell viability was measured by CCK-8 assay. Inflammatory factors and matrix protease levels were examined using ELISA kits, and cell apoptosis was assessed by measuring the levels of apoptosis-related proteins. A dual luciferase assay was used to verify the regulatory relationship between miR-126a-5p and ADAMTS-4. H1N1 influenza virus reduced fibroblast viability, inhibited miR-126a-5p expression, and promoted ADAMTS-4 expression. Overexpression of miR-126a-5p attenuated the cellular inflammatory response, apoptosis, matrix protease secretion, and virus replication. Luciferase reporter assays revealed that miR-126a-5p inhibited ADAMTS-4 expression by targeting ADAMTS-4 mRNA. Further experiments showed that overexpression of ADAMTS-4 significantly reversed the inhibitory effects of miR-126a-5p on fibroblast inflammation, apoptosis, matrix protease secretion, and virus replication. Upregulation of miR-126a-5p inhibits H1N1-induced apoptosis, inflammatory factors, and matrix protease secretion, as well as virus replication in lung fibroblasts.
Subject(s)
ADAMTS4 Protein , Apoptosis , Fibroblasts , Inflammation , Influenza A Virus, H1N1 Subtype , Lung , MicroRNAs , MicroRNAs/genetics , MicroRNAs/metabolism , Fibroblasts/virology , Fibroblasts/metabolism , Humans , Lung/virology , Lung/pathology , ADAMTS4 Protein/genetics , ADAMTS4 Protein/metabolism , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Inflammation/genetics , Cell Survival , Virus Replication , Influenza, Human/virology , Influenza, Human/genetics , Influenza, Human/metabolism , Cell LineABSTRACT
Tuft cells are a group of rare epithelial cells that can detect pathogenic microbes and parasites. Many of these cells express signaling proteins initially found in taste buds. It is, however, not well understood how these taste signaling proteins contribute to the response to the invading pathogens or to the recovery of injured tissues. In this study, we conditionally nullified the signaling G protein subunit Gγ13 and found that the number of ectopic tuft cells in the injured lung was reduced following the infection of the influenza virus H1N1. Furthermore, the infected mutant mice exhibited significantly larger areas of lung injury, increased macrophage infiltration, severer pulmonary epithelial leakage, augmented pyroptosis and cell death, greater bodyweight loss, slower recovery, worsened fibrosis and increased fatality. Our data demonstrate that the Gγ13-mediated signal transduction pathway is critical to tuft cells-mediated inflammation resolution and functional repair of the damaged lungs.To our best knowledge, it is the first report indicating subtype-specific contributions of tuft cells to the resolution and recovery.
Subject(s)
Influenza A Virus, H1N1 Subtype , Signal Transduction , Animals , Mice , Influenza A Virus, H1N1 Subtype/physiology , Orthomyxoviridae Infections , Lung Injury/metabolism , Lung/pathology , Inflammation , Epithelial Cells/metabolism , Mice, Knockout , Disease Models, AnimalABSTRACT
Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LCâMS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.
Subject(s)
Mitochondria , Viral Proteins , Virus Replication , Humans , Mitochondria/metabolism , Mitochondria/virology , Viral Proteins/metabolism , Viral Proteins/genetics , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/genetics , Influenza A virus/physiology , Influenza A virus/genetics , Influenza A virus/pathogenicity , Influenza A virus/metabolism , Host-Pathogen Interactions , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/physiology , Influenza A Virus, H3N2 Subtype/metabolism , Autophagy , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H1N1 Subtype/pathogenicity , HEK293 Cells , Influenza, Human/virology , Influenza, Human/metabolism , A549 Cells , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Tandem Mass SpectrometryABSTRACT
Qingke Pingchuan granules (QPGs), which contain Houttuynia cordata Thunb, Fritillaria cirrhosa, fired licorice, and fired bitter almonds, among other components, can clear heat and ventilate the lungs, relieving cough and asthma. Clinically, QPGs are mainly used to treat cough, asthma, fever and other discomforts caused by acute or chronic bronchitis. In this study, the antiviral activity of QPGs against respiratory syncytial virus (RSV), influenza A virus A/FM/1/47 (H1N1), oseltamivir-resistant H1N1, A/Beijing/32/92 (H3N2), Sendai virus, and human adenovirus type 3 in Hep-2 or MDCK cells was evaluated using the CCK-8 method, and the cytotoxicity of QPGs to these two cell lines was tested. The effect of QPGs on mice infected with influenza A virus A/FM/1/47 (H1N1) was evaluated by measuring body weight, survival time, and survival rate, as well as virus titers and lesions in the lungs and levels of inflammatory factors in serum. In addition, the expression of TLR-7-My88-NF-κB signaling pathway-related proteins in lung tissues was analyzed by Western blotting and qRT-PCR. The results showed that QPGs had a potent inhibitory effect on the six viruses tested in vitro. Interestingly, QPGs also displayed particularly pronounced antiviral activity against H1N1-OC, similar to that of oseltamivir, a well-known antiviral drug. QPGs effectively protected mice from infection by H1N1, as indicated by significantly increased body weights, survival times, and survival rates and reduced lung virus titers of inflammatory factors and lung tissue injury. The levels of TLR-7-MyD88-NF-κB-pathway-related proteins in the lung tissue of infected mice were found to be decreased after QPG treatment, thereby alleviating lung injury caused by excessive release of inflammatory factors. Taken together, these findings indicate that QPGs have satisfactory activity against influenza virus infection.
Subject(s)
Antiviral Agents , Drugs, Chinese Herbal , Influenza A Virus, H1N1 Subtype , Orthomyxoviridae Infections , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Mice , Drugs, Chinese Herbal/pharmacology , Humans , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Dogs , Madin Darby Canine Kidney Cells , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/physiology , Mice, Inbred BALB C , Lung/virology , Lung/drug effects , Lung/pathology , Cell Line , Houttuynia/chemistry , Influenza, Human/drug therapy , Influenza, Human/virology , NF-kappa B/metabolism , Female , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/physiologyABSTRACT
Pregnancy heightens susceptibility to influenza A virus (IAV) infection, thereby increasing the risk of severe pneumonia and maternal mortality. It also raises the chances of adverse outcomes in offspring, such as fetal growth restriction, preterm birth, miscarriage, and stillbirth in offsprings. However, the underlying mechanisms behind these effects remain largely unknown. Syncytiotrophoblast cells, crucial in forming the placental barrier, nutrient exchange and hormone secretion, have not been extensively studied for their responses to IAV. In our experiment, we used Forskolin-treated BeWo cells to mimic syncytiotrophoblast cells in vitro, and infected them with H1N1, H5N1 and H7N9 virus stains. Our results showed that syncytiotrophoblast cells, with their higher intensity of sialic acid receptors, strongly support IAV infection and replication. Notably, high-dose viral infection and prolonged exposure resulted in a significant decrease in fusion index, as well as gene and protein expression levels associated with trophoblast differentiation, ß-human chorionic gonadotropin secretion, estrogen and progesterone biosynthesis, and nutrient transport. In pregnant BALB/c mice infected with the H1N1 virus, we observed significant decreases in trophoblast differentiation and hormone secretion gene expression levels. IAV infection also resulted in preterm labor, fetal growth restriction, and increased maternal and fetal morbidity and mortality. Our findings indicate that IAV infection in syncytiotrophoblastic cells can result in adverse pregnancy outcomes by altering trophoblast differentiation, suppressing of ß-hCG secretion, and disrupting placental barrier function.
Subject(s)
Influenza A Virus, H1N1 Subtype , Mice, Inbred BALB C , Orthomyxoviridae Infections , Pregnancy Outcome , Trophoblasts , Female , Trophoblasts/virology , Pregnancy , Animals , Humans , Influenza A Virus, H1N1 Subtype/physiology , Mice , Orthomyxoviridae Infections/virology , Influenza, Human/virology , Cell Line , Influenza A Virus, H5N1 Subtype/physiology , Influenza A Virus, H7N9 Subtype/physiology , Influenza A Virus, H7N9 Subtype/pathogenicity , Pregnancy Complications, Infectious/virology , Placenta/virology , Virus ReplicationABSTRACT
Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human , Orthomyxoviridae Infections , Virus Replication , Animals , Swine , Orthomyxoviridae Infections/virology , Humans , Risk Assessment , Influenza, Human/virology , Influenza, Human/epidemiology , Cell Line , Influenza A Virus, H1N1 Subtype/physiology , Swine Diseases/virology , Disease Models, Animal , Influenza A Virus, H1N2 Subtype/genetics , Pandemics , Mice , Dogs , Epithelial Cells/virology , FemaleABSTRACT
In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.
Subject(s)
COVID-19 Vaccines , Influenza Vaccines , Animals , Humans , Mice , Antibodies, Neutralizing , Antibodies, Viral , Antibody Formation , COVID-19/immunology , COVID-19/prevention & control , Influenza A Virus, H1N1 Subtype/physiology , Influenza Vaccines/immunology , Nucleoproteins , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , COVID-19 Vaccines/immunology , Influenza, Human/immunology , Influenza, Human/prevention & controlABSTRACT
Expulsions of virus-laden aerosols or droplets from the oral and nasal cavities of an infected host are an important source of onward respiratory virus transmission. However, the presence of infectious influenza virus in the oral cavity during infection has not been widely considered, and thus, little work has explored the environmental persistence of influenza virus in oral cavity expulsions. Using the ferret model, we detected infectious virus in the nasal and oral cavities, suggesting that the virus can be expelled into the environment from both anatomical sites. We also assessed the stability of two influenza A viruses (H1N1 and H3N2) in droplets of human saliva or respiratory mucus over a range of relative humidities. We observed that influenza virus infectivity decays rapidly in saliva droplets at intermediate relative humidity, while viruses in airway surface liquid droplets retain infectivity. Virus inactivation was not associated with bulk protein content, salt content, or droplet drying time. Instead, we found that saliva droplets exhibited distinct inactivation kinetics during the wet and dry phases at intermediate relative humidity, and droplet residue morphology may lead to the elevated first-order inactivation rate observed during the dry phase. Additionally, distinct differences in crystalline structure and nanobead localization were observed between saliva and airway surface liquid droplets. Together, our work demonstrates that different respiratory fluids exhibit unique virus persistence profiles and suggests that influenza viruses expelled from the oral cavity may contribute to virus transmission in low- and high-humidity environments.IMPORTANCEDetermining how long viruses persist in the environment is important for mitigating transmission risk. Expelled infectious droplets and aerosols are composed of respiratory fluids, including saliva and complex mucus mixtures, but how well influenza viruses survive in such fluids is largely unknown. Here, we find that infectious influenza virus is present in the oral cavity of infected ferrets, suggesting that saliva-containing expulsions can play a role in onward transmission. Additionally, influenza virus in droplets composed of saliva degrades more rapidly than virus within respiratory mucus. Droplet composition impacts the crystalline structure and virus localization in dried droplets. These results suggest that viruses from distinct sites in the respiratory tract could have variable persistence in the environment, which will impact viral transmission fitness.
Subject(s)
Influenza A Virus, H1N1 Subtype , Animals , Humans , Humidity , Influenza A Virus, H1N1 Subtype/physiology , Saliva , Influenza A Virus, H3N2 Subtype/physiology , Seasons , Ferrets , Mucus , AerosolsABSTRACT
Influenza A virus (IAV) is one of the major global public health concerns but the emerging resistance of IAV to currently available antivirals requires the need to identify potential alternatives. Polyphenol rich sugarcane extract (PRSE) is an extract prepared from the sugarcane plant Saccharum Officinarum. Herein we aimed to determine if PRSE had antiviral activity against IAV. We showed that treatment of IAV-infected cells with PRSE results in a dose-dependent inhibition of virus infection at concentrations that were non-cytotoxic. PRSE treatment limited the early stages of infection, reducing viral genome replication, mRNA transcription and viral protein expression. PRSE did not affect the ability of IAV to bind sialic acid or change the morphology of viral particles. Additionally, PRSE treatment attenuated the replication of multiple IAV strains of the H3N2 and H1N1 subtype. In conclusion, we show that PRSE displays antiviral activity against a broad range of IAV strains, in vitro.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Saccharum , Humans , Polyphenols/pharmacology , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H3N2 Subtype , Virus Replication , Plant Extracts/pharmacology , Antiviral Agents/pharmacologyABSTRACT
Swine flu caused by swine influenza A virus (swIAV) is an acute respiratory viral disease that is spreading in swine herds worldwide. Although the effect of some host factors on replication of swIAV has been identified, the role of CD46 in this process is unclear. Here, we report that CD46 inhibits the replication of swIAV by promoting the production of type I interferons (IFNs) in porcine kidney (PK-15) cells. CD46 knockout (CD46-KO) and stably expressing (CD46-overexpression) PK-15 cells were prepared using lentivirus-mediated CRISPR/Cas9 gene editing and seamless cloning technology. The results of virus infection in CD46-overexpression PK-15 cells showed that the replication of H1N1 and H3N2 swIAVs were inhibited, and the production of type I IFNs (IFN-α, IFN-ß), interferon regulatory factor (IRF) 3, and mitochondrial antiviral-signaling protein (MAVS) was enhanced. Virus infection in CD46-KO PK-15 cells showed the opposite results. Further results showed that CD46-KO PK-15 cells have a favorable ability to proliferate influenza viruses compared to Madin-Darby canine kidney (MDCK) and PK-15 cells. These findings indicate that CD46 acts as promising target regulating the replication of swIAV, and help to develop new agents against infection and replication of the virus.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Interferon Type I , Swine Diseases , Virus Diseases , Animals , Dogs , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H3N2 Subtype , Interferon Type I/genetics , Swine , Virus Diseases/veterinary , Virus Replication/geneticsABSTRACT
In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Orthomyxoviridae Infections , Animals , Mice , Humans , Influenza A Virus, H1N1 Subtype/physiology , NLR Family, Pyrin Domain-Containing 3 Protein , Virulence , InflammasomesABSTRACT
Influenza A virus is an important respiratory pathogen that poses serious threats to human health. Owing to the high mutation rate of viral genes, weaker cross-protection of vaccines, and rapid emergence of drug resistance, there is an urgent need to develop new antiviral drugs against influenza viruses. Taurocholic acid is a primary bile acid that promotes digestion, absorption, and excretion of dietary lipids. Here, we demonstrate that sodium taurocholate hydrate (STH) exhibits broad-spectrum antiviral activity against influenza strains H5N6, H1N1, H3N2, H5N1, and H9N2 in vitro. STH significantly inhibited the early stages of influenza A virus replication. The levels of influenza virus viral RNA (vRNA), complementary RNA (cRNA), and mRNA were specifically reduced in virus-infected cells following STH treatment. In vivo, STH treatment of infected mice alleviated clinical signs and reduced weight loss and mortality. STH also reduced TNF-α, IL-1ß, and IL-6 overexpression. STH significantly inhibited the upregulation of TLR4 and the NF-kB family member p65, both in vivo and in vitro. These results suggest that STH exerts a protective effect against influenza infection via suppression of the NF-kB pathway, highlighting the potential use of STH as a drug for treating influenza infection.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A Virus, H5N1 Subtype , Influenza A Virus, H9N2 Subtype , Influenza, Human , Humans , Animals , Mice , Influenza, Human/drug therapy , NF-kappa B/metabolism , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/metabolism , Taurocholic Acid , Inflammation/drug therapy , Virus Replication , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic useABSTRACT
Respiratory viruses can be transmitted by multiple modes, including contaminated surfaces, commonly referred to as fomites. Efficient fomite transmission requires that a virus remain infectious on a given surface material over a wide range of environmental conditions, including different relative humidities. Prior work examining the stability of influenza viruses on surfaces has relied upon virus grown in media or eggs, which does not mimic the composition of virus-containing droplets expelled from the human respiratory tract. In this study, we examined the stability of the 2009 pandemic H1N1 (H1N1pdm09) virus on a variety of nonporous surface materials at four different humidities. Importantly, we used virus grown in primary human bronchial epithelial cell (HBE) cultures from different donors to recapitulate the physiological microenvironment of expelled viruses. We observed rapid inactivation of H1N1pdm09 on copper under all experimental conditions. In contrast to copper, viruses were stable on polystyrene plastic, stainless steel, aluminum, and glass, at multiple relative humidities, but greater decay on acrylonitrile butadiene styrene (ABS) plastic was observed at short time points. However, the half-lives of viruses at 23% relative humidity were similar among noncopper surfaces and ranged from 4.5 to 5.9 h. Assessment of H1N1pdm09 longevity on nonporous surfaces revealed that virus persistence was governed more by differences among HBE culture donors than by surface material. Our findings highlight the potential role of an individual's respiratory fluid on viral persistence and could help explain heterogeneity in transmission dynamics. IMPORTANCE Seasonal epidemics and sporadic pandemics of influenza cause a large public health burden. Although influenza viruses disseminate through the environment in respiratory secretions expelled from infected individuals, they can also be transmitted by contaminated surfaces where virus-laden expulsions can be deposited. Understanding virus stability on surfaces within the indoor environment is critical to assessing influenza transmission risk. We found that influenza virus stability is affected by the host respiratory secretion in which the virus is expelled, the surface material on which the droplet lands, and the ambient relative humidity of the environment. Influenza viruses can remain infectious on many common surfaces for prolonged periods, with half-lives of 4.5 to 5.9 h. These data imply that influenza viruses are persistent in indoor environments in biologically relevant matrices. Decontamination and engineering controls should be used to mitigate influenza virus transmission.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Humans , Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/epidemiology , Humidity , Copper , Plastics , LungABSTRACT
Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.IMPORTANCEThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.
Subject(s)
Coinfection , Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Animals , Humans , Streptococcus pneumoniae/physiology , Ferrets , Influenza A Virus, H1N1 Subtype/physiology , Respiratory Aerosols and DropletsABSTRACT
Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Animals , Dogs , Mice , Carcinogenesis/genetics , Cell Proliferation , Influenza A Virus, H1N1 Subtype/physiology , Madin Darby Canine Kidney Cells , Mice, Nude , Protein Glutamine gamma Glutamyltransferase 2/metabolismABSTRACT
Given an infected host, estimating the time that has elapsed since initial exposure to the pathogen is an important problem in public health. In this paper we use longitudinal gene expression data from human challenge studies of viral respiratory illnesses for building predictive models to estimate the time elapsed since onset of respiratory infection. We apply sparsity driven machine learning to this time-stamped gene expression data to model the time of exposure by a pathogen and subsequent infection accompanied by the onset of the host immune response. These predictive models exploit the fact that the host gene expression profile evolves in time and its characteristic temporal signature can be effectively modeled using a small number of features. Predicting the time of exposure to infection to be in first 48 h after exposure produces BSR in the range of 80-90% on sequestered test data. A variety of machine learning experiments provide evidence that models developed on one virus can be used to predict exposure time for other viruses, e.g., H1N1, H3N2, and HRV. The interferon [Formula: see text] signaling pathway appears to play a central role in keeping time from onset of infection. Successful prediction of the time of exposure to a pathogen has potential ramifications for patient treatment and contact tracing.
Subject(s)
Influenza A Virus, H1N1 Subtype , Respiratory Tract Infections , Virus Diseases , Humans , Influenza A Virus, H3N2 Subtype/physiology , Influenza A Virus, H1N1 Subtype/physiology , Machine LearningABSTRACT
OBJECTIVE: Influenza pandemic of the human lung was caused by the Influenza A (H1N1) over 100 years ago in 1918, but it recurred in pandemic fashion in 2009. Understanding the pathobiology of this infectious agent in the human lung could lead to adjuvant therapies that are relatively non-toxic and reduce the mortality of the human host. Overall, our objective was to apply morphoproteomics to pulmonary lung sections from an autopsied victim so that we may better define its biology from the perspective of its interaction with the host and provide options for therapeutic targets. METHODS: Morphoproteomic analysis from a case study of this Influenza A (H1N1) pulmonary infection included immunohistochemical probes to detect the expressions of fatty acid synthase (FAS), CD163+ (M2 polarized monocytes/macrophages), and programmed death-ligand 1 (PD-L1) expression as part of the host response to interaction with the Influenza A (H1N1) virus. RESULTS: Representative sections of the Influenza A (H1N1) victim's lung showed: cytoplasmic expression of FAS in most of the sloughed and atypical alveolar pneumocytes; abundance of intra-alveolar and alveolar interstitial CD163+ macrophages/monocytes; and PD-L1 expression on occasional macrophages, and focally on collections of alveolar pneumocytes and the alveolar interstitium. CONCLUSION: Morphoproteomics and microanatomical features coincide with the etiopathogenic features of pulmonary Influenza A (H1N1) infection and the host response. This plus data mining of the medical literature suggests that adjunctive, targeted therapy such as metformin and vitamin D3 could address the biology of Influenza A (H1N1) pneumonia, enhance the host immune response, and prevent its progression to a life-threatening, ventilator-dependent clinical situation.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human , Humans , Influenza A Virus, H1N1 Subtype/physiology , B7-H1 Antigen , Lung/pathology , Alveolar Epithelial CellsABSTRACT
Several small animal models, including mice, Syrian hamsters, guinea pigs, and ferrets are used to study the pathogenicity, transmissibility, and antigenicity of seasonal and pandemic influenza viruses. Moreover, animal models are essential for vaccination and challenge studies to evaluate the immunogenicity and protective efficacy of new vaccines. However, authentic human influenza viruses do not always replicate efficiently in these animal models. Previously, we developed a high-yield A/Puerto Rico/8/34 (PR8-HY) vaccine virus backbone that conferred an increased virus yield to several seasonal influenza vaccines in eukaryotic cells and embryonated chicken eggs. Here, we show that this PR8-HY genetic backbone also increases the replication of several seasonal influenza viruses in Syrian hamsters compared to the authentic viruses. Therefore, the PR8-HY backbone is useful for animal studies to assess the biological properties of influenza viral HA and NA.
Subject(s)
Influenza A Virus, H1N1 Subtype , Animals , Cricetinae , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Mesocricetus , Orthomyxoviridae Infections , Reassortant Viruses/genetics , Virus ReplicationABSTRACT
Age-induced dysregulation of the immune response is a major contributor to the morbidity and mortality related to influenza a virus infections. Experimental data have shown substantial changes to the activation and maintenance of the immune response will occur with age, but it remains unclear which of these many interrelated changes are most critical to controlling the survival of the host during infection. To ascertain which mechanisms are predominantly responsible for the increased morbidity in elderly hosts, we developed an ordinary differential equation model to simulate the immune response to pandemic H1N1 infection. We fit this model to experimental data measured in young and old macaques. We determined that the severity of the infection in the elderly hosts is caused by a dysregulation in the innate immune response. We also simulated CD8+ T cell exhaustion, a common consequence of chronic and extensive infections. Our simulations indicate that while T cell exhaustion is possible in both age groups, its effects are more severe in the elderly population, as their dysregulated immune response cannot easily compensate for the exhausted T cells. Finally, we explore a therapeutic approach to reversing T cell exhaustion through an inflammatory stimulus. A controlled increase in inflammatory signals can lead to a higher chance of surviving the infection, but excess inflammation will likely lead to septic death. These results indicate that our model captures distinctions in the predominant mechanisms controlling the immune response in younger and older hosts and allows for simulations of clinically relevant therapeutic strategies post-infection.