Mechanical compression attenuates normal human bronchial epithelial wound healing

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Identification of major histocompatibility complex class I C molecule as an attachment factor that facilitates coronavirus HKU1 spike-mediated infection

Human coronavirus HKU1 (HCoV-HKU1) is a recently discovered human coronavirus associated with respiratory tract infections worldwide. In this study, we have identified the major histocompatibility complex class I C molecule (HLA-C) as an attachment factor in facilitating HCoV-HKU1 spike (S)-mediated infection. HCoV-HKU1 S pseudotyped virus was assembled using a human immunodeficiency virus type 1-derived reporter virus harboring the human codon-optimized spike of HCoV-HKU1. We identified human alveolar epithelial A549 cells as the most susceptible cell line among those tested to infection by HCoV-HKU1 S pseudotypes. A549 cells were shown to bind purified soluble HCoV-HKU1 S(1-600) glycopeptide. To search for the functional receptor for HCoV-HKU1, an A549 cDNA expression library was constructed and transduced into the nonpermissive, baby hamster kidney cells line BHK-21. Transduced cells that bind soluble HCoV-HKU1 S(1-600) glycoprotein with C-terminal FLAG were sorted. Sequencing of two independent clones revealed cDNA inserts encoding HLA-C. Inhibition of HLA-C expression or function by RNAi silencing and anti-HLA-C antibody decreased HCoV-HKU1 S pseudotyped virus infection of A549 cells by 62 to 65%, whereas pretreatment of cells with neuraminidase decreased such infection by only 13%. When HLA-C was constitutively expressed in another nonpermissive cell line, NIH-3T3, quantitative PCR showed that the binding of HCoV-HKU1 S pseudotyped virus to cell surfaces was increased by 200-fold, but the cells remained nonsusceptible to HCoV-HKU1 S pseudotyped virus infection. Our data suggest that HLA-C is involved in the attachment of HCoV-HKU1 to A549 cells and is a potential candidate to facilitate cell entry. However, other unknown surface proteins on A549 cells may be concomitantly utilized by S glycoprotein of HCoV-HKU1 during viral entry. Further studies are required to elucidate other putative receptors or coreceptors for HCoV-HKU1 and the mechanism of HCoV-HKU1 S-mediated cell entry.     

Comparison of Different Cell Substrates on the Measurement of Human Influenza Virus Neutralizing Antibodies

Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log₂ lower) or superior (>1 log₂ higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.     

人呼吸道禽流感病毒受体的分布趋势

禽类流感病毒和人类流感病毒具有很强的受体识别特异性,分别与唾液酸α-2,3Gal和α-2,6Gal受体分子结合而感染各自的宿主细胞．这种受体结合特异性是流感病毒在禽类和人类之间跨种属传递的主要障碍．应用凝集素组织化学染色技术,探讨人呼吸道各解剖学部位流感病毒唾液酸受体的分布特征．结果显示,唾液酸α-2,3Gal受体, 即禽类流感受体,主要分布在下呼吸道的呼吸部即呼吸细支气管和肺泡, 而在主气管、支气管和细支气管仅少量分布．相反,人类流感病毒受体,唾液酸α-2,6Gal受体在气管、支气管呈高密度分布,随着支气管分级逐渐降低分布减少,至肺泡分布最少．但比较人呼吸道发育成熟过程中,唾液酸α-2,3Gal和α-2,6Gal受体的表达,未发现明显差别．禽流感H5N1病毒体外感染人呼吸道组织试验结果表明,肺泡上皮较支气管和气管上皮易感染,与唾液酸α-2,3Gal受体分布特点相符合．结果提示,人呼吸道可被禽流感病毒感染,目前H5N1病毒极少发生人传人的特点,可能与个体间上呼吸道唾液酸α-2,3Gal受体表达差异有关．     

Viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza H5N1 and H1N1 viruses

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.     

Culturing the Unculturable: Human Coronavirus HKU1 Infects,Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.About 335 new or emerging infectious diseases have been identified since 1940 (23), and while many threaten human health, the global economy, and national security, respiratory pathogens are of particular public health concern. Using modern methods, several previously unknown viruses have been identified, including respiratory pathogens (1, 18, 27, 54, 57), yet research remains restricted to prevalence and disease association studies since a virus culture system is oftentimes lacking. Immortalized tissue culture cells are adapted to growth in laboratory conditions and, as such, display altered gene expression patterns, which may not be optimal for the replication of fastidious viruses. Primary cell-differentiated culture models provide alternative in vitro model systems closer in nature to the in vivo host tissue environment for infection studies and amplification of pathogens for further characterization. Here, we use an in vitro model of human ciliated airway epithelial cell cultures (HAE) that mimic the properties of the cartilaginous airway epithelium (17) to culture the previously unculturable human coronavirus HKU1 (HCoV-HKU1).Coronaviruses are important pathogens of humans and animals, causing a range of symptoms depending on the host. Following the severe acute respiratory syndrome (SARS)-CoV epidemic, several new strains of human coronaviruses were identified by molecular techniques, including HCoV-NL63, identified in the Netherlands from an infant with bronchiolitis (54), and HCoV-HKU1, identified in an adult patient with severe pneumonia in Hong Kong (57). HCoV-NL63 has been demonstrated to infect and replicate in both conventional immortalized cells and human ciliated airway cell cultures, producing sufficient amounts of virus for characterization studies of viral replication and pathogenesis and the successful development of an infectious clone (3, 13, 22, 41). In contrast, little is known about HCoV-HKU1, as no in vitro replication model has been identified to date, limiting further investigations of the virus.Clinical isolates of previously isolated human coronaviruses have been adapted to replicate in standard transformed cell culture; for example, SARS-CoV and HCoV-NL63 replicate efficiently in epithelial monkey kidney cells (VeroE6 and LLC-MK2), HCoV-OC43 in BHK21 cells, and HCoV-229E in MRC5 cells (14, 24, 35, 47, 54, 59). Despite the successful amplification of these human coronaviruses in cell lines, all attempts to date to culture a clinical isolate of HCoV-HKU1 have failed. No HCoV-HKU1 genomic replication was observed after inoculation of standard cell lines previously utilized for virus propagation, including RD (human rhabdomyosarcoma cells), HRT-18 (colorectal adenocarcinoma cells), HEp-2 (human epithelial carcinoma cells), MRC-5 (human lung fibroblast cells), A549 (human lung epithelial adenocarcinoma cells), Caco2 (human colorectal adenocarcinoma cells), Huh-7 (human hepatoma cells), B95a (marmoset B-lymphoblastoid cells), mixed neuron-glia culture, LLC-MK2 (rhesus monkey kidney cells), FRhK-4 (rhesus monkey kidney cells), BSC-1 (African green monkey kidney cells), Vero E6 (African green monkey kidney cells), MDCK (Madin-Darby canine kidney cells), I13.35 (murine macrophage cells), and L929 (murine fibroblast cells) (57).Here, we use human ciliated airway epithelial cell cultures to successfully propagate HCoV-HKU1 for the first time in vitro. In this culture model, HCoV-HKU1 genome copy numbers increased by several logs over the initial three-day incubation period and electron micrograph, Northern blot, and immunofluorescence data confirmed HKU1 replication in HAE and that ciliated cells were the preferential target for virus infection, the same cell type infected by all human coronaviruses tested so far in these model systems.     

Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells

Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an alpha2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an alpha2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the alpha2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The alpha2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, alpha2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the alpha2,3-linked SA receptor.     

Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

Systems-Level Comparison of Host-Responses Elicited by Avian H5N1 and Seasonal H1N1 Influenza Viruses in Primary Human Macrophages

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-α genes. A network-based analysis suggests that the synergy between IFN-β and TNF-α results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.     

The role of antibodies in respiratory viral immunity

Antibodies protect against disease caused by viruses that infect the lower respiratory tract, and contribute to the resolution of established infection by these pathogens. The kinetics and specificities of antibodies secreted in response to respiratory virus infections are described, however the mechanisms by which antibodies prevent or resolve infection are less clear. Recent studies of virus neutralization in cell culture, the immunobiology of respiratory virus infections in animal models, and passive immunoprophylaxis of human patients are beginning to better define the role of antibodies in immunity to respiratory viruses.     

Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells: THE IMPORTANCE OF NeuAc��2�C6 GLYCANS*

To better understand influenza virus infection of pigs, we examined primary swine respiratory epithelial cells (SRECs, the primary target cells of influenza viruses in vivo), as a model system. Glycomic profiling of SRECs by mass spectrometry revealed a diverse range of glycans terminating in sialic acid or GalαGal. In terms of sialylation, α2–6 linkage was more abundant than α2–3, and NeuAc was more abundant than NeuGc. Virus binding and infection experiments were conducted to determine functionally important glycans for influenza virus infection, with a focus on recently emerged swine viruses. Infection of SRECs with swine and human viruses resulted in different infectivity levels. Glycan microarray analysis with a high infectivity “triple reassortant” virus ((A/Swine/MN/593/99 (H3N2)) that spread widely throughout the North American swine population and a lower infectivity human virus isolated from a single pig (A/Swine/ONT/00130/97 (H3N2)) showed that both viruses bound exclusively to glycans containing NeuAcα2–6, with strong binding to sialylated polylactosamine and sialylated N-glycans. Treatment with mannosamine precursors of sialic acid (to alter NeuAc/NeuGc abundances) and linkage-specific sialidases prior to infection indicated that the influenza viruses tested preferentially utilize NeuAcα2–6-sialylated glycans to infect SRECs. Our data indicate that NeuAcα2–6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs. As NeuAcα2–6 polylactosamine glycans play major roles in human virus infection, the importance of these receptor components in virus infection of swine cells has implications for transmission of viruses between humans and pigs and for pigs as possible adaptation hosts of novel human influenza viruses.     

The role of human rhinovirus in immunology, COPD, and corresponding treatments

The common cold is most often a result of human rhinovirus (HRV) infection. Common cold symptoms including rhinorrhea and nasal obstruction frequently occur during HRV infection of the upper respiratory tract. Conversely, HRV may also infect the epithelial cells of the lower respiratory tract. Symptom severity associated with HRV infection ranges from mild to potentially serious depending on a person’s susceptibility and pre-existing condition, such as chronic obstructive pulmonary disease. An over active host immune response is believed to be the primary contributor to HRV pathogenesis. Enhanced activity of various host cell cytokines and granulocytes mediate specific cellular pathways inducing many of the symptoms associated with HRV infection. There are over 100 serotypes of HRV which can be further categorized based on the specific characteristics of each type. The two main categories of HRV consist of the major and minor groups. The unique host cell receptor is the distinguishing factor between these two groups. Yet, these viruses may also differ in mechanism of infection and replication. Due to the high frequency of hospital and clinical visits and the corresponding economic burden, novel therapies are of interest. Several different treatment options varying from herbal remedies to anti-viral drugs have been studied. However, the vast number of HRV serotypes complicates the progress of developing a universal treatment for attenuating HRV infection.     

Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.     

Human immunodeficiency virus type 1 infection of cells and tissues from the upper and lower human female reproductive tract.

Viable tissue sections and isolated cell cultures from the human fallopian tube, uterus, cervix, and vaginal mucosa were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1). We examined infectivity by using the monocytotropic strain HIV-1(JR-FL) and several primary isolates of HIV-1 obtained from infected neonates. HIV-1 infection was measured by p24 production in short-term culture and by immunofluorescence detection of HIV-1 Nef and p24 proteins by laser scanning confocal microscopy. Three-color immunofluorescence was used to phenotype HIV-infected cells within tissue sections from each site. Our findings indicate that epithelial, stromal, and dendritic cells and cells with CD14+ CD4+, CD14-CD4-, and CD4+ CD14- phenotypes from the female reproductive tract are infectable with HIV-1. Of importance is the finding that tissues from the upper reproductive tract are susceptible to infection with HIV-1. Moreover, tissue samples from women in all stages of the menstrual cycle, including postmenopausal women (inactive), could be infected with HIV-1. Female reproductive tract cells required a minimum of 60 min of exposure to HIV-1 in order for infection to occur, in contrast to peripheral blood lymphocytes, which became infected after being exposed to HIV-1 for only 1 min. These findings demonstrate that HIV-1 can infect cells and tissues from different sites within the female reproductive tract and suggest that multiple cell types, including epithelial cells, may be targets for the initial infection by HIV-1.     

Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment.     

Hemagglutinin-Dependent Tropism of H5N1 Avian Influenza Virus for Human Endothelial Cells

Although current H5N1 highly pathogenic avian influenza viruses (HPAIV) are inefficiently transmitted to humans, infected individuals can suffer from severe disease, often progressing rapidly to acute respiratory distress syndrome and multiorgan failure. This is in contrast with the situation with human influenza viruses, which in immunocompetent individuals usually cause only a respiratory disease which is less aggressive than that observed with avian H5N1 viruses. While the biological basis of inefficient transmission is well documented, the mechanisms by which the H5N1 viruses cause fatal disease remain unclear. In the present study, we demonstrate that human pulmonary microvascular endothelial cells (hPMEC) had a clearly higher susceptibility to infection by H5N1 HPAIV than to infection by human influenza viruses. This was measurable by de novo intracellular nucleoprotein production and virus replication. It was also related to a relatively higher binding capacity to cellular receptors. After infection of hPMEC, cell activation markers E-selectin and P-selectin were upregulated, and the proinflammatory cytokines interleukin-6 and beta interferon were secreted. H5N1 virus infection was also associated with an elevated rate of cell death. Reverse genetics analyses demonstrated a major role for the viral hemagglutinin in this cell tropism. Overall, avian H5N1 viruses have a particular receptor specificity targeting endothelial cells that is different from human influenza viruses, and this H5N1 receptor specificity could contribute to disease pathogenesis.Certain highly pathogenic avian influenza viruses (HPAIV) expressing the H5 and H7 hemagglutinins (HA) have acquired the capacity to infect humans. Particularly, HPAIV with the H5 HA and the neuraminidase (NA) type 1 (H5N1) can cause severe disease, often with a fatal outcome in humans and other mammals (27). With such infections in humans, there are two striking differences compared to infection by human influenza A viruses (IAV). First, bird-to-human and human-to-human transmission has been considered inefficient, and second, the mortality rate of H5N1 virus infections has been unexpectedly high. There is a lot of experimental evidence that inefficient transmission rate is related to several viral gene products not optimally adapted to facilitate infection and replication in the primary target cells, the epithelial cells of the respiratory tract. Of particular importance is the HA determining receptor specificity with human viruses preferentially recognizing sialic acid (SA)-α-2,6-Gal-terminated saccharides (α-2,6-SA), abundantly expressed in the upper respiratory tract, and avian viruses preferentially binding to α-2,3-SA, expressed mainly in the lower respiratory tract and on ciliated epithelial cells (23, 33, 39). In addition, the viral polymerases determining the rate of replication as well as the NS1 protein involved in multiple processes enabling efficient viral replication and evasion of cellular antiviral responses are of importance in determining host tropism (17, 26).However, in contrast to infections with human influenza viruses, avian H5N1 virus infections more often cause severe pneumonia. These are associated with high levels of proinflammatory cytokines and chemokines in the respiratory tract, severe inflammatory reactions, and infiltration of leukocytes. Furthermore, a generalized inflammatory reaction with elevated cytokine and chemokine levels in the circulation, together with leukopenia and multiorgan failure, indicates that an aberrant immunological reaction is an important factor contributing to the fatality of H5N1 virus infections (19). This is supported by in vitro studies of human macrophages, dendritic cells, and epithelial cells, in which it was demonstrated that H5N1 viruses can induce higher levels of inflammatory cytokine and chemokine responses than human IV isolates (2, 3, 37). Based on this, it was proposed that factors of the innate and adaptive immune response are of central importance for the outcome of disease (8, 26).Endothelial cells (EDC) are abundant in all organs, particularly the lung, and play an important role in inflammatory processes through the regulation of leukocyte extravasation, the production of inflammatory cytokines and chemokines, and the regulation of coagulation (4). During systemic disease in chickens infected with H5N1 isolates, the cardiovascular system can be affected with coagulopathy and viral antigen detectable in EDC (15, 25, 36). This also relates to a report demonstrating a targeted infection of EDC in chicken embryo by A/FPV/Rostock/34 (H7N1) virus (6). In this study, the infection of human umbilical vein EDC is also reported. Finally, in humans, various degrees of hemorrhages as well as signs of disseminated intravascular coagulation have been found (1).Accordingly, the present study compared influenza virus isolates of avian and human origin with respect to their characteristics of interaction with human EDC. To this end, we infected primary human lung EDC with different naturally occurring virus isolates as well as viruses created by reverse genetics. Viruses expressing the H5 clearly possessed the greatest potency to infect and replicate in EDC, resulting in activation and inflammatory responses.     

Influenza virus receptors in the human airway

Avian influenza A (H5N1) virus infections have resulted in more than 100 human deaths; yet, human-to-human transmission is rare. We demonstrated that the epithelial cells in the upper respiratory tract of humans mainly possess sialic acid linked to galactose by alpha 2,6 linkages (SA alpha 2,6Gal), a molecule preferentially recognized by human viruses. However, many cells in the respiratory bronchioles and alveoli possess SA alpha 2,3Gal, which is preferentially recognized by avian viruses. These facts are consistent with the observation that H5N1 viruses can be directly transmitted from birds to humans and cause serious lower respiratory tract damage in humans. Furthermore, this anatomical difference in receptor prevalence may explain why the spread of H5N1 viruses among humans is limited. However, since some H5N1 viruses isolated from humans recognize human virus receptors, additional changes must be required for these viruses to acquire the ability for efficient human-to-human transmission.