The Role of Cytokine-Inducible SH2 Domain-Containing Protein (CISH) in the Regulation of Basal and Cytokine-Mediated Myelopoiesis.

Cytokine-inducible SH2 domain-containing protein (CISH) is a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators shown to play crucial roles in lymphoid cell development and function as well as appetite regulation. It has also been implicated in the control of signaling downstream of the receptors for the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in myeloid cells. To investigate the physiological role of CISH in myelopoiesis, mice deficient in CISH were analyzed basally and in response to administration of these cytokines. CISH knockout (KO) mice possessed basally elevated neutrophils in the blood, bone marrow, and spleen compared to wild-type (WT) mice. During GM-CSF-induced myelopoiesis, the frequency of neutrophils, myeloid dendritic cells (DCs), and CFU-M in the bone marrow was higher in the KO, as were the neutrophils and CFU-G in the spleen. In contrast, no differences were observed between KO and WT mice during G-CSF-induced myelopoiesis apart from an elevated frequency of CFU-G and CFU-M in the spleen. This work has identified a role for CISH in the negative regulation of granulopoiesis, including that mediated by GM-CSF.

Correction: ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

[This corrects the article DOI: 10.1371/journal.pbio.1002580.].

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames.

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.

ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality.

Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats.IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing awareness of the contribution of neuraminidase (NA) to influenza virus vaccine efficacy. Although NA is immunologically subdominant to HA, and clinical studies have shown variable NA responses to vaccination, in this study, we show that vaccination with a parainfluenza virus 5 recombinant vaccine candidate expressing NA (PIV5-NA) from a pandemic influenza (pdmH1N1) virus or highly pathogenic avian influenza (H5N1) virus elicits robust, cross-reactive protection from influenza virus infection in two animal models. New vaccination strategies incorporating NA, including PIV5-NA, could improve seasonal influenza virus vaccine efficacy and provide protection against emerging influenza viruses.

Virologic Differences Do Not Fully Explain the Diversification of Swine Influenza Viruses in the United States.

Influenza A(H1N1) viruses entered the U.S. swine population following the 1918 pandemic and remained genetically stable for roughly 80 years. In 1998, there was an outbreak of influenza-like illness among swine that was caused by A(H3N2) viruses containing the triple reassortant internal gene (TRIG) cassette. Following the TRIG cassette emergence, numerous reassortant viruses were isolated in nature, suggesting that the TRIG virus had an enhanced ability to reassort compared to the classical swine virus. The present study was designed to quantify the relative reassortment capacities of classical and TRIG swine viruses. Reverse genetic viruses were generated from the classical H1N1 virus A/swine/MN/37866/1999 (MN/99), the TRIG virus A/swine/NC/18161/2002 (NC/02), and a seasonal human H3N2 virus, A/TX/6/1996 (TX/96), to measure in vitro reassortment and growth potentials. After coinfection with NC/02 or MN/99 plus TX/96, H1/H3 double-positive cells were identified. Delayed TX/96 infection was fully excluded by both swine viruses. We then analyzed reassortant H3 viruses. Seventy-seven of 81 (95.1%) TX/96-NC/02 reassortants contained at least one polymerase gene segment from NC/02, whereas only 34 of 61 (55.7%) MN/99-TX/96 reassortants contained at least one polymerase gene segment from MN/99. Additionally, 38 of 81 (46.9%) NC/02-TX/96 reassortants contained all NC/02 polymerase gene segments, while none of the MN/99-TX/96 reassortants contained all MN/99 polymerase genes. There were 21 H3 reassortants between MN/99 and TX/96, compared to only 17 H3 reassortants between NC/02 and TX/96. Overall, the results indicate that there are no distinct differences in the ability of the TRIG to reassort with a human virus compared to the classical swine virus. IMPORTANCE: There appear to be no differences in the abilities of classical swine and TRIG swine viruses to exclude a second virus, suggesting that under the right circumstances both viruses have similar opportunities to reassort. The increased percentage of TRIG polymerase gene segments in reassortant H3 viruses indicates that these viruses may be more compatible with gene segments from other viruses; however, this needs to be investigated further. Nevertheless, the classical swine virus also showed the ability to reassort, suggesting that factors other than reassortment capacity alone are responsible for the different epidemiologies of TRIG and classical swine viruses. The post-TRIG diversity was likely driven by increased intensive farming practices rather than virologic properties. Our results indicate that host ecology can be a significant factor in viral evolution.

Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression.

UNLABELLED: Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE: Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

OBJECTIVE AND DESIGN: Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. MATERIAL OR SUBJECTS: Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. TREATMENT: A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. METHODS: Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. RESULTS: Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. CONCLUSIONS: Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Novel H7N9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model.

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.

Role of Cytokine-Inducible SH2 Domain-Containing (CISH) Protein in the Regulation of Erythropoiesis.

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

Passage of low-pathogenic avian influenza (LPAI) viruses mediates rapid genetic adaptation of a wild-bird isolate in poultry.

For a better understanding of evolution of influenza viruses, a chicken-origin and wild-bird-origin low-pathogenic avian influenza virus (LPAI) was serially passaged in chickens. Sequences of the hemagglutinin (HA) and neuraminidase (NA) genes at each passage level were compared to those of the parental virus. Multiple mutations occurring early during passage were detected, but these were maintained during passages. Interestingly, a number of the observed mutations already existed in the parental virus, as indicated by the presence of single-nucleotide polymorphisms. The greatest numbers of mutations occurred during passage of wild-bird-origin LPAI, where a 20-amino-acid deletion in the NA gene that was observed during the first passage was maintained during subsequent passages. Subsequent experiments showed that this NA deletion was already present as a minority population in the parental virus. These results showed that a selection process favoring a viral subpopulation had occurred.

Extracellular Matrix Enzymes and Immune Cell Biology.

Remodelling of the extracellular matrix (ECM) by ECM metalloproteinases is increasingly being associated with regulation of immune cell function. ECM metalloproteinases, including Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinases (ADAMs) and ADAMs with Thombospondin-1 motifs (ADAMTS) play a vital role in pathogen defence and have been shown to influence migration of immune cells. This review provides a current summary of the role of ECM enzymes in immune cell migration and function and discusses opportunities and limitations for development of diagnostic and therapeutic strategies targeting metalloproteinase expression and activity in the context of infectious disease.

Production of H5-specific monoclonal antibodies and the development of a competitive enzyme-linked immunosorbent assay for detection of H5 antibodies in multiple species.

The hemagglutinin gene of an avian influenza virus (AIV) A/duck/NC/674964/07 (H5N2) was cloned and expressed in a baculovirus system (H5-Bac). In parallel, a recombinant hemagglutinin of A/Vietnam/1203/04 (H5N1) was expressed in mammalian cells, purified, and used for generation of H5-specific monoclonal antibodies (MAb). The purified H5-Bac was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) to detect H5 antibodies in a species-independent approach using one of the established H5-specific MAbs as the competitor antibody. The cELISA performed with influenza antibody-free sera or with sera of animals infected with other than H5-encoding AIV showed no significant inhibition of H5-MAb binding, indicating high test specificity. In contrast, sera of poultry (chickens, turkeys, ducks) experimentally infected with H5-encoding AIV were able to significantly inhibit the binding of the MAb in a species-independent approach. Comparison of the results of the cELISA with results obtained by a hemagglutination inhibition assay showed a gradient of the sensitivity (turkeys > ducks > chicken). The described results show that H5-specific antibodies in sera can be detected in a species-independent approach by using a recombinant protein.

Enhanced immunogenicity following miR-155 incorporation into the influenza A virus genome.

Influenza A vaccine efficacy in the elderly is generally poor and so identification of novel molecular adjuvants to improve immunogenicity is important to reduce the overall burden of disease. Short non-coding RNAs, known as microRNAs (miRNAs) are known to regulate gene expression and have the potential to influence immune responses. One such miRNA, miR-155, has been shown to modulate T and B cell development and function. We incorporated miR-155 into the influenza A virus (IAV) genome creating a self-adjuvanting 'live vaccine' with the ability to modify immunogenicity. Infection of mice with a recombinant influenza virus encoding miR-155 in the NS gene segment altered epitope-specific expansion of influenza-specific CD8+ T cells and induced significantly higher levels of neutralising antibody.

Efficacy of a parainfluenza virus 5 (PIV5)-based H7N9 vaccine in mice and guinea pigs: antibody titer towards HA was not a good indicator for protection.

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.

Polymerase discordance in novel swine influenza H3N2v constellations is tolerated in swine but not human respiratory epithelial cells.

Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼ 80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-ß and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection.

Bat cells from Pteropus alecto are susceptible to influenza A virus infection and reassortment.

Waterfowl are primary hosts for influenza A viruses (IAVs); however, there is sporadic infection of swine and other species that pose a risk of zoonotic spread. Yellow-shouldered bats were shown to be hosts of an IAV, thereby constituting a potential novel reservoir. We show that Pteropus alecto kidney cells (PaKi) are susceptible to infection and sustain replication of A/WSN/33 (H1N1) and A/Vietnam/1203/04 (H5N1). Importantly, we show that co-infection of PaKi cells results in novel reassortants.

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.