Louis M. Schwartzman

An Overlapping Protein-Coding Region in Influenza A Virus Segment 3 Modulates the Host Response

Influenzas Cryptic Constraint Because of the well-known pandemic potential of influenza viruses, it is important to understand the range of molecular interactions between the virus and its host. Despite years of intensive research on the virus, Jagger et al. (p. 199, published online 28 June; see the Perspective by Yewdell and Ince) have found that the influenza A virus has been hiding a gene in its small negative-sense RNA genome. An overlapping open reading frame was found contained in the PA viral RNA polymerase gene, which is accessed by ribosomal frameshifting to produce a fusion protein containing the N-terminal messenger RNA (mRNA) endonuclease domain of PA and an alternative C-terminal X domain. The resulting polypeptide, PA-X, selectively degrades host mRNAs and, in a mouse model of infection, modulated cellular immune responses, thus limiting viral pathogenesis. A previously unidentified influenza protein, partly old and partly new, turns off the expression of host genes. Influenza A virus (IAV) infection leads to variable and imperfectly understood pathogenicity. We report that segment 3 of the virus contains a second open reading frame (“X-ORF”), accessed via ribosomal frameshifting. The frameshift product, termed PA-X, comprises the endonuclease domain of the viral PA protein with a C-terminal domain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model, acting to decrease pathogenicity. Loss of PA-X expression leads to changes in the kinetics of the global host response, which notably includes increases in inflammatory, apoptotic, and T lymphocyte–signaling pathways. Thus, we have identified a previously unknown IAV protein that modulates the host response to infection, a finding with important implications for understanding IAV pathogenesis.

Identification of a Novel Splice Variant Form of the Influenza A Virus M2 Ion Channel with an Antigenically Distinct Ectodomain

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

Lethal Synergism of 2009 Pandemic H1N1 Influenza Virus and Streptococcus pneumoniae Coinfection Is Associated with Loss of Murine Lung Repair Responses

ABSTRACT Secondary bacterial infections increase disease severity of influenza virus infections and contribute greatly to increased morbidity and mortality during pandemics. To study secondary bacterial infection following influenza virus infection, mice were inoculated with sublethal doses of 2009 seasonal H1N1 virus (NIH50) or pandemic H1N1 virus (Mex09) followed by inoculation with Streptococcus pneumoniae 48 h later. Disease was characterized by assessment of weight loss and survival, titration of virus and bacteria by quantitative reverse transcription-PCR (qRT-PCR), histopathology, expression microarray, and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09 plus S. pneumoniae showed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium, and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50 plus S. pneumoniae showed increased weight loss, 100% survival, and slightly enhanced lung pathology. Mex09-S. pneumoniae coinfection also resulted in increased S. pneumoniae replication in lung and bacteremia late in infection. Global gene expression profiling revealed that Mex09-S. pneumoniae coinfection did not induce significantly more severe inflammatory responses but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09-S. pneumoniae-infected mice. This study demonstrates that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in more severe disease and loss of lung repair responses than did seasonal influenza viral and bacterial coinfection. Moreover, this study provides novel insights into influenza virus and bacterial coinfection by showing correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses. IMPORTANCE Secondary bacterial pneumonias lead to increased disease severity and have resulted in a significant percentage of deaths during influenza pandemics. To understand the biological basis for the interaction of bacterial and viral infections, mice were infected with sublethal doses of 2009 seasonal H1N1 and pandemic H1N1 viruses followed by infection with Streptococcus pneumoniae 48 h later. Only infection with 2009 pandemic H1N1 virus and S. pneumoniae resulted in severe disease with a 100% fatality rate. Analysis of the host response to infection during lethal coinfection showed a significant loss of responses associated with lung repair that was not observed in any of the other experimental groups. This group of mice also showed enhanced bacterial replication in the lung. This study reveals that the extent of lung damage during viral infection influences the severity of secondary bacterial infections and may help explain some differences in mortality during influenza pandemics. Secondary bacterial pneumonias lead to increased disease severity and have resulted in a significant percentage of deaths during influenza pandemics. To understand the biological basis for the interaction of bacterial and viral infections, mice were infected with sublethal doses of 2009 seasonal H1N1 and pandemic H1N1 viruses followed by infection with Streptococcus pneumoniae 48 h later. Only infection with 2009 pandemic H1N1 virus and S. pneumoniae resulted in severe disease with a 100% fatality rate. Analysis of the host response to infection during lethal coinfection showed a significant loss of responses associated with lung repair that was not observed in any of the other experimental groups. This group of mice also showed enhanced bacterial replication in the lung. This study reveals that the extent of lung damage during viral infection influences the severity of secondary bacterial infections and may help explain some differences in mortality during influenza pandemics.

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice.

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.

Obese mice have increased morbidity and mortality compared to non-obese mice during infection with the 2009 pandemic H1N1 influenza virus

Please cite this paper as: Easterbrook et al. (2011) Obese mice have increased morbidity and mortality compared to non‐obese mice during infection with the 2009 pandemic H1N1 influenza virus. Influenza and Other Respiratory Viruses 5(6), 418–425.

An Intranasal Virus-Like Particle Vaccine Broadly Protects Mice from Multiple Subtypes of Influenza A Virus

ABSTRACT Influenza virus infections are a global public health problem, with a significant impact of morbidity and mortality from both annual epidemics and pandemics. The current strategy for preventing annual influenza is to develop a new vaccine each year against specific circulating virus strains. Because these vaccines are unlikely to protect against an antigenically divergent strain or a new pandemic virus with a novel hemagglutinin (HA) subtype, there is a critical need for vaccines that protect against all influenza A viruses, a so-called “universal” vaccine. Here we show that mice were broadly protected against challenge with a wide variety of lethal influenza A virus infections (94% aggregate survival following vaccination) with a virus-like particle (VLP) vaccine cocktail. The vaccine consisted of a mixture of VLPs individually displaying H1, H3, H5, or H7 HAs, and vaccinated mice showed significant protection following challenge with influenza viruses expressing 1918 H1, 1957 H2, and avian H5, H6, H7, H10, and H11 hemagglutinin subtypes. These experiments suggest a promising and practical strategy for developing a broadly protective “universal” influenza vaccine. IMPORTANCE The rapid and unpredictable nature of influenza A virus evolution requires new vaccines to be produced annually to match circulating strains. Human infections with influenza viruses derived from animals can cause outbreaks that may be associated with high mortality, and such strains may also adapt to humans to cause a future pandemic. Thus, there is a large public health need to create broadly protective, or “universal,” influenza vaccines that could prevent disease from a wide variety of human and animal influenza A viruses. In this study, a noninfectious virus-like particle (VLP) vaccine was shown to offer significant protection against a variety of influenza A viruses in mice, suggesting a practical strategy to develop a universal influenza vaccine. The rapid and unpredictable nature of influenza A virus evolution requires new vaccines to be produced annually to match circulating strains. Human infections with influenza viruses derived from animals can cause outbreaks that may be associated with high mortality, and such strains may also adapt to humans to cause a future pandemic. Thus, there is a large public health need to create broadly protective, or “universal,” influenza vaccines that could prevent disease from a wide variety of human and animal influenza A viruses. In this study, a noninfectious virus-like particle (VLP) vaccine was shown to offer significant protection against a variety of influenza A viruses in mice, suggesting a practical strategy to develop a universal influenza vaccine.

Analysis by Single-Gene Reassortment Demonstrates that the 1918 Influenza Virus Is Functionally Compatible with a Low-Pathogenicity Avian Influenza Virus in Mice

ABSTRACT The 1918-1919 “Spanish” influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight “7:1” chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.

Recombinant H7 hemagglutinin forms subviral particles that protect mice and ferrets from challenge with H7N9 influenza virus.

A novel avian-origin influenza A H7N9 virus emerged in China in 2013 and continues to cause sporadic human infections with mortality rates approaching 35%. Currently there are no approved human vaccines for H7N9 virus. Recombinant approaches including hemagglutinin (HA) and virus-like particles (VLPs) have resulted in experimental vaccines with advantageous safety and manufacturing characteristics. While high immunogenicity of VLP vaccines has been attributed to the native conformation of HA arranged in the regular repeated patterns within virus-like structures, there is limited data regarding molecular organization of HA within recombinant HA vaccine preparations. In this study, the full-length recombinant H7 protein (rH7) of A/Anhui/1/2013 (H7N9) virus was expressed in Sf9 cells. We showed that purified full-length rH7 retained functional ability to agglutinate red blood cells and formed oligomeric pleomorphic subviral particles (SVPs) of ∼20nm in diameter composed of approximately 10 HA0 molecules. No significant quantities of free monomeric HA0 were observed in rH7 preparation by size exclusion chromatography. Immunogenicity and protective efficacy of rH7 SVPs was confirmed in the mouse and ferret challenge models suggesting that SVPs can be used for vaccination against H7N9 virus.

Contemporary Avian Influenza A Virus Subtype H1, H6, H7, H10, and H15 Hemagglutinin Genes Encode a Mammalian Virulence Factor Similar to the 1918 Pandemic Virus H1 Hemagglutinin

ABSTRACT Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. IMPORTANCE Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks. Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks.

1918 pandemic influenza virus and Streptococcus pneumoniae co‐infection results in activation of coagulation and widespread pulmonary thrombosis in mice and humans

To study bacterial co‐infection following 1918 H1N1 influenza virus infection, mice were inoculated with the 1918 influenza virus, followed by Streptococcus pneumoniae (SP) 72 h later. Co‐infected mice exhibited markedly more severe disease, shortened survival time and more severe lung pathology, including widespread thrombi. Transcriptional profiling revealed activation of coagulation only in co‐infected mice, consistent with the extensive thrombogenesis observed. Immunohistochemistry showed extensive expression of tissue factor (F3) and prominent deposition of neutrophil elastase on endothelial and epithelial cells in co‐infected mice. Lung sections of SP‐positive 1918 autopsy cases showed extensive thrombi and prominent staining for F3 in alveolar macrophages, monocytes, neutrophils, endothelial and epithelial cells, in contrast to co‐infection‐positive 2009 pandemic H1N1 autopsy cases. This study reveals that a distinctive feature of 1918 influenza virus and SP co‐infection in mice and humans is extensive expression of tissue factor and activation of the extrinsic coagulation pathway leading to widespread pulmonary thrombosis. Copyright