Critical insights into the pathogenesis of clinical isolates of pandemic influenza A(H1N1) 2009 virus in mouse and ferret models.

Influenza A virus (IAV) is a minus-sense, segmented, single-stranded RNA virus that infects the respiratory tract of humans and can cause severe illness. Novel IAV variants perpetually emerge on every continent, and the emergence of variants with increased transmissibility and/or pathogenesis in the human population is a serious concern for global public health. Infection with IAV typically causes an acute, self-limiting upper respiratory tract disease. However, severe IAV disease is characterized by infection of the lower respiratory tract which can lead to pneumonia and may result in the development of acute respiratory distress syndrome (ARDS). Viral and host contributions to the development of ARDS are poorly understood, however IAV pathogenesis has been linked mutations in the receptor binding protein and the viral polymerase. Ferrets and mice are two important laboratory animal models for studying IAV pathogenesis. In 2009, a novel H1N1 subtype IAV (H1N1pdm) emerged in the human population and displayed variable pathology in humans. Using a mouse model, we show the variability of clinical isolates of H1N1pdm is driven by viral mutations, and that the timing of the inflammatory response is correlated with disease severity. To investigate spatiotemporal aspects of potential host and viral contributions to influenza pathogenesis, we developed a live imaging platform for ferrets infected with a human clinical isolate of H1N1pdm. We detected an early recruitment of neutrophils into ferret lungs following infection, which accumulated at foci of H1N1pdm infection within specific anatomical regions of the lung by 24 hours post-infection. The neutrophil response was biphasic, characterized by the recruitment of two populations with differing gene expression profiles, and baseline neutrophil levels were increased throughout the entire lung, including areas with no apparent viral infection. Changes in the viral microenvironment resulted in the regeneration of lung epithelium during recovery phase of infection, and this was imaged with PET-CT using a radiolabeled glucose analog. In summary, these data illustrate critical features of the immune response to IAV, and emphasize important considerations about the timing and accuracy of innate immune responses in studying viral pathogenesis

Receptor binding properties, cell tropism and transmission of influenza A virus

The first influenza pandemic of the 21st century was caused by the influenza A (H1N1) 2009 virus (A(H1N1)pdm09) that emerged from a swine-origin source. Although human infections with swine-origin influenza have been reported intermittently in the past decade, none went on to cause a pandemic or sustained human-to-human transmission. In previous pandemics, specific residues in the receptor binding site of the haemagglutinin (HA) protein of influenza have been associated with the ability of the virus to transmit between humans. In this thesis the effect of mutations at residue 227 in HA on cell tropism and transmission of A(H1N1)pdm09 is described. In A(H1N1)pdm09 and previous seasonal H1N1 viruses this residue is glutamic acid (E), whereas in swine influenza it is alanine (A). Using human airway epithelium, a differential cell tropism of A(H1N1)pdm09 compared to A(H1N1)pdm09 E227A and swine influenza was shown suggesting this residue may alter the sialic acid conformer binding preference of the HA. Furthermore, multi-cycle viral growth of both A(H1N1)pdm09 E227A and swine influenza was found to be attenuated in comparison to A(H1N1)pdm09 in human airway epithelium. However this altered tropism and viral growth in human airway epithelium did not abrogate respiratory droplet transmission of A(H1N1)pdm09 E227A in ferrets. This suggests that acquisition of 227E was not solely responsible for the ability of A(H1N1)pdm09 to transmit between humans. Because the work with the E227A mutant showed that small differences in cell tropism that may affect influenza virus transmissibility could be detected in human airway cells, a receptor binding assay was developed for laboratory surveillance using commercial human airway epithelium cultures, to screen for zoonotic influenza strains of particular concern for human health. To further investigate adaptations by influenza virus for infection of the human host, the cell tropism determined by the HA protein of an avian H7N7 and human H7N3 isolate was compared. Binding to non-ciliated human airway cells was increased for the human isolate. This human case of H7N3 infection yielded two isolates from different sites (eye and throat) from the same patient on the same day. A whole genome sequencing assay was designed for H7 isolates and both eye and throat isolate were fully sequenced. One synonymous nucleotide change was found in the NS gene segment and one synonymous and two non-synonymous nucleotide changes were found in the PB2 gene segment. Comparison of the non-synonymous changes in the protein sequence of PB2 to available avian and human virus PB2 sequences revealed that the substitutions in the eye isolate were comparatively uncommon. Interestingly, these changes resulted in an increased viral growth in human airway epithelial cells at 32ºC when compared to the throat isolate, a phenomenon which was not observed at 37ºC. Finally, the use of a lung model maintained by the ex-vivo lung perfusion (EVLP) technique for study of virus infection was tested. This technique allows the use of both human and porcine lungs up to 24 hours after abstraction and is a potential model for respiratory pathogens and novel treatments. Porcine lungs were infected with A(H1N1)pdm09. Physiological and virological parameters were measured in two separate experiments and infection was demonstrated by increased viral loads in samples obtained at late time points after infection

The development and application of pyrosequencing assays to determine antiviral susceptibility of Influenza A to neuraminidase inhibitors.

Influenza A can result in complicated disease and death in high risk individuals, and can be treated with the neuraminidase inhibitors oseltamivir and zanamivir. During the 2007-2008 season a rise in oseltamivir resistance in H1N1 viruses was observed worldwide. This resistance was caused by a point mutation in the neuraminidase gene segment at position 274 causing an amino acid change from histidine to tyrosine. Other point mutations have also been observed conferring resistance to neuraminidase inhibitors in H1N1 and H3N2 viruses, such as D151, E119V, R292K and N294S. Assays for the detection of resistance to the neuraminidase inhibitors have been well documented, the most novel being pyrosequencing. The aim of this study was to optimise pyrosequencing assays for routine use in a diagnostic laboratory. Once optimisation and validation were achieved using reference strains of influenza A, clinical validation was performed on influenza A isolates collected during the 2008-2009 season. Optimisation and clinical validation of a SNP pyrosequencing assay for the detection of the H274Y mutation in H1N1pdm09 isolates was also performed on isolates collected during the 2009-2010 and 2010-2011 seasons. Routine diagnostic assays were optimised for the H274Y mutation in pre 2009 H1N1 viruses and H1N1pdm09 viruses, and for the E119V mutation in H3N2 viruses. Only SQA assays were able to be optimised for the D151, R292K and N294S mutations in H3N2 viruses. Clinical validation showed that all seasonal H1N1 viruses isolated before April 2009 possessed the H274Y mutation at nearly 100%. The E119V mutation was detected in nearly 50% of H3N2 samples tested at varying levels from 1% to 31%. The D151, R292K and N294S mutations were not detected in any of the samples and the majority of the H1N1pdm09 samples contained a low level (1-10%) of H274Y mutation in the viral quasi-species

The development of a novel vaccine against avian influenza H7N9 virus

Influenza A viruses (IAV) are classified into the Orthomyxoviridae family and are composed of segmented, negative-sense, single-stranded ribonucleic acid (ssRNA) genomes in an enveloped particle. IAV is capable of infecting a wide variety of species, including but not limited to humans, birds, pigs, bats, and sea mammals. Up until 2013, H7N9 IAV was only prevalent in poultry; however, at this time H7N9 began infecting humans in China. Since 2013, China has seen six epidemic waves of the H7N9 virus, with human cases and deaths totaling 1,568 and 616, respectively, as of March 4, 2020. Although this virus presents high morbidity and mortality rates in humans, the majority of human cases have been a result of close contact to poultry in live poultry markets (LPMs). Fortunately, the change for sustained human-to-human transmission has not yet been acquired in this virus. However, due to IAVs evolutionary mechanisms of antigenic shift and antigenic drift, H7N9 could gain sustained transmission among humans at any time, which poses a severe threat to public health. Therefore, preventive control methods must be developed in an effort to control the spread of this influenza virus. Vaccination is currently the main method for controlling the spread and preventing influenza infection. Currently, the two available types of vaccines are the inactivated influenza vaccines (IIV) and the live attenuated influenza vaccines (LAIV). IIVs are composed of either whole influenza virions or portions of its virion produced in large volumes, followed by inactivation of the virus with either β-propiolactone or formaldehyde. IIVs are commonly administered intramuscularly, and sometimes include adjuvants to boost their immune responses. IIVs have been countlessly demonstrated to be highly safe for all populations. LAIVs on the other hand, are composed of live viruses of which their virulence is reduced through limited replication in the vaccinated host. LAIVs are administered intranasally and do not require an adjuvant because they are capable of stimulating stronger immune responses compared to IIVs. However, one common drawback towards the use of LAIVs is the possibility of virulence reversion. On the contrary, replication-defective virus vaccines are made up of viruses defective in either viral replication, synthesis, or assembly. These replication-defective virus vaccines, therefore, consist of very limited replication in the vaccinated host and have been found to possess the advantages of both IIVs and LAIVs. These advantages include the high safety profile due to the low risk of virulence reversion, as well as the ability to induce a strong immune response. To date, no commercial vaccine is available for the H7N9 influenza viruses. The first step in the influenza replication cycle is the binding of the virus to the host cell, which is followed by receptor-mediated endocytosis. After endocytosis occurs, in order for influenza virus to become infective, cleavage of the hemagglutinin (HA) precursor form, HA0, into HA1 and HA2 must occur. This cleavage is most often mediated by trypsin-like host proteases, inducing fusion between the viral and endosomal membranes. Therefore, this particular step is essential for determining viral pathogenicity. For this Masters project, the goal was to generate a replication-defective virus vaccine derived from H7N9 IAV, that is composed of an altered HA cleavage site that can only be cleaved and thus activated in vitro by the exogenous protease elastase which is not readily available in the respiratory tract. This replication-defective virus vaccine would, therefore, be inactive during natural infection, but active in vitro if the appropriate protease was provided. Previous studies have proven this replication-defective nature through the mutation of the HA cleavage site from a trypsin-sensitive motif to an elastase-sensitive motif. However, these studies have only been performed with the swine influenza virus (SIV) H1N1, a human-derived H7N7 HPAI, a mouse-adapted human-derived H1N1 virus, as well as an influenza B virus (IBV) (Babiuk et al., 2011; Gabriel et al., 2008; Mamerow et al., 2019; Masic et al., 2009; Masic et al., 2010; Masic et al., 2013; Stech et al., 2011; Stech et al., 2005). Using the technique of reverse genetics, we generated a recombinant mutant H7N9 virus, BC15-HA/QTV/NA (PR8), derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)] with a backbone from A/Puerto Rico/8 (H1N1) [PR8 (H1N1)]. This recombinant mutant BC15-HA/QTV/NA (PR8) virus possesses a mutant HA composed of three mutations at the HA cleavage site: lysine to glutamine at amino acid (aa) 337 (Lys-Gln), glycine to threonine at aa 338 (Gly-Thr), and arginine to valine at aa 339 (Arg-Val). In addition to the mutant HA, this recombinant mutant BC15-HA/QTV/NA (PR8) virus also contains the neuraminidase (NA) from BC15 (H7N9) and the six internal proteins from PR8 (H1N1). In the first part of our study, we established a mouse model of BC15 (H7N9) influenza virus. BALB/c mice were intranasally infected with various doses of BC15 (H7N9) (103 PFU, 104 PFU, and 105 PFU), and were monitored daily for 14 days post-infection (d.p.i.). In this study, we found BC15 (H7N9) to affect mice in a dose-dependent manner: the 103 dose killing all mice by 8 d.p.i.; the 104 dose by 6 d.p.i.; and the 105 dose by 5 d.p.i. In addition, all doses were capable of inducing high viral replication, pathology, and proinflammatory cytokine induction in the mouse lung. From this study, we concluded 103 PFU to be the chosen dose for future experiments. In the second part of this study, we developed and characterized the recombinant mutant BC15-HA/QTV/NA (PR8) virus, which showed this virus to be entirely dependent on elastase for its replication, contain similar growth properties to its wild-type counterpart, and be genetically stable in vitro. In addition, when this recombinant mutant BC15-HA/QTV/NA (PR8) virus was intranasally administered in BALB/c mice, it was found to be non-virulent and replication-defective, evident by a lack of body weight loss, 100% survival rate, and no viral replication detected in the mouse lung. Since we established this recombinant mutant BC15-HA/QTV/NA (PR8) virus to be replication-defective in mice, in order to consider this virus as a replication-defective virus vaccine candidate, we needed to test the immunogenicity and protective efficacy in BALB/c mice. To do this, we intranasally vaccinated mice twice with this recombinant mutant BC15-HA/QTV/NA (PR8) virus and then challenged the mice with a lethal dose of BC15 (H7N9). In this study, we reported that the intranasally administered BC15-HA/QTV/NA (PR8) virus induced significantly elevated levels of antigen-specific IFN-γ and IL-5 secreting cells in the splenocytes, which is evidence of a strong cell-mediated response. In addition, this virus increased the levels of neutralizing antibodies in the mouse serum, evident by both the hemagglutinin inhibition (HAI) and serum virus neutralization (SVN) assays, as well as heightened the levels of antigen-specific IgG, IgG1, and IgG2a in the mouse serum. Once the mice were challenged with BC15 (H7N9), our data showed that two intranasal vaccinations with BC15-HA/QTV/NA (PR8) were sufficient to provide complete protection of the mice from a homologous challenge. This complete protection was evident by the lack of body weight loss, 100% survival rate, lack of viral replication detected in the mouse lung, as well as the complete abolishment of proinflammatory cytokine induction in the mouse lung associated with the influenza disease. Taken together, this study demonstrates the strong potential the BC15-HA/QTV/NA (PR8) virus possesses to serve as a replication-defective virus vaccine candidate against H7N9 influenza viruses

Evaluation of how pandemic preparedness activities aided the response to influenza A (H1N1) pandemic in 2009: a qualitative analysis in seven countries within the WHO European Region

Background: The 2009 influenza A (H1N1) pandemic was the first pandemic in the era of modern pandemic planning and preparedness. Although the mortality and morbidity caused by the pandemic was low compared with the previous pandemics, it gave the first opportunity for member states to implement an actual pandemic response reflecting on years of pandemic preparedness and planning. The objective of this study was to evaluate the usefulness of pandemic preparedness activities as well as to identify challenges and activities that require further improvement. Methods: The study was conducted in seven countries within the WHO regional office for Europe; six of them were identified through a stratified random sampling in order to get a representative sample across different levels of preparedness within the WHO European Region. These were Armenia, Bosnia and Herzegovina, Denmark (pilot country for the study), Germany, Portugal, Switzerland and Uzbekistan. Research teams visited each country and interviewed six key stakeholder groups at different administrative levels. These were Ministry of Health (MOH), National Public Health Authority (NPHA), Civil Emergency Response (CER) representatives, Sub- National Government Authority, and primary and secondary healthcare workers (HCWs). Focus group interviews were conducted using open-ended questions in semi-structured interview guides. Results: Six recurring themes were identified as essential aspects of pandemic planning activities. These were communication, coordination, capacity building, mutual support, leadership, and flexibility. The following aspects of pandemic planning activities were found to be inadequate and should be improved in the future: risk communication with the public and healthcare workers, coordination of vaccine logistics, flexibility and adaptability of pandemic plans, and surveillance in the secondary healthcare setting. Conclusions: Stakeholders interviewed reported that the pandemic preparedness activities were worthwhile and appropriate for the response measures carried out during the pandemic influenza (H1N1) in 2009. However, the findings identified areas of under planning that were common to most of the participating countries