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H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza. H5 influenza is considered a potential pandemic threat. Using deep mutational scanning, this work reveals how the >10,000 different possible amino-acid mutations in hemagglutinin affect cell entry, receptor preference, protein stability and neutralization by antibodies, which can inform real-time interpretation of viral mutations observed during surveillance of H5 influenza. [ABSTRACT FROM AUTHOR]

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-β. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to sOH/04 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an overexpression of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages. Author summary: Human-to-swine FLUAV spillover events are relatively common and have led to the establishment of multiple evolutionarily distinct virus lineages in the swine population worldwide, leading to important economic losses in the pork industry. This also increases the risk of reassortment and emergence of novel FLUAV that may retain the ability to infect and transmit in humans, an event exemplified by the H1N1 pandemic in 2009. However, the molecular mechanisms driving FLUAV adaptation to pigs following the introduction of a human virus are still not fully understood. In this study we found that a point mutation in a human-origin HA improved infection and transmissibility in pigs characterized by an enhanced lung replication and the disruption of lung-resident immune cell populations. This relationship between improved replication and infectivity of lung-resident cells provides insights into host barriers limiting human FLUAV adaptation to pigs and the complexities associated with the host immune response that influenza A viruses must overcome to become established in the swine population. [ABSTRACT FROM AUTHOR]

Introduction: UniSA's Invictus Pathways Program (IPP), a service program, was originally developed to assist veterans to train for and participate in the Invictus Games. More recently, the scope of the IPP has widened to support and improve wellbeing and facilitate post traumatic growth and recovery among participants who are living with physical and mental health injuries and conditions. This paper describes the components of the IPP and reports its process evaluation. Methods: Underpinned by a pragmatic approach, data related to participant and student involvement in the IPP, the number of participant training sessions, session attendance, program activities and events, and program fidelity were compiled from process documentation that had been collected between 2017 and 2020, inclusive. Following ethics approval, semi-structured interviews were conducted with participants of the IPP, members of their family support network, and university staff to understand the operations of the IPP and satisfaction with the program. Findings: There was high fidelity for the student-led exercise training aspects of IPP; however, data collection relevant to participants' psychological outcomes, and non-training IPP events and activities did not always occur as intended. Between 2017 and 2020, 53 veterans had participated in or were still participating in the IPP, and 63 allied health students had completed placements as student trainers. Fifty-three individual training sessions were delivered in 2017, increasing to 1,024 in 2020. Seventy-one interviews were completed with key IPP stakeholders. The qualitative analysis identified four higher order themes: Implementation and fidelity of the IPP, Satisfaction with the IPP, Areas of the IPP requiring improvement and suggestions for change, and Sustainability of the IPP. Satisfaction was generally high for the IPP, although there were factors that negatively impacted the experience for some participants and their family support network. Suggestions for improvement to program components and delivery aspects were made during the interviews, and the precariousness of IPP funding and sustainability was raised as an ongoing concern. Conclusion: This process evaluation has demonstrated that the physical activity training components of the IPP were delivered with high levels of fidelity, and that satisfaction with the IPP is mostly high, although there are areas that could be improved. There is a need for a more structured approach to the ongoing evaluation of the IPP. This includes ensuring that program staff have a shared understanding of the purpose of evaluation activities and that these activities occur as intended. Beyond this is the need to secure funding to support the sustainability of the IPP, so that it can continue to contribute to the wellbeing of veterans living with physical and mental health conditions, and their families. [ABSTRACT FROM AUTHOR]

Introduction: UniSA's Invictus Pathways Program (IPP) is motivated by the spirit of the Invictus Games to mobilise the benefits of sport to aid physical, psychological, and social wellbeing. Originally developed to assist veterans to train for and participate in the Invictus Games, the program has extended its scope to promote recovery and wellbeing for all veterans through physical activity. This paper describes the expectations and experiences of the IPP from the perspective of program participants. Methods: Objective measures of physical and psychological wellbeing were collected by survey, to enable description of the participating veterans' wellbeing status. Semi-structured interviews were conducted with 15 participants of the IPP who had not participated in an Invictus Games or Warrior Games. Reflexive thematic analysis was used to analyse the interview data. Coding and themes were developed through a mixture of inductive and deductive approaches to analysis. Initial themes related to previous life experience, expectations of participation, and outcomes of participation were preconceived, but the analysis provided scope for an inductive approach to formulate additional themes. Findings: Five of the participants had very high K10 scores, and scores above the norm for PCL-C, whilst one would be classified with an alcohol disorder. The qualitative analysis identified five higher order themes: Life experiences prior to participation in the IPP, Making a choice to participate in the IPP, Expectations of participation in the IPP, Impact of participation in the IPP, and Future Plans. There were perceptions that the IPP was beneficial for the participating veterans, irrespective of their physical and psychological health status. Participants described the positive impact of the IPP on their physical fitness, their social engagement, and their sense of belonging within the IPP and the university. Participants perceived the IPP to be an opportunity for them to 'give back' by contributing to the education of the students delivering the IPP. Participants reported the intention to continue being physically active. For some, this meant selection in an Invictus Games team, for others, this meant getting involved in community sporting organisations. Conclusion: The Invictus Pathways Program has been shown to have a positive impact on the physical and psychological wellbeing of the veterans who participated in its initial stages. As the program evolves, the longitudinal impact of participation, for veterans and their families, will be assessed. [ABSTRACT FROM AUTHOR]

The 2009 H1N1 pandemic (pdm09) lineage of influenza A virus (IAV) crosses interspecies barriers with frequent human-to-swine spillovers each year. These spillovers reassort and drift within swine populations, leading to genetically and antigenically novel IAV that represent a zoonotic threat. We quantified interspecies transmission of the pdm09 lineage, persistence in swine, and identified how evolution in swine impacted zoonotic risk. Human and swine pdm09 case counts between 2010 and 2020 were correlated and human pdm09 burden and circulation directly impacted the detection of pdm09 in pigs. However, there was a relative absence of pdm09 circulation in humans during the 2020–21 season that was not reflected in swine. During the 2020–21 season, most swine pdm09 detections originated from human-to-swine spillovers from the 2018–19 and 2019–20 seasons that persisted in swine. We identified contemporary swine pdm09 representatives of each persistent spillover and quantified cross-reactivity between human seasonal H1 vaccine strains and the swine strains using a panel of monovalent ferret antisera in hemagglutination inhibition (HI) assays. The swine pdm09s had variable antigenic reactivity to vaccine antisera, but each swine pdm09 clade exhibited significant reduction in cross-reactivity to one or more of the human seasonal vaccine strains. Further supporting zoonotic risk, we showed phylogenetic evidence for 17 swine-to-human transmission events of pdm09 from 2010 to 2021, 11 of which were not previously classified as variants, with each of the zoonotic cases associated with persistent circulation of pdm09 in pigs. These data demonstrate that reverse-zoonoses and evolution of pdm09 in swine results in viruses that are capable of zoonotic transmission and represent a potential pandemic threat. Author summary: The diversity and evolution of influenza A virus (IAV) in pigs is linked to the emergence of IAV with zoonotic potential. Human-to-swine transmission of the 2009 H1N1 pandemic (pdm09) IAV lineage repeatedly occurred across the past decade and has increased genetic diversity in pigs: sporadic swine-to-human cases are associated with these viruses. We measured the frequency of human-to-swine transmission of the H1N1 pandemic IAV lineage between 2009 and 2021 and determined how this affected the diversity of IAV in swine and zoonotic risk. We detected 370 separate human-to-swine spillovers, with the frequency of interspecies transmission increasing when the burden of IAV was highest in the human population. Most spillovers were single events without sustained transmission, but a small subset resulted in the emergence, persistence, and cocirculation of different pdm09 genetic clades in US pigs. Each of the pdm09 representative of different persistent spillovers was genetically and antigenically different from human seasonal vaccine strains. The persistence of pdm09 within pigs resulted in at least five recent swine-to-human transmission events. These data suggest that controlling IAV infection in humans working with swine can minimize spillover into pigs, reduce resulting genetic diversity of IAV in pigs, and proactively reduce the potential for swine-to-human transmission of IAV with zoonotic potential. [ABSTRACT FROM AUTHOR]

Family carers of veterans have a tendency not to seek support for their own wellbeing concerns. Understanding the barriers and enablers that family carers face in attending to their own wellbeing and in their caring role generally, is key to supporting family carers of veterans. This qualitative study sought to explore family carers' experiences and perceptions of their caring role, using semi-structured interviews. Questions were designed to capture concepts related to the barriers and enablers family carers face in attending to their own wellbeing. Twenty-two family carers participated in interviews. Thematic analysis facilitated the identification of key themes including the impact of the caring role; a perceived lack of recognition or appreciation of the caring role; expressed preferences for support; and consideration of the family unit. Findings suggest a need for accessible and multi-faceted support services for family carers of veterans, that target the drivers of physical and psychological wellbeing. [ABSTRACT FROM AUTHOR]

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance. [ABSTRACT FROM AUTHOR]

Objectives: Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted disease, but there is limited data on its epidemiology among urban populations. The urban Emergency Department (ED) is a potential venue for surveillance as it predominantly serves an inner city minority population. We evaluate the seroprevalence and factors associated with HSV-2 infection among patients attending the Johns Hopkins Hospital Adult Emergency Department (JHH ED). Methods: An identity unlinked-serosurvey was conducted between 6/2007 and 9/2007 in the JHH ED; sera were tested by the Focus HerpeSelect ELISA. Prevalence risk ratios (PRR) were used to determine factors associated with HSV-2 infection. Results: Of 3,408 serum samples, 1,853 (54.4%) were seropositive for HSV-2. Females (adjPRR  = 1.47, 95% CI 1.38–1.56), non-Hispanic blacks (adjPRR  = 2.03, 95% CI 1.82–2.27), single (adjPRR  = 1.15, 95% CI 1.07–1.25), divorced (adjPRR  = 1.28, 95% CI 1.15–1.41), and unemployed patients (adjPRR  = 1.13, 95% CI 1.05–1.21) had significantly higher rates of HSV-2 infection. Though certain zip codes had significantly higher seroprevalence of HSV-2, this effect was completely attenuated when controlling for age and gender. Conclusions: Seroprevalence of HSV-2 in the JHH ED was higher than U.S. national estimates; however, factors associated with HSV-2 infection were similar. The high seroprevalence of HSV-2 in this urban ED highlights the need for targeted testing and treatment. Cross-sectional serosurveys in the urban ED may help to examine the epidemiology of HSV-2. [ABSTRACT FROM AUTHOR]