Hemagglutinin Stability Determines Influenza A Virus Susceptibility to a Broad-Spectrum Fusion Inhibitor Arbidol.

Understanding mechanisms of resistance to antiviral inhibitors can reveal nuanced features of targeted viral mechanisms and, in turn, lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum antiviral that binds to and prevents the fusion-associated conformational changes in the trimeric influenza A virus (IAV) hemagglutinin (HA). The rate-limiting step during the HA-mediated membrane fusion is the release of the hydrophobic fusion peptides from a conserved pocket on HA. Here, we investigated how destabilizing or stabilizing mutations in or near the fusion peptide affect viral sensitivity to Arbidol. The degree of sensitivity was proportional to the extent of fusion-peptide stability on the prefusion HA: stabilized mutants were more sensitive, and destabilized ones were resistant to Arbidol. Single-virion membrane fusion experiments for representative wild-type (WT) and mutant viruses demonstrated that resistance is a direct consequence of fusion-peptide destabilization not requiring reduced Arbidol binding to HA. Our results support the model whereby the probability of individual HAs extending to engage the target membrane is determined by the composite of two critical forces: a "tug" on the fusion peptide by HA rearrangements near the Arbidol binding site and the key interactions stabilizing the fusion peptide in the prefusion pocket. Arbidol increases and destabilizing mutations decrease the free-energy cost for fusion-peptide release, accounting for the observed resistance. Our findings have broad implications for fusion inhibitor design, viral mechanisms of resistance, and our basic understanding of HA-mediated membrane fusion.

Implementation Evaluation of HUGS/Abrazos During the COVID-19 Pandemic: A Program to Foster Resiliency in Pregnancy and Early Childhood.

Early life adversity can significantly impact child development and health outcomes throughout the life course. With the COVID-19 pandemic exacerbating preexisting and introducing new sources of toxic stress, social programs that foster resilience are more necessary now than ever. The Helping Us Grow Stronger (HUGS/Abrazos) program fills a crucial need for protective buffers during the COVID-19 pandemic, which has escalated toxic stressors affecting pregnant women and families with young children. HUGS/Abrazos combines patient navigation, behavioral health support, and innovative tools to ameliorate these heightened toxic stressors. We used a mixed-methods approach, guided by the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework, to evaluate the implementation of the HUGS/Abrazos program at Massachusetts General Hospital from 6/30/2020-8/31/2021. Results of the quality improvement evaluation revealed that the program was widely adopted across the hospital and 392 unique families were referred to the program. The referred patients were representative of the communities in Massachusetts disproportionately affected by the COVID-19 pandemic. Furthermore, 79% of referred patients followed up with the initial referral, with sustained high participation rates throughout the program course; and they were provided with an average of four community resource referrals. Adoption and implementation of the key components in HUGS/Abrazos were found to be appropriate and acceptable. Furthermore, the implemented program remained consistent to the original design. Overall, HUGS/Abrazos was well adopted as an emergency relief program with strong post-COVID-19 applicability to ameliorate continuing toxic stressors while decreasing burden on the health system.

Fostering Resilience in Pregnancy and Early Childhood During the COVID-19 Pandemic: The HUGS/Abrazos Program Design and Implementation.

Pregnancy and early childhood pose unique sensitivity to stressors such as economic instability, poor mental health, and social inequities all of which have been magnified by the COVID-19 pandemic. In absence of protective buffers, prolonged exposure to excessive, early adversity can lead to poor health outcomes with significant impact lasting beyond the childhood years. Helping Us Grow Stronger (HUGS/Abrazos) is a community-based program, designed and launched at the time of the COVID-19 surge in the Spring of 2020, that combines emergency relief, patient navigation, and direct behavioral health support to foster family resilience and mitigate the negative impacts of COVID-related toxic stress on pregnant women and families with children under age 6. Through a targeted referral process, community health workers provide resource navigation for social needs, and a social worker provides behavioral health support. The use of innovative tools such as a centralized resource repository, community health workers with specialized knowledge in this age range, and a direct referral system seeks to assist in streamlining communication and ensuring delivery of quality care. We aim to serve over 300 families within the 1st year. The HUGS/Abrazos program aims to fill an important void by providing the necessary tools and interventions to support pregnant women and young families impacted by adversity exacerbated by the COVID-19 pandemic.

The shape of pleomorphic virions determines resistance to cell-entry pressure.

Many enveloped animal viruses produce a variety of particle shapes, ranging from small spherical to long filamentous types. Characterization of how the shape of the virion affects infectivity has been difficult because the shape is only partially genetically encoded, and most pleomorphic virus structures have no selective advantage in vitro. Here, we apply virus fractionation using low-force sedimentation, as well as antibody neutralization coupled with RNAScope, single-particle membrane fusion experiments and stochastic simulations to evaluate the effects of differently shaped influenza A viruses and influenza viruses pseudotyped with Ebola glycoprotein on the infection of cells. Our results reveal that the shape of the virus particles determines the probability of both virus attachment and membrane fusion when viral glycoprotein activity is compromised. The larger contact interface between a cell and a larger particle offers a greater probability that several active glycoproteins are adjacent to each other and can cooperate to induce membrane merger. Particles with a length of tens of micrometres can fuse even when 95% of the glycoproteins are inactivated. We hypothesize that non-genetically encoded variable particle shapes enable pleomorphic viruses to overcome selective pressure and may enable adaptation to infection of cells by emerging viruses such as Ebola. Our results suggest that therapeutics targeting filamentous virus particles could overcome antiviral drug resistance and immune evasion in pleomorphic viruses.

Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice.

Reporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus with NanoLuc luciferase inserted into the 5' end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we compared in vivo replication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations and PB2 noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-like in vivo fitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCE Influenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.