Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Functional Landscape of SARS-CoV-2 Cellular Restriction.

A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals.

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

Implementing California Senate Bill 493 in an Outpatient Pharmacy Within an Integrated Health System: Evaluation of the Operational and Clinical Effect of Pharmacist-Ordered Laboratory Tests.

BACKGROUND: Under California Senate Bill 493, pharmacists can order patient laboratory tests (labs). Currently, it is unknown if this service affects patient outcomes or pharmacy operations. Does lab ordering by pharmacists improve access to care, improve quality outcomes, and/or affect pharmacy operational functions? PROGRAM DESCRIPTION: A 13-month pilot study was conducted at 2 Kaiser Permanente (KP) outpatient pharmacies where pharmacists provided extended adherence consultations and ordered hemoglobin A1c (HbA1c) labs for patients nonadherent to their oral diabetic medications with an HbA1c ≥ 8% or missing annual labs. Clinical outcomes of the pilot study were compared with a similar patient population at KP who concurrently received lab orders from their primary providers, defined here as the "usual care model." OBSERVATION: Of the 793 HbA1c lab orders, 87 (11.0%) were generated by a pharmacist, and 706 (89.0%) were generated by the usual care group. Forty-three (49.4%) patients in the pharmacist group completed their labs compared with 279 (39.5%) patients in the usual care group (P = 0.10). A significantly greater proportion of patients in the pharmacist group achieved an HbA1c < 8% within the follow-up period of 30-180 days (34.9%), compared with the usual care group (12.2%, P < 0.01). Of the patients who completed labs during the evaluation period, 38 (43.7%) patients in the pharmacist group and 111 (15.7%) patients in the usual care group had prelaboratory values ≥ 8% within the previous 12 months. The average pre-HbA1c value was 9.47% in the pharmacist group, and the average post-HbA1c value was 8.68% (P < 0.01). For the usual care group, the average pre-HbA1c value was 9.70%, and the average post-HbA1c value was 9.43% (P = 0.06). When comparing the difference in HbA1c reduction between the 2 groups, there was a larger decrease in HbA1c in the pharmacist group, but this difference was not significantly different (P = 0.06). The pilot study added an average of 5 minutes per patient encounter to the pharmacy workflow but did not affect overall patient wait times for receiving outpatient prescriptions. IMPLICATIONS: Laboratory ordering by pharmacists in the outpatient setting improved access to care, improved quality outcomes, and did not adversely affect pharmacy operations. DISCLOSURES: No outside funding supported this project. The authors have nothing to disclose.

Adaptation for Growth Via Learning New Skills as a Means to Long-Term Functional Independence in Older Adulthood: Insights From Emerging Adulthood.

Maintenance of functional independence, or the ability to perform daily tasks independently, is a hallmark of successful aging. Healthy older adults are considered functionally independent if they pass a short survey consisting of relatively simple daily activities, including grocery shopping and managing finances. We argue that aging research often has overlooked an important factor for long-term functional independence in a dynamic environment: adaptation for growth via learning new skills. Previous research has focused primarily on compensation and mitigating decline rather than growth. Given that adaptation for growth is at the core of intelligence, resilience, and neuroplasticity, we suggest that functional independence research with older adults could integrate adaptation for growth into the construct, following research on adolescent autonomy and emerging adulthood. After briefly reviewing research on functional independence and compensation in older adulthood, we offer suggestions to push forward gerontological research linking adaptation for growth and functional independence.

Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells.

Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, as well as sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to the growth of human H1N1 and H3N2 viruses and low-pathogenic avian influenza (LPAI) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) H5N1 virus successfully propagated in HeLa cells to levels comparable to those in a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 virus growth, suggesting the absence of a host factor(s) required for the replication of H1N1, but not H5N1, viruses in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, as well as deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from an H5N1 virus was necessary and sufficient to enable H1N1 virus growth. Overall, this study suggests that the absence of one or more cellular factors in HeLa cells results in abortive replication of H1N1, H3N2, and LPAI viruses, which can be circumvented upon the introduction of H5N1 virus NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into the virus-host interactions that underlie IAV pathogenesis and tropism.IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk that these zoonotic viruses may acquire adaptive mutations enabling them to propagate efficiently and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested the growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon the introduction of H5N1 virus HA and NP. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 virus pathogenesis and tropism.

Evaluation of an outpatient pharmacy clinical services program on adherence and clinical outcomes among patients with diabetes and/or coronary artery disease.

BACKGROUND: Poor medication adherence among patients with chronic diseases can result in complications and increased health care expenditures. An outpatient pharmacy clinical service (OPCS) program targeted nonadherent diabetes mellitus (DM) and/or coronary artery disease (CAD) patients with hemoglobin A1c (HbA1c) and/or low-density lipoprotein cholesterol (LDL-C) outside clinical goals. Pharmacists engaged identified patients with a face-to-face B-SMART consult, a consultation methodology to identify Barriers to medication adherence, work on Solutions to identified barriers, Motivate patients, recommend Adherence tools, reinforce the pharmacist-patient Relationship, and Triage if needed, to other services such as health education to improve outcomes.  OBJECTIVES: To (a) assess rates of medication adherence and clinical outcomes in the OPCS program compared with usual care in an integrated health care system and (b) estimate return-on-investment (ROI) from this intervention.  METHODS: This retrospective cohort study used data from the Kaiser Permanente Southern California region to identify patients who received OPCS consultations and usual care patients from March 2009 through December 2010, with 1 year of follow-up from the initial consult (index date). Four patients from usual care were matched to each patient in the OPCS program and were assigned the same index date as the matching OPCS patient. Additional selection criteria were applied after matching. All patients were required to have a medication possession ratio (MPR) of less than 0.80 for their diabetes or dyslipidemia oral medications 1 year prior to the index date, indicating lower adherence to the prescribed therapy. Diabetic patients or dyslipidemic patients had to have a HbA1c or LDL-C lab result outside of clinical goals prior to the index date to be included in the study, respectively. Adherence outcomes as well as clinical outcomes were measured 12 months after the index date using chi-square tests for differences in percentages and t-tests for differences in means. The ROI was based on a cost-avoidance model that compared the cost of the OPCS program with the cost savings gained through reduced hospitalizations and emergency department (ED) visits. The diabetes and dyslipidemia cohorts were combined for the ROI analysis. RESULTS: Demographic and clinical characteristics at baseline were similar between the OPCS group (n = 1,480) and usual care group (n = 1,477). Among patients with diabetes, a higher percentage in the OPCS group than in the usual care group were adherent with their diabetes medications (53.5% vs. 37.4%, P = 0.001). There was no significant difference in average MPR between groups. However, patients in the OPCS group had a greater increase in mean MPR (0.19 vs. 0.15, P = 0.024); were less likely to discontinue taking their diabetes medications (11.7% vs. 35.5%, P = 0.001); and were more likely to have a timely first fill after the index date (34.8% vs. 12.9%, P = 0.001). The average number of days to the first fill after the index date was significantly shorter for the OPCS group (79.3 vs. 156.3, P = 0.001). Regarding clinical outcomes, patients with diabetes in the OPCS group had a lower mean HbA1c (8.48 vs. 8.80, P = 0.024) and a greater reduction in HbA1c (-1.25 vs. -0.75, P = 0.001) than in the usual care group. They were also less likely to have an ED visit (1.67% vs. 4.21%, P = 0.040), but there was no significant difference in the percentage of patients with a hospital admission. Among patients with dyslipidemia, the mean MPR was significantly lower for the OPCS group than the usual care group (0.70 vs. 0.74, P = 0.003). There were no significant differences in the percentage of adherent patients or the change in mean MPR from baseline. However, the OPCS group was significantly less likely to discontinue dyslipidemia medications (21.1% vs. 35.4%, P less than 0.001) and more likely to have a timely fill (28.3% vs. 15.1%, P less than 0.001). The average days to first fill after the index date was 106.9 for the OPCS group, compared with 162.6 for the usual care group (P less than 0.001). The OPCS group had a lower mean LDL-C (105.1 vs. 110.4, P = 0.001) and a greater reduction in LDL-C (-30.5 vs. -22.4, P = 0.001) than the usual care group. There were no significant differences in the percentage of patients with an ED visit or a hospital admission. In terms of ROI, assuming that 58% of hospitalizations and 8.5% of ED visits incurred in the usual care group were avoidable, approximately $5.79 could be saved for every dollar spent on the OPCS program.  CONCLUSION: By engaging nonadherent patients to restart their DM or lipid medications during a face-to-face consult, the OPCS pharmacist was able to influence and improve medication adherence and clinical outcomes, particularly among patients with diabetes. A positive ROI was demonstrated.