Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission.

Baloxavir marboxil (BXM) is approved for treating uncomplicated influenza. The active metabolite baloxavir acid (BXA) inhibits cap-dependent endonuclease activity of the influenza virus polymerase acidic protein (PA), which is necessary for viral transcription. Treatment-emergent E23G or E23K (E23G/K) PA substitutions have been implicated in reduced BXA susceptibility, but their effect on virus fitness and transmissibility, their synergism with other BXA resistance markers, and the mechanisms of resistance have been insufficiently studied. Accordingly, we generated point mutants of circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses carrying E23G/K substitutions. Both substitutions caused 2- to 13-fold increases in the BXA EC50. EC50s were higher with E23K than with E23G and increased dramatically (138- to 446-fold) when these substitutions were combined with PA I38T, the dominant BXA resistance marker. E23G/K-substituted viruses exhibited slightly impaired replication in MDCK and Calu-3 cells, which was more pronounced with E23K. In ferret transmission experiments, all viruses transmitted to direct-contact and airborne-transmission animals, with only E23K+I38T viruses failing to infect 100% of animals by airborne transmission. E23G/K genotypes were predominantly stable during transmission events and through five passages in vitro. Thermostable PA-BXA interactions were weakened by E23G/K substitutions and further weakened when combined with I38T. In silico modeling indicated this was caused by E23G/K altering the placement of functionally important Tyr24 in the endonuclease domain, potentially decreasing BXA binding but at some cost to the virus. These data implicate E23G/K, alone or combined with I38T, as important markers of reduced BXM susceptibility, and such mutants could emerge and/or transmit among humans.

Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Circulating Globally in 2022-2023.

The antiviral susceptibility of currently circulating (2022-2023) highly pathogenic avian influenza (HPAI) A(H5N1) viruses was assessed by genotypic and phenotypic approaches. The frequency of neuraminidase (NA) and polymerase acidic (PA) substitutions associated with reduced inhibition by NA inhibitors (NAIs) (21/2698, 0.78%) or by the PA inhibitor baloxavir (14/2600, 0.54%) was low. Phenotypic testing of 22 clade 2.3.2.1a and 2.3.4.4b viruses revealed broad susceptibility to NAIs and baloxavir concluding that most contemporary HPAI A(H5N1) viruses retain susceptibility to antiviral drugs. Novel NA-K432E and NA-T438I substitutions (N2 numbering) were identified at elevated frequencies (104/2698, 3.85%) and caused reduced zanamivir and peramivir inhibition.

Swine H1N1 Influenza Virus Variants with Enhanced Polymerase Activity and HA Stability Promote Airborne Transmission in Ferrets.

Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor hemagglutinin (HA) stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits. IMPORTANCE Diverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage as a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both α-2,6- and α-2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.

Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility.

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

Circulating tumor DNA (ctDNA) to evaluate minimal residual disease (MRD), treatment response, and posttreatment prognosis in pancreatic adenocarcinoma.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a blood-based test with multiple utilities in oncology. In the past few years, multiple studies of varying designs, methods, and quality have emerged which show promise for ctDNA as a tool to assess response to treatment and detect minimal residual disease (MRD) across various gastrointestinal (GI) malignancies. We aim to review the current literature for ctDNA as it pertains to assessing treatment response, MRD, prognosis, and risk of recurrence for pancreatic adenocarcinoma. METHODS: PubMed was queried with a combination of terms regarding pancreatic adenocarcinoma, minimal residual disease, resection, and prognosis. All resultant articles were reviewed by the authors for appropriate fit with scope. RESULTS: Fourteen articles were identified that fit with the scope of this review. CONCLUSIONS: Detectable ctDNA after definitive resection, specifically mutated KRAS, correlates with shorter recurrence-free survival (RFS), overall survival (OS), and overall prognosis. Limited data also suggests ctDNA may provide a noninvasive means to assess response to chemotherapy. Whether this information is actionable in terms of altering neoadjuvant or postresection treatment regimens remains an open question requiring further study.

The role of circulating tumor DNA in evaluating minimal residual disease in luminal gastrointestinal malignancies.

The analysis of circulating tumor DNA (ctDNA) has multiple uses in oncology. In the past few years, studies with varying designs, methods, and quality have emerged that show promise for the use of ctDNA as a tool to detect minimal residual disease (MRD) across luminal gastrointestinal malignancies. This review of the current literature looks at ctDNA in relation to detecting MRD, predicting patient prognosis, and assessing risk for recurrence.

Baloxavir Treatment Delays Influenza B Virus Transmission in Ferrets and Results in Limited Generation of Drug-Resistant Variants.

Clinical efficacy of the influenza antiviral baloxavir marboxil (baloxavir) is compromised by treatment-emergent variants harboring a polymerase acidic protein I38T (isoleucine-38-threonine) substitution. However, the fitness of I38T-containing influenza B viruses (IBVs) remains inadequately defined. After the pharmacokinetics of the compound were confirmed in ferrets, animals were injected subcutaneously with 8 mg/kg of baloxavir acid (BXA) at 24 h postinoculation with recombinant BXA-sensitive (BXA-Sen, I38) or BXA-resistant (BXA-Res, I38T) B/Brisbane/60/2008 (Victoria lineage) virus. BXA treatment of donor ferrets reduced virus replication and delayed transmission of the BXA-Sen but not the BXA-Res IBV. The I38 genotype remained dominant in the BXA-Sen-infected animals, even with BXA treatment. In competitive-mixture experiments, no transmission to aerosol contacts was seen from BXA-treated donors coinfected with the BXA-Sen and BXA-Res B/Brisbane/60/2008 viruses. However, in parallel mixed infections with the B/Phuket/3073/2013 (Yamagata lineage) virus background, BXA treatment failed to block airborne transmission of the BXA-Res virus, and the I38T genotype generally predominated. Therefore, the relative fitness of BXA-Res IBVs is complex and dependent on the virus backbone and within-host virus competition. BXA treatment of single-virus-infected ferrets hampers aerosol transmission of the BXA-Sen virus and does not readily generate BXA-Res variants, whereas mixed infections may result in propagation of BXA-Res IBVs of the Yamagata lineage. Our findings confirm the antiviral potency of baloxavir against IBVs, while supporting optimization of the dosing regimen to maximize clinical benefit.

Multiple polymerase acidic (PA) I38X substitutions in influenza A(H1N1)pdm09 virus permit polymerase activity and cause reduced baloxavir inhibition.

BACKGROUND: Baloxavir marboxil is an antiviral drug that targets the endonuclease activity of the influenza virus polymerase acidic (PA) protein. PA I38T/M/F substitutions reduce its antiviral efficacy. OBJECTIVES: To understand the effects of the 19 possible amino acid (AA) substitutions at PA 38 on influenza A(H1N1)pdm09 polymerase activity and inhibition by baloxavir acid, the active metabolite of baloxavir marboxil. METHODS: Influenza A(H1N1)pdm09 viral polymerase complexes containing all 19 I38X AA substitutions were reconstituted in HEK293T cells in a mini-replicon assay. Polymerase complex activity and baloxavir inhibitory activity were measured in the presence or absence of 50 nM baloxavir acid. RESULTS: Only three substitutions (R, K, P) reduced polymerase activity to <79% of I38-WT. When compared with the prototypical baloxavir marboxil resistance marker T38, 5 substitutions conferred 10%-35% reductions in baloxavir acid inhibitory activity (M, L, F, Y, C) and 11 substitutions conferred >50% reductions (R, K, S, N, G, W, A, Q, E, D, H), while two substitutions (V, P) maintained baloxavir acid inhibitory activity. CONCLUSIONS: Most PA 38 substitutions permit a functional replication complex retaining some drug resistance in the mini-replicon assay. This study provides a targeted approach for virus rescue and analysis of novel baloxavir marboxil reduced-susceptibility markers, supports the consideration of a broader range of these markers during antiviral surveillance and adds to the growing knowledge of baloxavir marboxil resistance profiles.

Synthesis, inhibitory activity and oral dosing formulation of AV5124, the structural analogue of influenza virus endonuclease inhibitor baloxavir.

BACKGROUND: The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. OBJECTIVES: To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. METHODS: The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. RESULTS: Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin-Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. CONCLUSIONS: Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.

The PA Endonuclease Inhibitor RO-7 Protects Mice from Lethal Challenge with Influenza A or B Viruses.

Current influenza treatment relies on a single class of antiviral drugs, the neuraminidase inhibitors (NAIs), raising concern over the potential emergence of resistant variants and necessitating the development of novel drugs. In recent years, investigational inhibitors targeting the endonuclease activity of the influenza acidic polymerase (PA) protein have yielded encouraging results, although there are only limited data on their in vivo efficacy. Here, we examined the antiviral potential of the PA endonuclease inhibitor RO-7 in prophylactic and therapeutic regimens in BALB/c mice inoculated with influenza A/California/04/2009 (H1N1)pdm09 or B/Brisbane/60/2008 viruses, which represent currently circulating antigenic variants. RO-7 was administered to mice intraperitoneally twice daily at dosages of 6, 15, or 30 mg/kg/day for 5 days, starting 4 h before or 24 or 48 h after virus inoculation, and showed no adverse effects. Prophylactic administration completely protected mice from lethal infection by influenza A or B virus. The level of therapeutic protection conferred depended upon the time of treatment initiation and RO-7 dosage, resulting in 60 to 100% and 80 to 100% survival with influenza A and B viruses, respectively. RO-7 treatment significantly decreased virus titers in the lung and lessened the extent and severity of lung damage. No PA endonuclease-inhibitor resistance was observed in viruses isolated from lungs of RO-7-treated mice, and the viruses remained susceptible to the drug at nanomolar concentrations in phenotypic assays. These in vivo efficacy results further highlight the potential of RO-7 for development as antiviral therapy for influenza A and B virus infections.

The Hemagglutinin Stem-Binding Monoclonal Antibody VIS410 Controls Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.

A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro.

Antiviral drugs are important in preventing and controlling influenza, particularly when vaccines are ineffective or unavailable. A single class of antiviral drugs, the neuraminidase inhibitors (NAIs), is recommended for treating influenza. The limited therapeutic options and the potential risk of antiviral resistance are driving the search for additional small-molecule inhibitors that act on influenza virus proteins. The acid polymerase (PA) of influenza viruses is a promising target for new antivirals because of its essential role in initiating virus transcription. Here, we characterized a novel compound, RO-7, identified as a putative PA endonuclease inhibitor. RO-7 was effective when added before the cessation of genome replication, reduced polymerase activity in cell-free systems, and decreased relative amounts of viral mRNA and genomic RNA during influenza virus infection. RO-7 specifically inhibited the ability of the PA endonuclease domain to cleave a nucleic acid substrate. RO-7 also inhibited influenza A viruses (seasonal and 2009 pandemic H1N1 and seasonal H3N2) and B viruses (Yamagata and Victoria lineages), zoonotic viruses (H5N1, H7N9, and H9N2), and NAI-resistant variants in plaque reduction, yield reduction, and cell viability assays in Madin-Darby canine kidney (MDCK) cells with nanomolar to submicromolar 50% effective concentrations (EC50s), low toxicity, and favorable selective indices. RO-7 also inhibited influenza virus replication in primary normal human bronchial epithelial cells. Overall, RO-7 exhibits broad-spectrum activity against influenza A and B viruses in multiple in vitro assays, supporting its further characterization and development as a potential antiviral agent for treating influenza.

Influenza A(H7N9) virus transmission between finches and poultry.

Low pathogenicity avian influenza A(H7N9) virus has been detected in poultry since 2013, and the virus has caused >450 infections in humans. The mode of subtype H7N9 virus transmission between avian species remains largely unknown, but various wild birds have been implicated as a source of transmission. H7N9 virus was recently detected in a wild sparrow in Shanghai, China, and passerine birds, such as finches, which share space and resources with wild migratory birds, poultry, and humans, can be productively infected with the virus. We demonstrate that interspecies transmission of H7N9 virus occurs readily between society finches and bobwhite quail but only sporadically between finches and chickens. Inoculated finches are better able to infect naive poultry than the reverse. Transmission occurs through shared water but not through the airborne route. It is therefore conceivable that passerine birds may serve as vectors for dissemination of H7N9 virus to domestic poultry.

Replication Capacity of Avian Influenza A(H9N2) Virus in Pet Birds and Mammals, Bangladesh.

Avian influenza A(H9N2) is an agricultural and public health threat. We characterized an H9N2 virus from a pet market in Bangladesh and demonstrated replication in samples from pet birds, swine tissues, human airway and ocular cells, and ferrets. Results implicated pet birds in the potential dissemination and zoonotic transmission of this virus.

Risk assessment of H2N2 influenza viruses from the avian reservoir.

H2N2 influenza A viruses were the cause of the 1957-1958 pandemic. Historical evidence demonstrates they arose from avian virus ancestors, and while the H2N2 subtype has disappeared from humans, it persists in wild and domestic birds. Reemergence of H2N2 in humans is a significant threat due to the absence of humoral immunity in individuals under the age of 50. Thus, examination of these viruses, particularly those from the avian reservoir, must be addressed through surveillance, characterization, and antiviral testing. The data presented here are a risk assessment of 22 avian H2N2 viruses isolated from wild and domestic birds over 6 decades. Our data show that they have a low rate of genetic and antigenic evolution and remained similar to isolates circulating near the time of the pandemic. Most isolates replicated in mice and human bronchial epithelial cells, but replication in swine tissues was low or absent. Multiple isolates replicated in ferrets, and 3 viruses were transmitted to direct-contact cage mates. Markers of mammalian adaptation in hemagglutinin (HA) and PB2 proteins were absent from all isolates, and they retained a preference for avian-like α2,3-linked sialic acid receptors. Most isolates remained antigenically similar to pandemic A/Singapore/1/57 (H2N2) virus, suggesting they could be controlled by the pandemic vaccine candidate. All viruses were susceptible to neuraminidase inhibitors and adamantanes. Nonetheless, the sustained pathogenicity of avian H2N2 viruses in multiple mammalian models elevates their risk potential for human infections and stresses the need for continual surveillance as a component of prepandemic planning.

Possible role of songbirds and parakeets in transmission of influenza A(H7N9) virus to humans.

Avian-origin influenza A(H7N9) recently emerged in China, causing severe human disease. Several subtype H7N9 isolates contain influenza genes previously identified in viruses from finch-like birds. Because wild and domestic songbirds interact with humans and poultry, we investigated the susceptibility and transmissibility of subtype H7N9 in these species. Finches, sparrows, and parakeets supported replication of a human subtype H7N9 isolate, shed high titers through the oropharyngeal route, and showed few disease signs. Virus was shed into water troughs, and several contact animals seroconverted, although they shed little virus. Our study demonstrates that a human isolate can replicate in and be shed by such songbirds and parakeets into their environment. This finding has implications for these birds' potential as intermediate hosts with the ability to facilitate transmission and dissemination of A(H7N9) virus.

Neuromodulation for intractable headaches.

Intractable chronic headaches are a major challenge for both patients and healthcare professionals. Over the last two decades, implantable electrical neuromodulators, previously established to manage other forms of chronic pain, have been used increasingly for intractable primary and secondary headache disorders. We review the current approaches to the management of refractory headaches using neuromodulation. Indications, operative considerations and complications are discussed based on our experience and a review of the literature. The field of neuromodulation has been rapidly advancing, with many new targets being discovered and novel devices being developed for treating craniofacial pain. We discuss some of these targets, detailing the latest advances in the area of neuromodulation for intractable headaches.

Factors Impacting Intent to Share Multigenic Cancer Testing Results in a Community Hospital Setting.

BACKGROUND/OBJECTIVES: Multi-gene, multi-cancer, hereditary cancer risk screenings may be useful in cancer prevention and treatment, not only for cancer patients but also for patients' family members. If genetic cancer screening is to have the widest possible benefit, it must be extended into diverse cancer care settings that serve diverse patient communities, providing cancer patients and their relatives with individualized cancer risk evaluations. Little research, to date, has examined the impact of extending multigenic cancer screening into diverse settings. Without empirical data characterizing the support needs of cancer patients and their family members, we may not adequately satisfy the needs of all patients and risk exacerbating existing disparities in cancer care and outcomes. METHODS: We examined patient perspectives on the sharing of genetic results with at-risk family members by surveying a racially diverse sample of cancer patients receiving a multi-gene, multi-cancer risk screen in a community hospital setting. RESULTS: In a survey of 230 cancer patients, we found that intent to share results with family members was high but varied across family member types. More respondents planned to disclose results to at least one sister (82.5%) compared to at least one brother (73.1%). Over one-fourth of participants (27.4%) were either uncertain about sharing or intended to withhold their genomic screening results from at least one at-risk family member eligible for cascade testing. Participants were more likely to withhold their results from a sibling than from a child. Furthermore, intent to share across all family member types was lower if probands failed to identify at least one benefit to sharing. CONCLUSIONS: Understanding factors associated with decisions to share results with at-risk relatives in diverse patient populations can help clinicians support cascade genetic cancer screenings in diverse communities and settings.

Genotypic and phenotypic susceptibility of emerging avian influenza A viruses to neuraminidase and cap-dependent endonuclease inhibitors.

Avian influenza outbreaks, including ones caused by highly pathogenic A(H5N1) clade 2.3.4.4b viruses, have devastated animal populations and remain a threat to humans. Risk elements assessed for emerging influenza viruses include their susceptibility to approved antivirals. Here, we screened >20,000 neuraminidase (NA) or polymerase acidic (PA) protein sequences of potentially pandemic A(H5Nx), A(H7Nx), and A(H9N2) viruses that circulated globally in 2010-2023. The frequencies of NA or PA substitutions associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NA inhibitors (NAIs) (oseltamivir, zanamivir) or a cap-dependent endonuclease inhibitor (baloxavir) were low: 0.60% (137/22,713) and 0.62% (126/20,347), respectively. All tested subtypes were susceptible to NAIs and baloxavir at sub-nanomolar concentrations. A(H9N2) viruses were the most susceptible to oseltamivir, with IC50s 3- to 4-fold lower than for other subtypes (median IC50: 0.18 nM; n = 22). NA-I222M conferred RI of A(H5N1) viruses by oseltamivir (with a 26-fold IC50 increase), but NA-S246N did not reduce inhibition. PA-E23G, PA-K34R, PA-I38M/T, and the previously unreported PA-A36T caused RI by baloxavir in all subtypes tested. Avian A(H9N2) viruses endemic in Egyptian poultry predominantly acquired PA-I38V, which causes only a <3-fold decrease in the baloxavir EC50 and fails to meet the RI criteria. PA-E199A/D in A(H7Nx) and A(H9N2) viruses caused a 2- to 4-fold decrease in EC50 (close to the borderline for RI) and should be closely monitored. Our data indicate antiviral susceptibility is high among avian influenza A viruses with pandemic potential and present novel markers of resistance to existing antiviral interventions.

Identifying Actionable Alterations in KRAS Wild-Type Pancreatic Cancer.

The 5-year relative survival rate for pancreatic cancer is currently the lowest among all cancer types with a dismal 13%. A Kirsten rat sarcoma virus (KRAS) gene mutation is present in approximately 90% of patients with pancreatic cancer; however, KRAS-specific drugs are not yet widely used in clinical practice for pancreatic cancer, specifically the KRASG12D variant. Advances in genomic testing revealed an opportunity to detect genetic alterations in a subset of patients with no KRAS mutation termed KRAS wild-type. Patients with KRAS wild-type tumors have a propensity to express driver alterations, hence paving the way for utilizing a targeted therapy approach either via clinical trials or standard-of-care drugs. These alterations include fusions, amplifications, translocations, rearrangements and microsatellite instability-high tumors and can be as high as 11% in some studies. Here, we discuss some of the most notable alterations in KRAS wild-type and highlight promising clinical trials.