Hepatocellular carcinoma after direct-acting antivirals for hepatitis C is associated with KIR-HLA types predicting weak NK cell-mediated immunity.

BACKGROUND AND AIMS: Second-generation direct-acting antivirals (2G DAA) to cure HCV have led to dramatic clinical improvements. HCV-associated hepatocellular carcinoma (HCC), however, remains common. Impaired immune tumor surveillance may play a role in HCC development. Our cohort evaluated the effects of innate immune types and clinical variables on outcomes including HCC. METHODS: Participants underwent full HLA class I/KIR typing and long-term HCV follow-up. RESULTS: A total of 353 HCV+ participants were followed for a mean of 7 years. Cirrhosis: 25% at baseline, developed in 12% during follow-up. 158 participants received 2G DAA therapy. HCC developed without HCV therapy in 20 subjects, 24 HCC after HCV therapy, and 10 of these after 2G DAA. Two predictors of HCC among 2G DAA-treated patients: cirrhosis (OR, 10.0, p = 0.002) and HLA/KIR profiles predicting weak natural killer (NK) cell-mediated immunity (NK cell complementation groups 6, 9, 11, 12, OR of 5.1, p = 0.02). Without 2G DAA therapy: cirrhosis was the main clinical predictor of HCC (OR, 30.8, p < 0.0001), and weak NK-cell-mediated immunity did not predict HCC. CONCLUSION: Cirrhosis is the main risk state predisposing to HCC, but weak NK-cell-mediated immunity may predispose to post-2G DAA HCC more than intermediate or strong NK-cell-mediated immunity.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

An RNA replicon system to investigate promising inhibitors of feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal feline disease, caused by a feline coronavirus (FCoV), namely feline infectious peritonitis virus (FIPV). We produced a baby hamster kidney 21 (BHK) cell line expressing a serotype I FCoV replicon RNA with a green fluorescent protein (GFP) reporter gene (BHK-F-Rep) and used it as an in vitro screening system to test different antiviral compounds. Two inhibitors of the FCoV main protease (Mpro), namely GC376 and Nirmatrelvir, as well as the nucleoside analog Remdesivir proved to be effective in inhibiting the replicon system. Different combinations of these compounds also proved to be potent inhibitors, having an additive effect when combined. Remdesivir, GC376, and Nirmatrelvir all have a 50% cytotoxic concentration (CC50) more than 200 times higher than their half-maximal inhibitory concentrations (IC50), making them important candidates for future in vivo studies as well as clinically implemented drug candidates. In addition, results were acquired with a virus infection system, where Felis catus whole fetus 4 (Fcwf-4) cells were infected with a previously described recombinant GFP-expressing FIPV (based on the laboratory-adapted serotype I FIPV strain Black) and treated with the most promising compounds. Results acquired with the replicon system were comparable to the results acquired with the virus infection system, demonstrating that we successfully implemented the FCoV replicon system for antiviral screening. We expect that this system will greatly facilitate future screens for anti-FIPV compounds and provide a non-infectious system to study and evaluate drug-resistant mutations that may emerge in the FIPV genome.IMPORTANCEFIPV is of great significance in the cat population around the world, causing 0.3%-1.4% of feline deaths in veterinary practices (2). As there are neither effective preventive measures nor approved treatment options available, there is an urgent need to identify antiviral drugs against FIPV. Our FCoV replicon system provides a valuable tool for drug discovery in vitro. Due to the lack of cell culture systems for serotype I FCoVs (the serotype most prevalent in the feline population) (2), a different system is needed to study these viruses. A viral replicon system is a valuable tool for studying FCoVs. Overall, our results demonstrate the utility of the serotype I feline coronavirus replicon system for antiviral screening as well as to study this virus in general. We propose several compounds representing promising candidates for future clinical trials and ultimately with the potential to save cats suffering from FIP.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Antiviral Approaches against Influenza Virus.

Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.

SARS-CoV-2 Antiviral Prescribing Gaps Among Nonhospitalized High-Risk Adults.

Within a multistate clinical cohort, SARS-CoV-2 antiviral prescribing patterns were evaluated from April 2022-June 2023 among nonhospitalized patients with SARS-CoV-2 with risk factors for severe COVID-19. Among 3247 adults, only 31.9% were prescribed an antiviral agent (87.6% nirmatrelvir/ritonavir, 11.9% molnupiravir, 0.5% remdesivir), highlighting the need to identify and address treatment barriers.

A Phase III Randomized Controlled Trial of Plitidepsin, a Marine-Derived Compound, in Hospitalized Adults With Moderate COVID-19.

BACKGROUND: Plitidepsin has shown potent preclinical activity against severe acute respiratory syndrome coronavirus 2 and was generally well tolerated in a phase I trial of hospitalized patients with coronavirus disease 2019 (COVID-19). NEPTUNO, a phase III, multicenter, randomized, controlled trial, was designed to evaluate the efficacy and safety of plitidepsin in the management of moderate COVID-19 in hospitalized adult patients. METHODS: Included patients had documented severe acute respiratory syndrome coronavirus 2 infection, required oxygen therapy, and had adequate organ function. The planned sample size was 609 patients. Patients were randomized 1:1:1 to at least 3 days of dexamethasone plus either plitidepsin (1.5 mg/day or 2.5 mg/day, for 3 days) or standard of care (control). The primary endpoint was the time to sustained withdrawal of supplemental oxygen. Secondary endpoints included time to sustained hospital discharge, clinical status, duration of oxygen support, percentage of patients requiring admission to the intensive care unit, and safety. RESULTS: After randomizing 205 patients, NEPTUNO was discontinued due to a notable drop in COVID-19-related hospitalizations. Available data suggest a 2-day improvement in the median time to sustained oxygen therapy discontinuation (5 vs 7 days) favoring both plitidepsin arms (hazard ratio, 1.37; 95% confidence interval, .96-1.96; P = .08 for plitidepsin 1.5 mg vs control; hazard ratio, 1.06; 95% confidence interval, .73-1.53; P = .78 for plitidepsin 2.5 mg vs control). Plitidepsin was generally well tolerated. CONCLUSIONS: Despite the trial limitations, these results suggest that plitidepsin may have a positive benefit-risk ratio in the management of patients requiring oxygen therapy. Further studies with plitidepsin, including those in immunosuppressed patients, are warranted.Results from this phase III trial suggest that plitidepsin, a first-in-class antiviral, may have a positive benefit-risk ratio in the management of hospitalized patients requiring oxygen therapy for moderate COVID-19.

Recent Advances in Enterovirus A71 Infection and Antiviral Agents.

Enterovirus A71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease (HFMD) that majorly affects children. Most of the time, HFMD is a mild disease but can progress to severe complications, such as meningitis, brain stem encephalitis, acute flaccid paralysis, and even death. HFMD caused by EV-A71 has emerged as an acutely infectious disease of highly pathogenic potential in the Asia-Pacific region. In this review, we introduced the properties and life cycle of EV-A71, and the pathogenesis and the pathophysiology of EV-A71 infection, including tissue tropism and host range of virus infection, the diseases caused by the virus, as well as the genes and host cell immune mechanisms of major diseases caused by enterovirus 71 (EV-A71) infection, such as encephalitis and neurologic pulmonary edema. At the same time, clinicopathologic characteristics of EV-A71 infection were introduced. There is currently no specific medication for EV-A71 infection, highlighting the urgency and significance of developing suitable anti-EV-A71 agents. This overview also summarizes the targets of existing anti-EV-A71 agents, including virus entry, translation, polyprotein processing, replication, assembly and release; interferons; interleukins; the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase B signaling pathways; the oxidative stress pathway; the ubiquitin-proteasome system; and so on. Furthermore, it overviews the effects of natural products, monoclonal antibodies, and RNA interference against EV-A71. It also discusses issues limiting the research of antiviral drugs. This review is a systematic and comprehensive summary of the mechanism and pathological characteristics of EV-A71 infection, the latest progress of existing anti-EV-A71 agents. It would provide better understanding and guidance for the research and application of EV-A71 infection and antiviral inhibitors.

Combination of ganciclovir and trifluridine prevents drug-resistance emergence in HSV-1.

Ocular herpes simplex virus 1 (HSV-1) infections can lead to visual impairment. Long-term acyclovir (ACV) prophylaxis reduces the frequency of recurrences but is associated with drug resistance. Novel therapies are needed to treat drug-resistant HSV-1 infections. Here, we describe the effects of trifluridine (TFT) in combination with ACV or ganciclovir (GCV) on HSV-1 replication and drug-resistance emergence. Wild-type HSV-1 was grown under increasing doses of one antiviral (ACV, GCV, or TFT) or combinations thereof (ACV + TFT or GCV + TFT). Virus cultures were analyzed by Sanger sequencing and deep sequencing of the UL23 [thymidine kinase (TK)] and UL30 [DNA polymerase (DP)] genes. The phenotypes of novel mutations were determined by cytopathic effect reduction assays. TFT showed overall additive anti-HSV-1 activity with ACV and GCV. Five passages under ACV, GCV, or TFT drug pressure gave rise to resistance mutations, primarily in the TK. ACV + TFT and GCV + TFT combinatory pressure induced mutations in the TK and DP. The DP mutations were mainly located in terminal regions, outside segments that typically carry resistance mutations. TK mutations (R163H, A167T, and M231I) conferring resistance to all three nucleoside analogs (ACV, TFT, and GCV) emerged under ACV, TFT, ACV + TFT pressure and under GCV + TFT pressure initiated from suboptimal drug concentrations. However, higher doses of GCV and TFT prevented drug resistance in the resistance selection experiments. In summary, we identified novel mutations conferring resistance to nucleoside analogs, including TFT, and proposed that GCV + TFT combination therapy may be an effective strategy to prevent the development of drug resistance.

The nucleoside analog 4'-fluorouridine suppresses the replication of multiple enteroviruses by targeting 3D polymerase.

Human enteroviruses are the major pathogens causing hand-foot-and-mouth disease in infants and young children throughout the world, and infection with enterovirus is also associated with severe complications, such as aseptic meningitis and myocarditis. However, there are no antiviral drugs available to treat enteroviruses infection at present. In this study, we found that 4'-fluorouridine (4'-FlU), a nucleoside analog with low cytotoxicity, exhibited broad-spectrum activity against infections of multiple enteroviruses with EC50 values at low micromolar levels, including coxsackievirus A10 (CV-A10), CV-A16, CV-A6, CV-A7, CV-B3, enterovirus A71 (EV-A71), EV-A89, EV-D68, and echovirus 6. With further investigation, the results indicated that 4'-FlU directly interacted with the RNA-dependent RNA polymerase of enterovirus, the 3D pol, and impaired the polymerase activity of 3D pol, hence inhibiting viral RNA synthesis and significantly suppressing viral replication. Our findings suggest that 4'-FlU could be promisingly developed as a broad-spectrum direct-acting antiviral agent for anti-enteroviruses therapy.

Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Practical updates in clinical antiviral resistance testing.

The laboratory diagnosis of antiviral resistance is a quickly changing field due to new drug availability, the sunsetting of older drugs, the development of novel technologies, rapid viral evolution, and the financial/logistic pressures of the clinical laboratory. This mini-review summarizes the current state of clinically available antiviral resistance testing in the United States in 2024, covering the most commonly used test methods, mechanisms, and clinical indications for herpes simplex virus, cytomegalovirus, human immunodeficiency virus, influenza, hepatitis B virus, and hepatitis C virus drug resistance testing. Common themes include the move away from phenotypic to genotypic methods for first-line clinical testing, as well as uncertainty surrounding the clinical meaningfulness of minority variant detection as next-generation sequencing methods have become more commonplace.

Targeting SARS-CoV-2 Proteins: In Silico Investigation with Polypyridyl-Based Zn(II)-Curcumin Complexes.

Herein, we have selected eight Zn(II)-based complexes viz., [Zn(bpy)(acac)Cl] (1), [Zn(phen)(acac)Cl] (2), [Zn(dppz)(acac)Cl] (3), [Zn(dppn)(acac)Cl] (4), [Zn(bpy)(cur)Cl] (5), [Zn(phen)(cur)Cl] (6), [Zn(dppz)(cur)Cl] (7), [Zn(dppn)(cur)Cl] (8), where bpy=2,2'-bipyridine, phen=1,10-phenanthroline, dppz=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine, dppn=naphtho[2,3-i]dipyrido[3,2-a:2',3'-c]phenazine, acac=acetylacetonate, cur=curcumin and performed in silico molecular docking studies with the viral proteins, i. e., spike protein (S), Angiotensin-converting enzyme II Receptor protein (ACE2), nucleocapsid protein (N), main protease protein (Mpro), and RNA-dependent RNA polymerase protein (RdRp) of SARS-CoV-2. The binding energy calculations, visualization of the docking orientation, and analysis of the interactions revealed that these complexes could be potential inhibitors of the viral proteins. Among complexes 1-8, complex 6 showed the strongest binding affinity with S and ACE2 proteins. 4 exerted better binding affinity in the case of the N protein, whereas 8 presented the highest binding affinities with Mpro and RdRp among all the complexes. Overall, the study indicated that Zn(II) complexes have the potential as alternative and viable therapeutic solutions for COVID-19.

Pseudorabies Virus Regulates the Extracellular Translocation of Annexin A2 To Promote Its Proliferation.

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

A realistic approach for evaluating antimicrobial surfaces for dry surface exposure scenarios.

The severe acute respiratory syndrome coronavirus 2 pandemic has raised public awareness about the importance of hygiene, leading to an increased demand for antimicrobial surfaces to minimize microbial contamination on high-touch surfaces. This is particularly relevant in public and private transportation settings, where surfaces frequently touched by individuals pose a significant, yet preventable, risk of infection transmission. Typically, the antimicrobial activity of surfaces is tested using test methods of the International Standards Organization, American Society for Testing and Materials, or Japanese Industrial Standards, which involve complete submersion in liquid, elevated temperature (37°C), and prolonged (24 h) contact periods. However, these conditions do not accurately represent real-world scenarios where surfaces are exposed to air. In this study, we propose a modified test method designed to better reflect real-life conditions in the intended end-use setting. The modifications included using deionized water instead of nutrient broth while preparing bacterial inoculum, applying a small test inoculum to the surface and allowing it to dry, maintaining ambient temperature and relative humidity throughout the contact period, and reducing the contact period to 4 h. With this modified approach, the antimicrobial activity of 20 samples was reassessed. This screening revealed that out of 20 samples, only 2 samples were effective against all species, while 8 samples demonstrated partial effectiveness against selected species, and 10 samples showed no significant effect. These findings highlight the inadequacy of the current test standard and emphasize the urgent necessity for revised and adapted testing method to ensure a reliable and accurate evaluation.IMPORTANCEThe recent severe acute respiratory syndrome coronavirus 2 pandemic has sparked increased demand for antimicrobial surfaces to mitigate the risk of fomites-transmitted infection in both indoors and confined spaces. Commonly, the antimicrobial activity of these surfaces is assessed using test standards established by national standards bodies, which do not distinguish between different application scenarios. While these test standards are suitable for surfaces intended for submerged application, they are inappropriate for antimicrobial surfaces designed for dry surface exposure. The usage of these standards can lead to an overestimation of antimicrobial efficacy. Thus, this study introduces a modified dry exposure test method aimed at better reflecting real-life conditions in the intended end-use setting. Our results revealed the subpar antimicrobial performance of numerous samples, highlighting the necessity to revise and tailor the universal test standard to real-world scenarios in order to ensure a reliable and accurate evaluation.

The role of treatment of hepatitis C with direct-acting antiviral agents on glycaemic control in diabetic patients: An updated systematic review and meta-analysis.

Recent studies suggested that successful clearance of chronic Hepatitis C Virus (HCV) by using direct-acting antiviral (DAA) agents could improve glycemic control in patients with diabetes; however, some studies failed to identify this benefit. We conducted a systematic review and meta-analysis to assess the impact of sustained virologic response (SVR) after treatment with DAA agents on glycemic control. Embase, Scopus and PubMed were searched through March 26th, 2023, for all studies evaluating whether eradication of HCV infection with DAAs is associated with an impact on glycemic control. Only studies with data on glycemic control, including haemoglobin A1c (HbA1c), fasting glucose, or Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), at least 12-week post-SVR were included. Sixteen studies met our eligibility criteria and were included in qualitative analysis. The mean HbA1c was 8.05% (95% CI: 7.79%-8.31%) before treatment and 7.19% (95% CI: 6.98%-7.39%) after treatment. There was a significant mean absolute reduction in HbA1c of 0.72% (95% CI: 0.52%-0.93%) with high heterogeneity between studies (I2 = 91.7%). The reduction in HbA1c remained significant in the subgroup analysis at 3 months follow up post SVR [0.74% (95% CI: 0.57%-0.91%)] and at least 6 months follow up [0.66% (95% CI: 0.23%-1.10%)]. We found a significant reduction in HbA1C after SVR in patients with type 2 diabetes mellitus, reflecting better glycemic control with HCV eradication. This data highlights an important extrahepatic benefit of HCV eradication.

A novel test and treat program for hepatitis C virus infection utilizing HCV core antigen testing, among police and general population, Islamabad, Pakistan, 2022.

Hepatitis C virus core antigen (HCVcAg) testing can simplify and decrease costs of HCV infection confirmation compared to molecular testing (nucleic acid testing). We piloted HCVcAg testing for the confirmation of active infection. The study was conducted during June through December 2022 among the police and the general population of Islamabad, Pakistan age 18 years and older. Initial screening for HCV antibody was conducted using a rapid diagnostic test (RDT) for all consenting participants. Those who tested positive had venous blood samples tested for HCVcAg, platelets and aspartate aminotransferase (AST). Persons with HCVcAg values ≥3 fmol/L were defined as viremic, and they were offered treatment with direct acting antiviral (DAA) medications, sofosbuvir and daclatasvir. Aspartate aminotransferase to platelet ratio index (APRI) was calculated for each HCV infected person, and those with an APRI score <1.5 received treatment for 12 weeks, while those with APRI ≥ to 1.5 received 24 weeks of treatment. A total of 15,628 persons were screened for anti-HCV using RDT and 643 (4.1%) tested positive. HCVcAg values of ≥3 fmol/L was found in 399/643 (62.1%), and all were offered and accepted treatment. Of those treated, 273/399 (68.4%) returned for a follow-up SVR and HCVcAg was not detected in 261/273, a 95.6% cure rate. The pilot study demonstrated the effectiveness of reaching and treating an urban population using RDT for screening and HCVcAg for confirmation of infection and test of cure.

A retrospective cohort study on early antibiotic use in vaccinated and unvaccinated COVID-19 patients.

The bacteriophage behavior of SARS-CoV-2 during the acute and post-COVID-19 phases appears to be an important factor in the development of the disease. The early use of antibiotics seems to be crucial to inhibit disease progression-to prevent viral replication in the gut microbiome, and control toxicological production from the human microbiome. To study the impact of specific antibiotics on recovery from COVID-19 and long COVID (LC) taking into account: vaccination status, comorbidities, SARS-CoV-2 wave, time of initiation of antibiotic therapy and concomitant use of corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs). A total of 211 COVID-19 patients were included in the study: of which 59 were vaccinated with mRNA vaccines against SARS-CoV-2 while 152 were unvaccinated. Patients were enrolled in three waves: from September 2020 to October 2022, corresponding to the emergence of the pre-Delta, Delta, and Omicron variants of the SARS-CoV-2 virus. The three criteria for enrolling patients were: oropharyngeal swab positivity or fecal findings; moderate symptoms with antibiotic intake; and measurement of blood oxygen saturation during the period of illness. The use of antibiotic combinations, such as amoxicillin with clavulanic acid (875 + 125 mg tablets, every 12 h) plus rifaximin (400 mg tablets every 12 h), as first choice, as suggested from the previous data, or azithromycin (500 mg tablets every 24 h), plus rifaximin as above, allows healthcare professionals to focus on the gut microbiome and its implications in COVID-19 disease during patient care. The primary outcome measured in this study was the estimated average treatment effect, which quantified the difference in mean recovery between patients receiving antibiotics and those not receiving antibiotics at 3 and 9 days after the start of treatment. In the analysis, both vaccinated and unvaccinated groups had a median illness duration of 7 days (interquartile range [IQR] 6-9 days for each; recovery crude hazard ratio [HR] = 0.94, p = 0.700). The median illness duration for the pre-Delta and Delta waves was 8 days (IQR 7-10 days), while it was shorter, 6.5 days, for Omicron (IQR 6-8 days; recovery crude HR = 1.71, p < 0.001). These results were confirmed by multivariate analysis. Patients with comorbidities had a significantly longer disease duration: median 8 days (IQR 7-10 days) compared to 7 days (IQR 6-8 days) for those without comorbidities (crude HR = 0.75, p = 0.038), but this result was not confirmed in multivariate analysis as statistical significance was lost. Early initiation of antibiotic therapy resulted in a significantly shorter recovery time (crude HR = 4.74, p < 0.001). Concomitant use of NSAIDs did not reduce disease duration and in multivariate analysis prolonged the disease (p = 0.041). A subgroup of 42 patients receiving corticosteroids for a median of 3 days (IQR 3-6 days) had a longer recovery time (median 9 days, IQR 8-10 days) compared to others (median 7 days, IQR 6-8 days; crude HR = 0.542, p < 0.001), as confirmed also by the adjusted HR. In this study, a statistically significant reduction in recovery time was observed among patients who received early antibiotic treatment. Early initiation of antibiotics played a crucial role in maintaining higher levels of blood oxygen saturation. In addition, it is worth noting that a significant number of patients who received antibiotics in the first 3 days and for a duration of 7 days, during the acute phase did not develop LC.