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1.
JACS Au ; 4(2): 607-618, 2024 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-38425896

RESUMEN

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biologically relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical biantennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by α2,3- or α2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH2 at one of the antennae, which temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of the evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the α1,3-antenna of N-glycans are critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the α1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses again agglutinate erythrocytes, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicate that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

2.
Lancet Infect Dis ; 2024 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-38330975

RESUMEN

BACKGROUND: Onradivir (ZSP1273) is a novel anti-influenza A virus inhibitor. Preclinical studies show that onradivir can inhibit influenza A H1N1 and H3N2 replication and increase the survival rate of infected animals. In this study, we aimed to evaluate the safety and efficacy of three onradivir dosing regimens versus placebo in outpatients with acute uncomplicated influenza A virus infection. METHODS: We did a multicentre, double-blind, randomised, placebo-controlled, phase 2 trial at 20 clinical sites in China. Eligible participants were adults (18-65 years) with an influenza-like illness screened by rapid antigen testing at the first clinical visit, had the presence of a fever (axillary temperature ≥38·0°C), and had the presence of at least one moderate systemic and one respiratory symptom within 48 h of symptom onset. Patients were excluded if they were pregnant, allergic to onradivir, or had received any influenza antiviral medication within 7 days before enrolment. Participants were randomly assigned (1:1:1:1) into four groups by an interactive web response system: onradivir 200 mg twice per day group, onradivir 400 mg twice per day group, onradivir 600 mg once per day group, and a matching placebo group. A 5-day oral treatment course was initiated within 48 h after symptoms onset. The primary outcome was the time to alleviate influenza symptoms in the modified intention-to-treat population. Safety was a secondary outcome. We evaluated the patients' self-assessed severity of seven influenza symptoms on a 4-point ordinal scale, and the treatment-emergent adverse events in all patients. This trial is registered with ClinicalTrials.gov, number NCT04024137. FINDINGS: Between Dec 7, 2019, and May 18, 2020, a total of 205 patients were screened; of whom, 172 (84%) were randomly assigned to receive onradivir (n=43 in the 200 mg twice per day group; n=43 in the 400 mg twice per day group; and n=43 in the 600 mg once per day group), or placebo (n=42). Median age was 22 years (IQR 20-26). All three onradivir groups showed decreased median time to alleviate influenza symptoms (46·92 h [IQR 24·00-81·38] in the 200 mg twice per day group, 54·87 h [23·67-110·62] in the 400 mg twice per day group, and 40·05 h [17·70-65·82] in the 600 mg once per day) compared with the placebo group (62·87 h [36·40-113·25]). The median difference between the onradivir 600 mg once per day group and the placebo group was -22·82 h (p=0·0330). The most frequently reported treatment-emergent adverse event was diarrhoea (71 [42%] of 171), ranging from 33-65% of the patients in onradivir-treated groups compared with 10% in the placebo group; no serious adverse events were observed. INTERPRETATION: Onradivir showed a safety profile comparable to placebo, as well as higher efficacy than placebo in ameliorating influenza symptoms and lowering the viral load in adult patients with uncomplicated influenza infection, especially the onradivir 600 mg once per day regimen. FUNDING: National Multidisciplinary Innovation Team Project of Traditional Chinese Medicine, National Natural Science Foundation of China, Guangdong Science and Technology Foundation, Guangzhou Science and Technology Planning Project, Emergency Key Program of Guangzhou Laboratory, Macao Science and Technology Development Fund, and Guangdong Raynovent Biotech.

3.
J Med Virol ; 96(3): e29484, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38402600

RESUMEN

Antiviral therapy based on neuraminidase (oseltamivir) or polymerase (baloxavir marboxil) inhibitors plays an important role in the management of influenza infections. However, the emergence of drug resistance and the uncontrolled inflammatory response are major limitations in the treatment of severe influenza disease. Protectins D1 (PD1) and DX (PDX), part of a family of pro-resolving mediators, have previously demonstrated anti-influenza activity as well as anti-inflammatory properties in various clinical contexts. Herein, we synthetized a series of simplified PDX analogs and assessed their in vitro antiviral activity against influenza A(H1N1) viruses, including oseltamivir- and baloxavir-resistant variants. In ST6GalI-MDCK cells, the PDX analog AN-137B reduced viral replication in a dose-dependent manner with IC50 values of 23.8 for A/Puerto Rico/8/1934 (H1N1) and between 32.6 and 36.7 µM for susceptible and resistant A(H1N1)pdm09 viruses. In MTS-based cell viability experiments, AN-137B showed a 50% cellular cytotoxicity (CC50 ) of 638.7 µM with a resulting selectivity index of 26.8. Of greater importance, the combination of AN-137B with oseltamivir or baloxavir resulted in synergistic and additive in vitro effects, respectively. Treatment of lipopolysaccharide (LPS)-stimulated macrophages with AN-137B resulted in a decrease of iNOS activity as shown by the reduction of nitrite production, suggesting an anti-inflammatory effect. In conclusion, our results indicate that the protectin analog AN-137B constitutes an interesting therapeutic modality against influenza A virus, warranting further evaluation in animal models.


Asunto(s)
Dibenzotiepinas , Ácidos Docosahexaenoicos , Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Morfolinas , Piridonas , Triazinas , Animales , Humanos , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Gripe Humana/tratamiento farmacológico , Antiinflamatorios/uso terapéutico , Farmacorresistencia Viral , Neuraminidasa
4.
Phytochemistry ; 220: 113996, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38311150

RESUMEN

The study entailed the investigation of the roots of Euphorbia wallichii, which resulted in the isolation of 29 ent-atisane diterpenoids (1-29), 14 of which were previously unknown. These previously undescribed ones were named euphorwanoids A-N (3-5, 7, 9, and 10-18). Various techniques, including comprehensive spectroscopic methods and calculated electronic circular dichroism, were employed to determine their molecular structures. Additionally, the absolute configurations of ten ent-atisane diterpenoids (1, 2, 5, 6, 8, 9, 11, 12, 14 and 16) were established through X-ray crystallographic analyses. All isolated compounds' potential to inhibit the influenza A virus in vitro were evaluated. Compounds 18, 20, and 24 exhibited notable antiviral activity against the A/Puerto Rico/8/1934 strain. Their effective concentrations for reducing viral activity (EC50 values) were found to be 8.56, 1.22, and 4.97 µM, respectively. An intriguing aspect of this research is that it marks the first instance of ent-atisane diterpenes displaying anti-H1N1 activity. Empirical NMR rules were established with Δδ to distinguish the R/S configurations of C-13 and C-16 in ent-atisanes.

5.
Nat Commun ; 15(1): 254, 2024 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-38177116

RESUMEN

Repeat vaccination with egg-based influenza vaccines could preferentially boost antibodies targeting the egg-adapted epitopes and reduce immunogenicity to circulating viruses. In this randomized trial (Clinicaltrials.gov: NCT03722589), sera pre- and post-vaccination with quadrivalent inactivated egg-based (IIV4), cell culture-based (ccIIV4), and recombinant (RIV4) influenza vaccines were collected from healthcare personnel (18-64 years) in 2018-19 (N = 723) and 2019-20 (N = 684) influenza seasons. We performed an exploratory analysis. Vaccine egg-adapted changes had the most impact on A(H3N2) immunogenicity. In year 1, RIV4 induced higher neutralizing and total HA head binding antibodies to cell- A(H3N2) virus than ccIIV4 and IIV4. In year 2, among the 7 repeat vaccination arms (IIV4-IIV4, IIV4-ccIIV4, IIV4-RIV4, RIV4-ccIIV4, RIV4-RIV4, ccIIV4-ccIIV4 and ccIIV4-RIV4), repeat vaccination with either RIV4 or ccIIV4 further improved antibody responses to circulating viruses with decreased neutralizing antibody egg/cell ratio. RIV4 also had higher post-vaccination A(H1N1)pdm09 and A(H3N2) HA stalk antibodies in year 1, but there was no significant difference in HA stalk antibody fold rise among vaccine groups in either year 1 or year 2. Multiple seasons of non-egg-based vaccination may be needed to redirect antibody responses from immune memory to egg-adapted epitopes and re-focus the immune responses towards epitopes on the circulating viruses to improve vaccine effectiveness.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Humanos , Anticuerpos Antivirales , Formación de Anticuerpos , Técnicas de Cultivo de Célula , Epítopos , Pruebas de Inhibición de Hemaglutinación , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana/prevención & control , Vacunación , Vacunas de Productos Inactivados
6.
J Virol Methods ; 323: 114838, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37914041

RESUMEN

In influenza A virus-infected cells, newly synthesized viral neuraminidases (NAs) transiently localize at the host cell Golgi due to glycosylation, before their expression on the cell surface. It remains unproven whether Golgi-localized intracellular NAs exhibit sialidase activity. We have developed a sialidase imaging probe, [2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenyl]-α-D-N-acetylneuraminic acid (BTP9-Neu5Ac). This probe is designed to be cleaved by sialidase activity, resulting in the release of a hydrophobic fluorescent compound, 2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenol (BTP9). BTP9-Neu5Ac makes the location of sialidase activity visually detectable by the BTP9 fluorescence that results from the action of sialidase activity. In this study, we established a protocol to visualize the sialidase activity of intracellular NA at the Golgi of influenza A virus-infected cells using BTP9-Neu5Ac. Furthermore, we employed this fluorescence imaging protocol to elucidate the intracellular inhibition of laninamivir octanoate, an anti-influenza drug. At approximately 7 h after infection, newly synthesized viral NAs localized at the Golgi. Using our developed protocol, we successfully histochemically stained the sialidase activity of intracellular viral NAs localized at the Golgi. Importantly, we observed that laninamivir octanoate effectively inhibited the intracellular viral NA, in contrast to drugs like zanamivir or laninamivir. Our study establishes a visualization protocol for intracellular viral NA sialidase activity and visualizes the inhibitory effect of laninamivir octanoate on Golgi-localized intracellular viral NA in infected cells.


Asunto(s)
Antivirales , Inhibidores Enzimáticos , Virus de la Influenza A , Neuraminidasa , Proteínas Virales , Humanos , Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , Neuraminidasa/análisis , Neuraminidasa/antagonistas & inhibidores , Imagen Óptica/métodos , Zanamivir/farmacología , Proteínas Virales/análisis , Proteínas Virales/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología
7.
Influenza Other Respir Viruses ; 17(12): e13231, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38098649

RESUMEN

Background: Respiratory complications often accompany influenza in patients with chronic obstructive pulmonary disease (COPD). In this retrospective study, we quantified the impact of antiviral therapy on exacerbations, healthcare resource utilization (HRU), and costs in patients with COPD across 5 influenza seasons. Methods: Using claims data from US MarketScan® databases, we identified patients with COPD who had an influenza diagnosis during the 2012-2016 influenza seasons. Patients who received a neuraminidase inhibitor within 48 h of diagnosis (N = 4134) were identified and propensity score-matched 1:1 to a comparator cohort of untreated patients. We determined COPD- and pneumonia-related HRU and costs during month 1, each subsequent quarter, and months 2-13. Results: Antiviral-treated patients had a significantly lower frequency of COPD-related outcomes than untreated patients during all periods (exacerbations: 10.4% vs 18.2% [month 1] and 17.7% vs 24.2% [months 2-13]; inpatient visit: 2.5% vs 7.9% [month 1] and 3.8% vs 6.7% [months 2-13]; P < 0.0001, all comparisons). Treated patients also had significantly lower outpatient and emergency department (ED) visits beyond month 1. Pneumonia-related inpatient, ED, and outpatient visits were significantly lower in antiviral-treated patients than in untreated patients over all periods (P < 0.0001, all comparisons). In all HRU categories, COPD- and pneumonia-related costs were significantly lower in treated patients over all periods (month-1 ED visit costs were higher). Conclusions: Antiviral treatment in patients with COPD and influenza is associated with significantly lower HRU and costs in the postinfection month and for an entire year following infection compared with untreated patients.


Asunto(s)
Gripe Humana , Neumonía , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Broncodilatadores/uso terapéutico , Estudios Retrospectivos , Gripe Humana/complicaciones , Gripe Humana/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Neumonía/complicaciones , Antivirales/uso terapéutico
8.
Lancet Infect Dis ; 2023 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-38006892

RESUMEN

BACKGROUND: Spread of SARS-CoV-2 led to a global pandemic, and there remains unmet medical needs in the treatment of Omicron infections. VV116, an oral antiviral agent that has potent activity against SARS-CoV-2, was compared with a placebo in this phase 3 study to investigate its efficacy and safety in patients with mild-to-moderate COVID-19. METHODS: This multicentre, double-blind, phase 3, randomised controlled study enrolled adults in hospitals for infectious diseases and tertiary general hospitals in China. Eligible patients were randomly assigned in a 1:1 ratio using permuted block randomisation to receive oral VV116 (0·6 g every 12 h on day 1 and 0·3 g every 12 h on days 2-5) or oral placebo (on the same schedule as VV116) for 5 days. Randomisation stratification factors included SARS-CoV-2 vaccination status and the presence of high-risk factors for progression to severe COVID-19. Inclusion criteria were a positive SARS-CoV-2 test, an initial onset of COVID-19 symptoms 3 days or less before the first study dose, and a score of 2 or more for any target COVID-19-related symptoms in the 24 h before the first dose. Patients who had severe or critical COVID-19 or who had taken any antiviral drugs were excluded from the study. The primary endpoint was the time to clinical symptom resolution for 2 consecutive days. Efficacy analyses were performed on a modified intention-to-treat population, comprising all patients who received at least one dose of VV116 or placebo, tested positive for SARS-CoV-2 nucleic acid, and did not test positive for influenza virus before the first dose. Safety analyses were done on all participants who received at least one dose of VV116 or placebo. This study was registered with ClinicalTrials.gov, NCT05582629, and has been completed. FINDINGS: A total of 1369 patients were randomly assigned to treatment groups and 1347 received either VV116 (n=674) or placebo (n=673). At the interim analysis, VV116 was superior to placebo in reducing the time to sustained clinical symptom resolution among 1229 patients (hazard ratio [HR] 1·21, 95% CI 1·04-1·40; p=0·0023). At the final analysis, a substantial reduction in time to sustained clinical symptom resolution was observed for VV116 compared with placebo among 1296 patients (HR 1·17, 95% CI 1·04-1·33; p=0·0009), consistent with the interim analysis. The incidence of adverse events was similar between groups (242 [35·9%] of 674 patients vs 283 [42·1%] of 673 patients). INTERPRETATION: Among patients with mild-to-moderate COVID-19, VV116 significantly reduced the time to sustained clinical symptom resolution compared with placebo, with no observed safety concerns. FUNDING: Shanghai Vinnerna Biosciences, Shanghai Science and Technology Commission, and the National Key Research and Development Program of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

9.
J Enzyme Inhib Med Chem ; 38(1): 2277135, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37955306

RESUMEN

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Oseltamivir/farmacología , Oseltamivir/química , Neuraminidasa , Simulación del Acoplamiento Molecular , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Glicósido Hidrolasas
10.
bioRxiv ; 2023 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-37986780

RESUMEN

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biological relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical bi-antennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by α2,3- or α2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH2 at one of the antennae that temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the α1,3-antenna of N-glycans is critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the α1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses agglutinate erythrocytes again, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicates that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

11.
BMC Infect Dis ; 23(1): 641, 2023 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-37784010

RESUMEN

BACKGROUND: Diagnosis of bacterial meningitis remains a challenge in most developing countries due to low yield from bacterial culture, widespread use of non-prescription antibiotics, and weak microbiology laboratories. The objective of this study was to compare the yield from standard bacterial culture with the multiplex nested PCR platform, the BioFire® FilmArray® Meningitis/Encephalitis Panel (BioFire ME Panel), for cases with suspected acute bacterial meningitis. METHODS: Following Gram stain and bacterial culture on cerebrospinal fluid (CSF) collected from children aged less than 5 years with a clinical suspicion of acute bacterial meningitis (ABM) as defined by the WHO guidelines, residual CSF specimens were frozen and later tested by BioFire ME Panel. RESULTS: A total of 400 samples were analyzed. Thirty-two [32/400 (8%)] of the specimens were culture positive, consisting of; three Salmonella spp. (2 Typhi and 1 non-typhi), three alpha hemolytic Streptococcus, one Staphylococcus aureus, six Neisseria meningitidis, seven Hemophilus influenzae, 11 Streptococcus pneumoniae and 368 were culture negative. Of the 368 culture-negative specimens, the BioFire ME Panel detected at least one bacterial pathogen in 90 (24.5%) samples, consisting of S. pneumoniae, N. meningitidis and H. influenzae, predominantly. All culture positive specimens for H. influenzae, N. meningitidis and S. pneumoniae also tested positive with the BioFire ME Panel. In addition, 12 specimens had mixed bacterial pathogens identified. For the first time in this setting, we have data on the viral agents associated with meningitis. Single viral agents were detected in 11 (2.8%) samples while co-detections with bacterial agents or other viruses occurred in 23 (5.8%) of the samples. CONCLUSIONS: The BioFire® ME Panel was more sensitive and rapid than culture for detecting bacterial pathogens in CSF. The BioFire® ME Panel also provided for the first time, the diagnosis of viral etiologic agents that are associated with meningoencephalitis in this setting. Institution of PCR diagnostics is recommended as a routine test for suspected cases of ABM to enhance early diagnosis and optimal treatment.


Asunto(s)
Encefalitis , Meningitis Bacterianas , Meningitis , Neisseria meningitidis , Niño , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Encefalitis/diagnóstico , Nigeria , Meningitis Bacterianas/diagnóstico , Meningitis/diagnóstico , Neisseria meningitidis/genética , Bacterias/genética , Haemophilus influenzae/genética , Streptococcus pneumoniae/genética , Líquido Cefalorraquídeo/microbiología
12.
ChemMedChem ; : e202300458, 2023 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-37864572

RESUMEN

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

13.
Clin Infect Dis ; 2023 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-37819010

RESUMEN

BACKGROUND: Influenza immunization programs aim to reduce the risk and burden of severe outcomes. To inform optimal program strategies, we monitored influenza hospitalizations over seven seasons, stratified by age, comorbidity and vaccination status. METHODS: We assembled data from four hospitals involved in an active surveillance network with systematic collection of nasal samples and PCR testing for influenza virus in all patients admitted through the emergency department with acute respiratory infection during 2012/13 to 2018/19 influenza seasons in Quebec, Canada. We estimated seasonal, population-based incidence of influenza-associated hospitalizations by subtype predominance, age, comorbidity and vaccine status, and derived the number-needed-to-vaccinate to prevent one hospitalization per stratum. RESULTS: The average seasonal incidence of influenza-associated hospitalization was 89/100,000 (95%CI: 86, 93), lower during A(H1N1) (49-82/100,000) than A(H3N2) seasons (73-143/100,000). Overall risk followed a J-shaped age pattern, highest among infants 0-5 months and adults ≥75 years. Hospitalization risks were highest for children <5 years during A(H1N1) but for adults ≥75 years during A(H3N2) seasons. Age-adjusted hospitalization risks were 7-fold higher among individuals with versus without comorbidities (214 versus 30/100,000). The number-needed-to-vaccinate to prevent hospitalization was 82-fold lower for ≥75-years-olds with comorbidity (n = 1,995), who comprised 39% of all hospitalizations, than for healthy 18-64-year-olds (n = 163,488), who comprised just 6% of all hospitalizations. CONCLUSIONS: In the context of broad-based influenza immunization programs (targeted or universal), severe outcome risks should be simultaneously examined by subtype, age, comorbidity, and vaccine status. Policymakers require such detail to prioritize promotional efforts and expenditures toward the greatest and most efficient program impact.

14.
Viruses ; 15(9)2023 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-37766287

RESUMEN

Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2.


Asunto(s)
COVID-19 , Canidae , Coinfección , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N8 del Virus de la Influenza A , Infecciones del Sistema Respiratorio , Perros , Animales , SARS-CoV-2/genética , Coinfección/diagnóstico , Coinfección/veterinaria , Subtipo H3N2 del Virus de la Influenza A , COVID-19/diagnóstico , COVID-19/veterinaria , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/veterinaria
15.
Nat Commun ; 14(1): 5785, 2023 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-37723184

RESUMEN

Soluble HMW1C-like N-glycosyltransferases (NGTs) catalyze the glycosylation of Asn residues in proteins, a process fundamental for bacterial autoaggregation, adhesion and pathogenicity. However, our understanding of their molecular mechanisms is hindered by the lack of structures of enzymatic complexes. Here, we report structures of binary and ternary NGT complexes of Aggregatibacter aphrophilus NGT (AaNGT), revealing an essential dyad of basic/acidic residues located in the N-terminal all α-domain (AAD) that intimately recognizes the Thr residue within the conserved motif Asn0-X+1-Ser/Thr+2. Poor substrates and inhibitors such as UDP-galactose and UDP-glucose mimetics adopt non-productive conformations, decreasing or impeding catalysis. QM/MM simulations rationalize these results, showing that AaNGT follows a SN2 reaction mechanism in which the acceptor asparagine uses its imidic form for catalysis and the UDP-glucose phosphate group acts as a general base. These findings provide key insights into the mechanism of NGTs and will facilitate the design of structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria.


Asunto(s)
Asparagina , Proteínas Bacterianas , Glicosilación , Proteínas Bacterianas/genética , Haemophilus influenzae , Glucosa , Uridina Difosfato
16.
Medicina (Kaunas) ; 59(9)2023 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-37763660

RESUMEN

Background and Objectives: Baloxavir marboxil is a novel cap-dependent endonuclease inhibitor prescribed for influenza treatment. Unlike neuraminidase inhibitors like oseltamivir, which impair viral release from infected host cells, baloxavir blocks influenza virus proliferation by inhibiting viral mRNA transcription. This study aimed to compare the effectiveness of baloxavir and oseltamivir for the treatment of early childhood influenza. Materials and Methods: Of 1410 patients diagnosed with influenza between 2015 and 2018 at a Japanese primary care outpatient clinic, 1111 pediatric patients aged 0-6 years who were treated with baloxavir (n = 555) or oseltamivir (n = 556) were enrolled retrospectively. The following clinical factors were compared between patients treated with baloxavir and oseltamivir: age, sex, time from fever onset to drug administration (<24 h or 24-48 h), time from drug administration to fever reduction, influenza type (A or B), and influenza vaccination before disease onset. The duration of the fever, which was used as an index of clinical effectiveness, was compared using the log-rank test. Clinical factors associated with fever duration were determined using multivariate logistic regression analysis. Results: Median age (3.0 vs. 2.5 years), influenza type A (99% vs. 47%), median duration from drug administration to fever resolution (1 day vs. 2 days), and influenza vaccination (done, 41% vs. not done, 65%) were significantly different between the baloxavir and oseltamivir groups (p < 0.001). The number of patients with a fever duration of one day was 553 (99.6%) in the baloxavir group and 6 (1.1%) in the oseltamivir group (p < 0.001). Baloxavir use was only significantly associated with fever duration in the multivariate analysis (odds ratio 50,201, p < 0.001). Apparent adverse effects were not observed in the baloxavir-treated group. Conclusions: Baloxavir treatment resulted in a shorter fever duration than oseltamivir treatment in early childhood influenza.


Asunto(s)
Dibenzotiepinas , Gripe Humana , Preescolar , Humanos , Niño , Oseltamivir/uso terapéutico , Gripe Humana/tratamiento farmacológico , Estudios Retrospectivos , Dibenzotiepinas/uso terapéutico , Fiebre/tratamiento farmacológico
17.
J Vis Exp ; (191)2023 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-37602881

RESUMEN

ARTICLES DISCUSSED: Festa, F., Labaer, J. Kinase inhibitor screening in self-assembled human protein microarrays. Journal of Visualized Experiments. 152, e59886 (2019). Stockman, B. J. et al. NMR-Based activity assays for determining compound inhibition, IC50 values, artifactual activity, and whole-cell activity of nucleoside ribohydrolases. Journal of Visualized Experiments. 148, e59928 (2019). Gao, S. et al. A high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of Caenorhabditis elegans. Journal of Visualized Experiments. 145, e59082 (2019). Axelsson, H., Almqvist, H., Seashore-Ludlow, B. Using high content imaging to quantify target engagement in adherent cells. Journal of Visualized Experiments. 141, e58670 (2018). Chorba, J. S., Galvan, A. M., Shokat, K. M. A high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease PCSK9. Journal of Visualized Experiments. 138, e58265 (2018). Sullivan, C. et al. Using zebrafish models of human influenza A virus infections to screen antiviral drugs and characterize host immune cell responses. Journal of Visualized Experiments. 119, e55235 (2017). Tiemann, K., Garri, C., Wang, J., Clarke, L., Kani, K. Assessment of resistance to tyrosine kinase inhibitors by an interrogation of signal transduction pathways by antibody arrays. Journal of Visualized Experiments. 139, e57779 (2018). Radnai, L., Stremel, R. F., Sellers, J. R., Rumbaugh, G., Miller, C. A. A semi-high-throughput adaptation of the NADH-coupled ATPase assay for screening small molecule inhibitors. Journal of Visualized Experiments. 150, e60017 (2019). Nandha Premnath, P., Craig, S., McInnes, C. Development of inhibitors of protein-protein interactions through REPLACE: Application to the design and development non-ATP competitive CDK inhibitors. Journal of Visualized Experiments. 104, e52441 (2015). Chen, E. W., Ke, C. Y., Brzostek, J., Gascoigne, N. R. J., Rybakin, V. Identification of mediators of T-cell receptor signaling via the screening of chemical inhibitor libraries. Journal of Visualized Experiments. 143, e58946 (2019). Takakusagi, Y. Biosensor-based high throughput biopanning and bioinformatics analysis strategy for the global validation of drug-protein interactions. Journal of Visualized Experiments. 166, e61873 (2020). Riching, K. M., Mahan, S. D., Urh, M., Daniels, D. L. High-Throughput cellular profiling of targeted protein degradation compounds using HiBiT CRISPR cell lines. Journal of Visualized Experiments. 165, e61787 (2020).


Asunto(s)
Proproteína Convertasa 9 , Pez Cebra , Humanos , Animales , Proteolisis , Aclimatación , Caenorhabditis elegans
18.
Biosensors (Basel) ; 13(8)2023 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-37622868

RESUMEN

In this study, we developed a highly sensitive and specific bimolecular fluorescence complementation (BiFC)-based influenza A virus (IAV)-sensing system by combining a galactose/glucose-binding protein (GGBP) with an N-terminal large domain (YN1-172) and a C-terminal small domain (YC173-239) made up of enhanced yellow fluorescence protein (eYFP). The GGBP-based BiFC reporter exhibits the fluorescence reconstitution as a result of conformational changes in GGBP when lactose, which was derived from 6'-silalyllactose and used as a substrate for neuraminidase (NA), binds to GGBP in the presence of IAV. The system showed a linear dynamic range extending from 1 × 100 to 1 × 107 TCID50/mL, and it had a detection limit of 1.1 × 100 TCID50/mL for IAV (H1N1), demonstrating ultra-high sensitivity. Our system exhibited fluorescence intensity enhancements in the presence of IAV, while it displayed weak fluorescence signals when exposed to NA-deficient viruses, such as RSV A, RSV B, adenovirus and rhinovirus, thereby indicating selective responses for IAV detection. Overall, our system provides a simple, highly sensitive and specific IAV detection platform based on BiFC that is capable of detecting ligand-induced protein conformational changes, obviating the need for virus culture or RNA extraction processes.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Fluorescencia , Glucosa
19.
Antiviral Res ; 217: 105701, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37567255

RESUMEN

Neuraminidase inhibitors (NAIs) are recommended for influenza treatment and prevention worldwide. The most widely prescribed NAI is oral oseltamivir, while inhaled zanamivir is less commonly used. Using phenotypic neuraminidase (NA) enzymatic assays and molecular modeling approaches, we examined the ability of the investigational orally-dosed NAI AV5080 to inhibit viruses of the influenza A(H1N1)pdm09, A(H3N2), A(H5N1), and A(H7N9) subtypes and the influenza B/Victoria- and B/Yamagata-lineages containing NA substitutions conferring oseltamivir or zanamivir resistance including: NA-R292K, NA-E119G/V, NA-H274Y, NA-I122L/N, and NA-R150K. Broadly, AV5080 showed enhanced in vitro efficacy when compared with oseltamivir and/or zanamivir. Reduced AV5080 inhibition was determined for influenza A viruses with NA-E119G and NA-R292K, and for B/Victoria-lineage viruses with NA-I122N/L and B/Yamagata-lineage virus with NA-R150K. Molecular modeling suggested loss of the short hydrogen bond to the carboxyl group of AV5080 affected inhibition of NA-R292K viruses, whereas loss of the salt bridge with the guanidine group of AV5080 affected inhibition of NA-E119G. The resistance profiles and predicted binding modes of AV5080 and zanamivir are most similar, but dissimilar to those of oseltamivir, in part because of a guanidine moiety compensatory binding effect. Overall, our data suggests that AV5080 is a promising orally-dosed NAI that exhibited similar or superior in vitro efficacy against viruses with reduced or highly reduced inhibition phenotypes with respect to currently approved NAIs.


Asunto(s)
Herpesvirus Cercopitecino 1 , Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Subtipo H7N9 del Virus de la Influenza A , Gripe Humana , Humanos , Antivirales/farmacología , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , Guanidina/metabolismo , Guanidinas/metabolismo , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana/virología , Neuraminidasa/genética , Oseltamivir/farmacología , Zanamivir/farmacología
20.
BMC Neurol ; 23(1): 308, 2023 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-37608315

RESUMEN

BACKGROUND: Persisting coma is a common complication in (neuro)intensive care in neurological disease such as acute ischemic stroke, intracerebral hemorrhage or subarachnoid hemorrhage. Amantadine acts as a nicotinic receptor antagonist, dopamine receptor agonist and non-competitive N-Methyl-D-aspartate receptor antagonist. Amantadine is a long-known drug, originally approved for treatment of influenza A and Parkinson`s Disease. It has been proven effective in improving vigilance after traumatic brain injury. The underlying mechanisms remain largely unknown, albeit anti-glutamatergic and dopaminergic effects might be most relevant. With limited evidence of amantadine efficacy in non-traumatic pathologies, the aim of our study is to assess the effects of amantadine for neuroenhancement in non-traumatic neurointensive patients with persisting coma. METHODS: An investigator-initiated, monocenter, phase IIb proof of concept open-label pilot study will be carried out. Based on the Simon design, 43 adult (neuro)intensive care patients who meet the clinical criteria of persisting coma not otherwise explained and < 8 points on the Glasgow Coma Scale (GCS) will be recruited. Amantadine will be administered intravenously for five days at a dosage of 100 mg bid. The primary endpoint is an improvement of at least 3 points on the GCS. If participants present as non-responders (increase < 3 points or decrease on the GCS) within the first 48 h, the dosage will be doubled from day three to five. Secondary objectives aim to demonstrate that amantadine improves vigilance via alternative scales. Furthermore, the incidence of adverse events will be investigated and electroencephalography (EEG) will be recorded at baseline and end of treatment. DISCUSSION: The results of our study will help to systematically assess the clinical utility of amantadine for treatment of persisting coma in non-traumatic brain injury. We expect that, in the face of only moderate treatment risk, a relevant number of patients will benefit from amantadine medication by improved vigilance (GCS increase of at least 3 points) finally leading to a better rehabilitation potential and improved functional neurological outcome. Further, the EEG data will allow evaluation of brain network states in relation to vigilance and potentially outcome prediction in this study cohort. TRIAL REGISTRATION: NCT05479032.


Asunto(s)
Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Accidente Cerebrovascular Isquémico , Adulto , Humanos , Amantadina/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Coma , Proyectos Piloto , Estudios Prospectivos , Prueba de Estudio Conceptual
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