Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. ABSTRACT The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC50) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.

The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir

Influenza A viruses (IAVs) of the H9 subtype are enzootic in Asia, the Middle East, and parts of North and Central Africa, where they cause significant economic losses to the poultry industry. Of note, some strains of H9N2 viruses have been linked to zoonotic episodes of mild respiratory diseases. Because of the threat posed by H9N2 viruses to poultry and human health, these viruses are considered of pandemic concern by the World Health Organization (WHO). H9N2 IAVs continue to diversify into multiple antigenically and phylogenetically distinct lineages that can further promote the emergence of strains with pandemic potential. Somewhat neglected compared with the H5 and H7 subtypes, there are numerous indicators that H9N2 viruses could be involved directly or indirectly in the emergence of the next influenza pandemic. The goal of this work is to discuss the state of knowledge on H9N2 IAVs and to provide an update on the contemporary global situation.

/pdf/h9-influenza-viruses-an-emerging-challenge-2r6r1hh29f.pdf

H9 Influenza Viruses: An Emerging Challenge.

Abstract The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with &gt; 20x increase in EC 50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Abstract Importance Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro . The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors. 

The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitorin vitroand confer resistance to nirmatrelvir

An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

/pdf/comparison-of-pathogenicity-of-subtype-h9-avian-influenza-1uudwpzx92.pdf

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

A core-shell AuNRs@Ag-enhanced and magnetic separation-assisted surface-enhanced Raman spectroscopy (SERS) immunosensing platform was developed for rapid, accurate and sensitive detection of amantadine (AMD) contaminant in animal-derived foods. This SERS immunosensing platform works in a competitive strategy, which used the 4-mercaptobenzoic acid (4-MBA) labeled AuNRs@Ag with AMD-BSA immobilized as a signal probe and magnetic beads (MBs) with mAbs covalently attached to the surface as a capture probe. It was found that the synthesized core-shell AuNRs@Ag could significantly enhance the SERS signal of the reporter 4-MBA (EF: 2.91 × 107), and the coating of Ag shell effectively avoided the shedding of 4-MBA, thereby improving the sensitivity and stability of SERS analysis. The use of MBs not only overcomes the shortcoming of mAbs inactivation, but also simplifies the detection procedure, improving the detection efficiency. This SERS immunosensing platform was demonstrated to have a linear response in a wide AMD concentration range of 0.01–50 μg/L with high sensitivity (limit of detection (LOD): 0.0038 μg/L) and specificity. For actual animal-derived foods (chicken, milk, egg), acceptable recoveries of AMD (82.0–106.0%) at three spiked levels (0.5, 5, 50 μg/kg) were obtained with relative standard deviation (RSD, n = 5) ranging from 4.7% to 9.6%. The whole detection procedure can be completed within 30 min. These results demonstrated that the proposed SERS immunosensing platform was reliable and sensitive in the quantitative analysis of AMD and could be applied as an effective tool for rapid screening for AMD-contaminated foods.

Core-shell AuNRs@Ag-enhanced and magnetic separation-assisted SERS immunosensing platform for amantadine detection in animal-derived foods

To understand the evolution and molecular characteristics of Jiangxi H9N2 viruses, we isolated 17 viruses in 2011 and analyzed their characteristics. Phylogenetic analyses revealed that their hemagglutinin genes originate from JS/1/00-like sublineage, neuraminidase genes originate from BJ/94-like sublineage, PB1, PA, NP, and NS genes all come from SH/F/98-like sublineage, PB2 genes originate from ST/163/04-like sublineage, while M genes come from G1-like sublineage. Genotype analysis showed that our isolates were classified as genotype 57. Molecular analyses indicated that our strains contained specific sites characteristic of low-pathogenic viruses. The current study once again highlights the necessity for continued surveillance of novel H9N2 viruses.

The evolution and molecular characteristics of H9N2 avian influenza viruses in Jiangxi of China.

H1N1 influenza is a kind of acute respiratory infectious disease that has a high socioeconomic and medical burden each year around the world. In the past decades, H1N1 influenza viruses have exhibited high resistance to adamantanes, which has become a serious issue. To understand the up-to-date distribution and evolution of H1N1 influenza viruses with adamantanes-resistant mutations, we conducted a deep analysis of 15875 M2 protein and 8351 MP nucleotides sequences. Results of the distribution analyses showed that 77.32% of H1N1 influenza viruses harbored-resistance mutations of which 73.52% were S31N, And the mutant variants mainly appeared in North America and Europe and H1N1 influenza viruses with S31N mutation became the circulating strains since 2009 all over the world. In addition, 80.65% of human H1N1 influenza viruses and 74.61% of swine H1N1 influenza viruses exhibited adamantanes resistance, while the frequency was only 1.86% in avian H1N1 influenza viruses. Studies from evolutionary analyses indicated that the avian-origin swine H1N1 influenza viruses replaced the classical human H1N1 influenza viruses and became the circulating strains after 2009; The interspecies transmission among avian, swine, and human strains over the past 20 years contributed to the 2009 swine influenza pandemic. Results of our study clearly clarify the historical drug resistance level of H1N1 influenza viruses around the world and demonstrated the evolution of adamantanes-resistant mutations in H1N1 influenza viruses. Our findings emphasize the necessity for monitoring the adamantanes susceptibility of H1N1 influenza viruses and draw attention to analyses of the evolution of drug-resistant H1N1 influenza variants.

Distribution and evolution of H1N1 influenza A viruses with adamantanes-resistant mutations worldwide from 1918 to 2019.

Introduction Infectious meningitis and encephalitis (ME) are life-threatening conditions are caused by various pathogens. Conventional laboratory tests with low sensitivity and specificity cannot help with early diagnosis. Methods A prospective study using the novel multiplex PCR detection for 18 pathogens of ME (MME-18) was conducted to investigate the clinical utilization and the epidemiology characteristics of ME in southwestern China. Patients with suspected intracranial infection were recruited between May and October 2019 at West China Hospital of Sichuan University. The MME-18 was used to detect cerebrospinal fluid, and conventional experiments including cryptococcal capsular antigen detection, GeneXpert, real-time PCR, and clinical feedback were used to verify the result of MME-18. Results Among 581 tested patients, 139 eligible individuals were enrolled in the study. Among them, Mycobacterium tuberculosis was the most common pathogen in mono-infection. Viruses and Cryptococcus neoformans were also frequently detected. Of 139 infected patients, 12 cases were diagnosed by MME-18 only, 57 patients by conventional testing only, and 70 cases by both comparator tests and MME-18. There were 96.3% (79/82) diagnoses made by MME-18 had a favorable outcome, and two of twelve diagnoses, made solely by MME-18, had a likely unclear clinical significance. Discussion The MME-18 showed satisfactory consistency with expert clinical consensus for patients presenting with ME. Combined with conventional testing and clinical suspicion, MME-18 may help clinicians with the early identification of pathogens.

Multiplex detection of meningitis and encephalitis pathogens: A study from laboratory to clinic

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The evolution and molecular characteristics of H9N2 avian influenza viruses in Jiangxi of China.

Distribution and evolution of H1N1 influenza A viruses with adamantanes-resistant mutations worldwide from 1918 to 2019.

Multiplex detection of meningitis and encephalitis pathogens: A study from laboratory to clinic