Rotavirus vaccine (Rotarix [RV1]) has reduced diarrhea-associated hospitalizations and deaths in Malawi. We examined the trends in circulating rotavirus genotypes in Malawi over a 22-year period to assess the impact of RV1 introduction on strain distribution.Data on rotavirus-positive stool specimens among children aged <5 years hospitalized with diarrhea in Blantyre, Malawi before  and after  RV1 introduction were analyzed. Rotavirus G and P genotypes were assigned using reverse-transcription polymerase chain reaction.P < .149; D′ P < .287). Overall, G1 (n = 268/924 [28.7%]), G2 (n = 308/924 [33.0%]), G3 (n = 72/924 [7.7%]), and G12 (n = 109/924 [11.8%]) were the most prevalent genotypes identified following RV1 introduction. The prevalence of G1P[8] and G2P[4] genotypes declined each successive year following RV1 introduction, and were not detected after 2018. Genotype G3 reemerged and became the predominant genotype from 2017 onward. No evidence of genotype selection was observed 7 years post–RV1 introduction.A rich rotavirus strain diversity circulated throughout the 22-year period; Shannon (H′) and Simpson diversity (D′) indices did not differ between the pre- and postvaccine periods (H′ Rotavirus strain diversity and genotype variation in Malawi are likely driven by natural mechanisms rather than vaccine pressure. Combined G and P genotype diversity did not significantly change following rotavirus vaccine introduction in Malawi, but P genotype diversity increased in children aged >12 months. G3 rotaviruses reemerged and replaced G1P[8] and G2P[4] as the predominant genotype from 2017 onward. There has been a notable reduction in global rotavirus diarrhea-associated mortality following vaccine introduction, from more than half a million deaths each year to an estimated 128 500 annual deaths among children <5 years of age. However, 258 million annual diarrhea episodes are still associated with rotavirus infection  rotavirus vaccine (RV1) into Malawi’s national immunization program on 29 October 2012 , there h rotaviruhttp://rega.kuleuven.be/cev/viralmetagenomics/virus-classification), of which G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] are the major global circulating strains in humans , G2P[4], G3[P4], G3P[8], G4P[8], G8P[6], G8P[8], and G12P[8] were the most frequently detected rotaviruses before and immediately following vaccine introduction ,http://www.nsomalawi.mw).We enrolled children <5 years old presenting to the QECH, Blantyre, with acute gastroenteritis . QECH is the main referral hospital in the southern region of Malawi that serves 23 government primary health centers within Blantyre city, which has a population of >1.25 million with a median age of 17.4 years according to the Malawi 2018 population census .A minimum 20% stool suspension prepared in Rotaclone kit diluent buffer was used to screen for the presence of group A rotaviruses using a commercially available enzyme immunoassay . Raw stool samples were stored at –80°C and were subsequently used to prepare 20% stool suspensions in phosphate-buffered saline for molecular assays. Rotavirus dsRNA was extracted from freshly prepared rotavirus-positive stool suspensions collected from 2011 to 2019 using the Viral RNA Mini-Kit . Rotavirus dsRNA was reverse transcribed to complementary DNA (cDNA) using random primers  and reverse transcriptase enzyme  . The cDNRotavirus genotype diversity during pre- and postvaccine periods was compared using the Simpson diversity index (D′) and Shannon diversity index (H′) . In thist test or Wilcoxon signed-rank test. The χ 2 test or Fisher exact test were used to measure associations. Indicator species analysis was conducted to identify the genotypes that were significantly associated with a given group  as the reference genotype. Model fitting included the following covariates: surveillance year (November–October), age in months, and sex. Multinomial odds ratios (MORs), 95% confidence intervals (CIs), and associated software .Ethical approval was obtained from the National Health Sciences Research Committee, Lilongwe, Malawi (number 867) and the Research Ethics Committee of the University of Liverpool, United Kingdom (000490).P = .034); however, no significant differences were observed in the overall genotype diversity index between the pre– and post–RV1 introduction periods  and after (29.6% [n = 934/3155]) RV1 introduction. The relative abundance of rotavirus genotypes varied annually . The num periods . The incWith the exception of G1 strains , that consistently circulated from 1997 to 2018, the distribution and relative proportion of other genotypes fluctuated over time . For insP < .001) and G3P[4]  was higher in the postvaccine period relative to wild-type G1P[8]. In contrast, cases due to G12P[6], G12P[8], G1P[6], G2P[4], and G2P[6] were lower in the postvaccine period relative to G1P[8], whereas cases due to mixed or other rarer genotypes were unchanged relative to G1P[8]  of infection caused by G3P[8] (aMOR, 1038 ; o G1P[8] .Among the 934 rotavirus-positive stool samples detected following RV1 introduction, 24 distinct genotypes were identified, of which G1P[8], G2P[4], G2P[6], G12P[6], G3P[4], G12P[8], G1P[6], and G3P[8] were the most common . OverallThe median age of children infected with any genotype, with the exception of genotype G9P[8], was higher after RV1 introduction . The genA rich diversity of rotavirus strains was detected among children hospitalized with diarrhea in Blantyre during >22 years of surveillance. While we observed differences in the prevalence of individual rotavirus genotypes between pre– and post–RV1 vaccine periods, overall combined G and P genotype diversity in the 2 periods was similar. Genotypes G1P[8] and G2P[4] predominated just prior to RV1 introduction and thereafter declined in each successive year until 2018, after which they were not detected. In contrast, rotavirus strains that prevailed in the 1990s reemerged post-2018, notably G3, which became the predominant genotype by the end of 2019.Unlike in most high-income countries, a wide rotavirus strain diversity circulates simultaneously in many LMICs , where aDespite genotype-specific differences in vaccine effectiveness, it seems unlikely that the changes in rotavirus genotype distributions following vaccine introduction in Malawi could be attributed to selective vaccine pressure. For example, with each successive postvaccination year, G1 and G2 rotaviruses declined in frequency and they were not detected after 2018; if the frequency of circulating strains was vaccine driven, a gradual and constant decline in the detection of G1 compared to G2 and G12 rotaviruses would have been expected. It is noteworthy, however, that changes in genotype distributions following vaccination have been observed in other settings, in particular a predominance of G2 strains following introduction of RV1 in South America, the Caribbean, Australia, and some European counties , 35, whiRegardless of the changes in genotype distributions, the overall prevalence of rotavirus has decreased substantially in all countries following rotavirus vaccine introduction. While in this study there was only a small decrease in the proportion of hospitalized diarrheal cases that tested positive for rotavirus following vaccine introduction among children aged <5 years, the greatest reduction in the prevalence of rotavirus in this population has been reported in those aged <12 months . FurtherOur study has some limitations. The diversity of genotypes that circulated prior to vaccine introduction could have been underestimated compared to the postvaccine years, as the RT-PCR assay used during the post–RV1 introduction period included additional genotype-specific primers. We did not determine the frequency of the occurrence of nonspecific PCR product or PCR artifacts resulting from primer cross-binding or mismatches, which could have potentially resulted in overestimation of the proportion of mixed infections during the postvaccine era due to additional primers included in the genotyping cocktail . It is lIn conclusion, our comprehensive rotavirus strain surveillance dataset spanning more than 2 decades demonstrates that genotype diversity did not significantly change following introduction of rotavirus vaccine. The overall variation in the prevalent rotavirus genotypes could not be attributed to vaccine introduction and most likely represents natural genotype oscillation. These data support continued use of rotavirus vaccination regardless of the circulating rotavirus genotypes. Nevertheless, continued strain surveillance is warranted to accompany ongoing vaccine impact evaluation and to monitor viral evolution.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiaa616_suppl_Supplementary_Table_1Click here for additional data file.jiaa616_suppl_Supplementary_Table_2Click here for additional data file.jiaa616_suppl_Supplementary_Figure_1Click here for additional data file.jiaa616_suppl_Supplementary_Figure_2Click here for additional data file.


Vibrio cholerae O139 was first reported in Bangladesh and India in 1992. To determine the genomic epidemiology and origins of O139 in China, we sequenced 104 O139 isolates collected from Zhejiang Province, China, during 1994–2018 and compared them with 57 O139 genomes from other countries in Asia. Most Zhejiang isolates fell into 3 clusters (C1–C3), which probably originated in India (C1) and Thailand (C2 and C3) during the early 1990s. Different clusters harbored different antimicrobial resistance genes and IncA/C plasmids. The integrative and conjugative elements carried by Zhejiang isolates were of a new type, differing from ICEVchInd4 and SXTMO10 by single-nucleotide polymorphisms and presence of genes. Quinolone resistance–conferring mutations S85L in parC and S83I in gyrA occurred in 71.2% of the Zhejiang isolates. The ctxB copy number differed among the 3 clusters. Our findings provided new insights for prevention and control of O139 cholera .Cholera caused by  Vibrio cholerae; serogroups O1 and O139 cause epidemic- and pandemic-level disease. Serogroup O139 first caused an outbreak in Bangladesh and India in 1992  plays a vital role in the management of cholera patients  gene profiles and the distribution of IncA/C plasmids to elucidate the evolution of virulence and AMR.In this study, we sequenced the genomes of 104 V. cholerae O139 isolates collected during 1994–2018 from the Zhejiang Provincial Center for Disease Control and Prevention (Zhejiang CDC). We downloaded 57 public V. cholerae genomes from countries in Asia (V. cholerae genomes from China from the European Nucleotide Archive database (https://www.ebi.ac.uk/ena) and identified them by searching for the O139 O-antigen–specific wbf gene using BLASTN version 2.9.0 (We recovered 104  in Asia  Table 1 https://www.illumina.com). We checked all input read sets for contamination by using kraken2 with a threshold of 10% for non–V. cholerae reads  by using a section of the SaRTree (https://github.com/tseemann/abricate) with the AMRFinderPlus gene database  was isolated in 1994 from the first clinical case-patient in Zhejiang but shared a common ancestor with 1 isolate from Shanghai (isolated in 1994) and was sibling to L2. We also constructed a phylogenetic tree of 349 genomes that included the 133 isolates from China without metadata  Figure 4V. cholerae virulence genes. We further confirmed by reads mapping any genomes with a negative result for any of these virulence genes. The presence of a gene was a combined result of ABRicate searches of assembled genomes and reads mapping. Four core CTX phage genes  and the repeat sequences were all present in 94 genomes. Three genomes  were all negative for these genes. The repeat sequences were not well assembled and on different contigs, whereas ctxAB, zot, and ace of 89/104 isolates were on the same contig.We searched the 104 genomes from this study by using ABRicate with our custom database of 67 known Vibrio pathogenicity island genes, 18 genes on 2 Vibrio seventh pandemic islands, and 19 type VI secretion system–related genes. All but 1 genome contained the intact repeats-in-toxin gene cluster.  (ctxB. Most C2 isolates (81.4% [48/59]) carried >2 copies (average 5 copies) of ctxB. In contrast, only 3 isolates (10.7% [3/28]) in C3 carried >1 copy (average 2.6 copies), and 6 isolates (54.5% [6/11]) in C1 carried 2 copies. Six outlier isolates carried multiple copies of ctxB (range 2–22 copies). All 101 ctxB–positive isolates contained ctxB genotype 3.Because a strain may contain multiple copies of the CTX phage ( copies)  Table 2,varG and catB9 in all isolates. floR, dfr18, sul2, aph(3′′)-lb, and aph-ld were in most of the isolates, including international isolates, and 8 AMR genes detected only in cluster 3 were present at low frequencies (3.13%–15.63%) -la, mph(E), msr(E), sul, dfrA12, tet(M), and tet(Y) only in isolates from China, including Shanghai isolates, whereas blaCMY-2, blaOXA-1, catB3, aac(6′)-lb-cr5, aadA3, ere(A), mph(A), mph(F), qnrA1, qnrA7, aar-3, dfrA27, dfrA32, tet(A), and tet(D) were in Zhejiang isolates only (aph(3′)-Ia and sul1 were common to C2 and C3, aacII, aadA2, tet(D), mph(E), msr(E), blaTEM-1, and catA2 were more common in C2, and tet(M), mph(A), and dfrA12 were more common in C3.We found the chromosomally encoded resistance genes –15.63%) . We fountes only  Table 4.parC and Ser83Ile in gyrA. Ten isolates had a mutation in Asp87 of gyrA, of which 6 had Asp87Tyr, 3 had Asp87Gly, and 1 had Asp87Asn.We also searched these isolates for quinolone-resistance mutations. Seventy-four isolates (74/104 [71.2%]) harbored mutations Ser85Leu in MO10 and ICEVchInd4) -Ib, aph-Id, and sul2, were probably carried by the ICE.We analyzed the integrative and conjugative elements (ICEs) carried by our isolates and compared them with the 2 known ICE variants in O139 (SXTVchInd4)  Figure 5V. cholerae plasmid pVC1447 of 160 kb , tet(R), mel, qacEdelta1, and folP genes , msr(E), and blaTEM. Seven C2 isolates lost >1 of the AMR genes. Isolate V29 and V30 lost sul1, tetD, mph(E), and msr(E) genes. The 2 C2 isolates without the pVC1447-like plasmid did not contain any of the pVC1447 AMR genes. All except 4 C3 isolates carried an IncA/C plasmid; 17 had the IncA/C_1_FJ705807 replicon type, and 5 had the IncA/C2_1_JN157804 replicon type. We further determined that the IncA/C_1_FJ705807 type plasmid is a novel plasmid that was most closely related to Aeromonas veronii plasmid p158496 (V. cholerae O139 pVC211 (GenBank accession no. KY399978.1). The p158496-like plasmid in the 17 C3 isolates shared an average nucleotide identity of 97.7% and length coverage of 82.89% with the 158 kb p158496 and probably carried aadA2, tet(D), tet(M), mph(A), dfrA12, and sul1 genes. However, more than half of these C3 isolates lost the tet(D) gene. Two outlier isolates (V17 and V18) also carried the p158496-like plasmid. The pVC211-like plasmid in the 5 C3 isolates shared an average nucleotide identity of 99.09% and length coverage of 92.46% with the 148 kb pVC211 plasmid and probably carried aadA16, tet(A), mph(A), dfrA27, qnrA1, and arr-3 resistance genes. Some isolates had further loss and gain of AMR genes.Eighty-three isolates carried an IncA/C plasmid. We found 2 IncA/C subtypes   Table 4.V. cholerae O139 isolate in Zhejiang Province was reported in September 1994, which was 16 months after the first O139 case reported in China  and tet(Y) were present only in isolates in China, and tet(A) and tet(D) were only present in Zhejiang isolates. tet(M) was found in 65.6% of C3 isolates; some C3 isolates carried both tet(D) and tet(M). Previous studies found that O139 isolates from 1991–2013 in Thailand and from 1997 in India were susceptible to tetracycline  was present in 68.8% of C3 isolates only. mph(A) conferring azithromycin resistance is plasmid-borne and rarely found in V. cholerae (mph(A) was first identified in an isolate in 1998, only 10 years after azithromycin was first used for treatment. The high percentage of mph(A) in O139 C3 isolates in this study is concerning. However, because cholera was relatively infrequent in China, the acquisition of such resistance may not be attributable to selection pressure from clinical antimicrobial treatment.C2 and C3 shared 2 nonsynonymous mutations, 1 each in the genes encoding for penicillin-binding protein 2 and a lytic murein transglycosylase with affinity to β-lactam antibiotic resistance , whereas C3 isolates acquired 2 different IncA/C plasmids, Although O139 spread to China in 1993  and mph(A) were only detected in C3 and dfrA12 was mainly present in C3. In addition, the AMR genes present at low frequency in different C3 isolates may have also collectively contributed to C3’s fitness. However, 63% of C2 isolates simultaneously carried blaTEM1, catA2, aac-lld, aadA2, aph(3′)-la, sul1, mph(E), and msr(E) genes, a pattern not present in other clusters. The epidemiologic pattern uncovered raises many interesting questions, most notably regarding what advantage did subsequent clusters have over their predecessor. C2 and C3 carried more resistance genes than C1. Although we have no AMR phenotypic data, difference in their AMR gene profiles suggests that AMR may have been the driver that caused C1 to be replaced by C2 and C3. C2 overall carried more copies of The persistence of each cluster for many years in Zhejiang is also intriguing. The clusters possibly were circulating in other parts of China and spread to Zhejiang. Most of the other isolates in China fell into C2, and Shanghai isolates were shown to be ancestral to some Zhejiang isolates within C2 and C3  Figure 2V. cholerae O139 isolates in Zhejiang grouped into 3 major clusters, which were probably derived from multiple independent importation events directly or indirectly from other countries in Asia and prevailed over the period 1994–2018, with one cluster replacing another sequentially. Variations in AMR gene content or resistance mutations suggest that acquisition of AMR probably has played a role in the succession of the V. cholerae O139 clusters in Zhejiang.This study describes the possible origin, evolution, and spread of O139 cholera in a single province, Zhejiang. Further studies are required to expand this analysis to the national level. Most Vibrio cholerae O139, Zhejiang Province, China, 1994–2018.Additional data for study of genomic epidemiology of Vibrio cholerae O139, Zhejiang Province, China, 1994–2018.Additional information about study of genomic epidemiology of


Recent studies unveiled a potential link between these two systems, implicating the potential mechanism of coordinated transcriptional and post-transcriptional regulation in porcine prenatal myogenesis and postnatal skeletal muscle growth.N6-methyladenosine  to identify the co-expression patterns of m6A and 5mC regulators and their potential role in pig myogenesis. Whole-genome bisulfite sequencing (WGBS) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were performed on the skeletal muscle samples from Landrace pigs at four postnatal growth stages .Immunofluorescence and co-IP assays were carried out between the 5mC writers and m6A writers and co-occurrence of 5mC and m6A modification were revealed from public datasets of C2C12 myoblasts. The protein-protein interactions between the DNA methylase and the m6A methylase were observed in mouse myoblast cells. Further, by analyzing transcriptome data comprising 81 pig skeletal muscle samples across 27 developmental stages, we identified a 5mC/m6A epigenetic module eigengene and decoded its potential functions in pre- or post-transcriptional regulation in postnatal skeletal muscle development and growth of pigs. Following integrative multi-omics analyses on the WGBS methylome data and MeRIP-seq data for both m6A and gene expression profiles revealed a genome/transcriptome-wide correlated dynamics and co-occurrence of 5mC and m6A modifications as a consequence of 5mC/m6A crosstalk in the postnatal myogenesis progress of pigs. Last, we identified a group of myogenesis-related genes collaboratively regulated by both 5mC and m6A modifications in postnatal skeletal muscle growth in pigs.Significantly correlated expression between 5mC writers and mOur study discloses a potential epigenetic mechanism in skeletal muscle development and provides a novel direction for animal breeding and drug development of related human muscle-related diseases.The online version contains supplementary material available at 10.1186/s40104-022-00791-3. The skeletal muscle is the largest organ of mammals and is crucial to the metabolism and energy balance of the whole body . Disrupt6A) are two typical epigenetic modifications that play essential roles in transcription and post-transcription regulation, respectively [6A regulators, which include a group of proteins responsible for 5mC and m6A modification addition (writers), identification (readers) and removal (erasers) from DNA or RNA sequence, based on analyzing the gene expression profiles of 11,080 samples from 33 kinds of human cancers in the TCGA database [6A modifications in the genome of tomatoes [The spatiotemporal expression of gene functions in skeletal muscle development and growth is tightly regulated by dynamic epigenetic modifications , 8. DNA ectively –11. Receectively , 13, micectively , 15, cirectively , 17, DNAectively , 18. Theectively , 20. Intdatabase . Anothertomatoes . These s6A) regulation of hub genes of porcine skeletal muscle development and growth are tightly coordinated via protein-protein interaction of 5mC and m6A methylases. To test this, we first demonstrated the genome/transcriptome-wide association between 5mC and m6A modification as well as their regulators’ expression, following with co-IP and immunofluorescence experiments to confirm the interaction between 5mC and m6A methylases, in the C2C12 myoblasts. Next, we evaluated the key 5mC/m6A regulators that played an important role in regulating prenatal and postnatal skeletal muscle development and identified a group of essential target genes whose transcription and translation are coordinately regulated by these regulators. Our study uncovered a novel epigenetic mechanism in skeletal muscle development and provided novel directions for investigating animal breeding, muscle biology, and related human diseases.Previously, we have characterized DNA methylome dynamics of 27 developmental stages in pig skeletal muscle development and revealed the involvement of the DNA methylation/SP1/IGF2BP3 axis in skeletal myogenesis . In addi2 at 37 °C. For co-IP assays, the cells were transferred to DMEM containing 2% horse serum  and induced to differentiate for 4 d.For immunofluorescence experiments, C2C12 cells were cultured in DMEM supplemented with 10% FBS  in a humidified incubator with 5% COC2C12 cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After permeabilization with 0.25% Triton X-100 for 10 min, the cells were blocked by using 1% bovine serum albumin for 30 min. Then, the cells were incubated overnight with Anti-METTL3 antibody , and Anti-DNMT3A antibody ,or Anti-DNMT3B antibody  diluted in PBST containing 1% bovine serum albumin at 4 °C. The cells were incubated with Alexa Fluor 647 donkey anti-mouse IgG (H + L),  or Alexa Fluor 488 donkey anti-rabbit IgG (H + L)  for 1 h at room temperature. Nuclei were stained with DAPI for 1 min. The cells observed microscopically using an A1HD25 confocal microscope .The differentiated C2C12 cells lyzed according to the instructions of the Co-Immunoprecipitation Kit . Anti-Mettl3 antibody (ab195352), Anti-METTL14 antibody (ab252562), Anti-WTAP antibody (ab195380), Anti-DNMT1 antibody (ab19905), Anti-DNMT3A antibody (ab228691) and Anti-DNMT3B antibody (ab227883) were used for IP . Nonspecific IgG antibodies  precipitated the complex as a control group. Total proteins from C2C12 cells were extracted and incubated overnight with IgG or the Co-IP antibodies (5 μg) at 4 °C, while the extracted proteins without any antibodies were used as Input control. Next, 30 μL of Protein A/G PLUS-Agarose  was added and incubated for 2 h at 4 °C to form an immune complex. Following centrifugation for 4 min at 3000 r/min at 4 °C, the Protein A/G Plus-Agarose beads were washed 4 times with 1 mL of lysate and boiled in the appropriate protein loading buffer for 5 min. Centrifugation at 3000 r/min again, the supernatants were collected to a new tube for Western blot analysis.The total proteins from C2C12 cells were lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail . The membranes were blocked with 5% BSA for 1 h at room temperature and subsequently probed with primary antibodies overnight at 4 °C. the following dilutions were used for each antibody: METTL3, METTL14, WTAP, DNMT1, DNMT3A and DNMT3B . The membranes were then washed with PBS-Tween and incubated for 50 min with horseradish peroxidase-conjugated secondary antibodies . Protein bands were detected after treatment with the SuperSignal west Femto agent .longissimus dorsi, LD) samples were collected from Landrace pigs at four developmental stages, including postnatal days 30, 60, 120, and 180 , as we previously reported [The skeletal muscle  combined with protein A beads  at room temperature for 1 h in immunoprecipitation purification (IPP) buffer . Bound mRNA was eluted from the beads with 0.5 mg/mL of m6A in IPP buffer. Eluted mRNA was precipitated using an ethanol-sodium acetate solution and glycogen  overnight at − 80 °C. mRNA was resuspended in water and used for library generation. Input mRNA and IP mRNA sequencing libraries were generated using a Vazyme mRNA-seq Kit for Illumina . Sequencing was performed using an Illumina HiSeq X Ten system .Total RNA was immediately isolated using Trizol reagent according to the manufacturer’s instructions . All samples were treated with DNase  to avoid DNA contamination. Profiling of mus study . For iso6A-modified RNA regions (m6A peaks) and differentially m6A methylated peaks were identified using exomePeak software (version 2.16.0) [6A peaks identified in two biological replicates were merged and regarded as highly enriched and selected for further analysis .For each sample, paired-end reads were used for bioinformatics analysis. Raw data quality control was performed using FastQC software (version 0.11.3). Sequencing data were trimmed using Trimmomatic (version 0.36)  with def 2.16.0) , 28. m6A6A regulators and epigenetic module eigengenes (EME) identification were described in Chen et al.’s paper [6A regulators for pig skeletal muscle, we introduced the concept of “module” from the weighted gene co-expression network analysis (WGCNA) algorithm and treated the 41 5mC and m6A regulators as a module [6A regulator and the module eigengene. Hub 5mC/m6A regulators were then defined as those that achieved a module membership greater than 0.7. The summary expression level of the identified hub 5mC/m6A regulators was again calculated as EME for pig skeletal muscle.Methods for hub 5mC/m’s paper . To idena module . The over > 0.8 and P-value < 0.05 were regarded as EME correlated co-expression modules for further analysis.Genes with the top 25% variant RNA expression level among all the 81 pig skeletal muscle samples were selected for co-expression analysis. Then we identified the co-expression modules by the WGCNA algorithm with default parameters. Correlation analysis was conducted between the EME vector and the identified co-expression modules. Modules with 6A interaction effects in the 80 up-regulated genes in the postnatal stages, we recruited the general linear model (GLM) using the postnatal days, 5mC methylation level, enrich score of m6A peaks and 5mC/m6A interaction item as explanatory variables:To find out 5mC/mS denotes the postnatal days, X is the enrich score of an m6A peak, Y is the 5mC methylation level of the m6A peak, X × Y is the interaction item, and ε is the random variable. Additionally, the enrich score of a specified m6A peak is calculated as:Here, Where:A is the read counts mapped to a specified peak region in a sample, and ∑A is the total mapped read counts in a sample. Finally, regions or genes with a P-value < 0.05 for the interaction item were selected as of 5mC/m6A modification interaction effect regions or genes. All the fitted GLM were constructed by the lm function in R.Here, IP is the MeRIP sample and Input is the corresponding control, In addition, we identified the protein-protein interactions among the co-expression genes in the EME negative correlated module based on the STRING database .P-value < 0.05 were considered nominally significant.Gene list enrichment analysis was performed using the online tool Enrichr . Enrichm6A writers  were extracted and followed with Pearson’s correlation analysis. The RRBS and MeRIP-seq data of C2C12 cells were also downloaded from GEO datasets with accession numbers GSE131842 and GSE154720, respectively. The CpG methylation matrix and the m6A peak tables were directly used in the further co-enrichment analysis. For 5mC/m6A co-occurrence analysis, we split the upstream 5 kb, the m6A peak region, and the downstream 5 kb sequence into 20, 10, and 20 bins with the same length, respectively. Then the bins covered with no less than 20 CpG sites were selected for average methylation level calculation and visualization. In total, 81 RNA-seq data from 27 prenatal and postnatal developmental stages  and 8 WGBS data from the four postnatal developmental stages  were obtained from a previously in-house study on pig skeletal muscle development [The RNA-seq data of C2C12 myoblasts were downloaded from GEO datasets (GSE131842). Expression levels of DNA 5mC writers  and RNA melopment . The who6A writers, we checked the patterns of expression correlation between DNA 5mC writers  and RNA m6A writers  in mouse myoblast cells (C2C12) after differentiation for 0, 12, 24, 48 and 60 h . Positive correlations were unveiled between the mRNA level of 5mC and m6A writers  and found a significantly higher methylation level in the m6A peaks than in the up- or down-stream regions  by Western blot analyses , while the Midnightblue module shows a significantly negative correlation . These results reveal that a group of genes might be under coordinated pre and post-transcriptional regulation by 5mC/m6A crosstalk. The expression heatmap indicated that these genes are highly divergent between prenatal and postnatal development and may be essential to embryonic or postnatal skeletal muscle development  . The DNA methylome data was generated in the previous study [To further address the 5mC and mus study . In the 6A modifications were enriched in CDS and 3’UTR regions, especially in the TSS site  and the m6A modified gene number (5324–7003 genes) showed a slight increase in postnatal 60 d and then a persistently decrease onto the postnatal 180 d  and erasers (ALKBH5 and FTO) reveals that the global m6A dynamic may be mainly driven by the cooperated regulation of METTL3 and ALKBH5 . In addition, we also observed significantly higher DNA methylation levels inside the m6A peaks than in the upstream and the downstream regions  to fit the 5mC/m6A interaction effects on mRNA expression for the Midnightblue module genes in the postnatal stages, under the hypothesis that the observed mRNA levels are affected by both 5mC and m6A modifications . Intriguingly, five of these genes  are involved in myogenesis and three of them  are involved in the glycogen metabolic process . Several previous studies indicated the essential roles of 5mC [6A regulation [6A methylation landscape during prenatal skeletal muscle development and revealed essential roles in the regulation of myogenesis in another recent study [6A) regulation of hub genes is coordinated via protein-protein interaction of 5mC and m6A methylases, and disclosed a cooperative regulation of 5mC and m6A modifications on spatiotemporal gene expression in myogenesis.Nucleotide methylation carries essential information for gene regulation, especially DNA 5-cytosine methylation (5mC) and RNA N6-adenosine methylation , MyHC IIa (fast-oxidative), MyHC IIx (intermediary to MyHC IIa and MyHC IIb), and MyHC IIb (fast-glycolytic) [The potential association of 5mC and mpression . Howevercolytic) . In pigscolytic) . After tcolytic) . Our res6A methylases  is a structural component of the sarcomeric Z line and is related to sarcopenia and physical fitness inactive older women [PHKB (phosphorylase kinase regulatory subunit beta) is involved in glycogen metabolism and associated with glycogen storage disease IXb, a disease also known as phosphorylase kinase deficiency in liver and muscle [Moreover, immunofluorescence colocalization and co-IP experiments confirmed the interaction between 5mC and mses Fig. . We furtl stages . These rocuments , 39. Theer women , 41. PHKd muscle .6A methylases, either directly or indirectly. We speculated that proteins like the histone deacetylase HDAC2 and the E3 ubiquitin protein ligase UHRF1 might act as adaptors to guide the interactions, as previous studies indicated they both could interact with the 5mC and m6A writers separately  EME positive correlated Lightyellow module and its gene expression heatmap. (B) GO process enrichment analysis of Midnightblue module genes. Fig. S5. Protein-Protein Interaction network among the Midnightblue module genes. Fig. S6. Distribution of m6A peaks across the whole transcript. Fig. S7. Dynamics of the DNA methylation during postnatal skeletal muscle development in pigs. (A) mRNA expression profiles of 5mC writers (DNMT1/DNMT3A/DNMT3B) and erasers (TET1/TET2/TET3). (B) Numbers of 5mC hypermethylated or hypomethylated genes from inter-stage comparisons (C) Top 5 GO terms enriched by the inter-stage differentially 5mC modification genes. Fig. S8. Possible indirect interactions between 5mC writers and m6A writers. (A) Protein-protein interaction network shows the proteins interacted with 5mC writers (DNMT1/3A/3B) in human beings. (B) Experiment evidence found in the published documents indicated the linker protein (UHRF1 and HDAC1) could interact with the m6A MTC proteins.Additional file 2: Table S1. Summary of MeRIP-seq data in this study. Table S2. Genes expressed under significant 5mC and m6A interaction effects in postnatal skeletal muscle development. Table S3–S6. RNA expression data were used in this study. Table S7–S10. Consensus m6A peak identification of the four postnatal stages by exomePeak.


NOC2 like nucleolar associated transcriptional repressor (NOC2L) was recently identified as a novel inhibitor of histone acetyltransferase (INHAT). NOC2L is found to have two INHAT function domains and regulates histone acetylation in a histone deacetylases (HDAC) independent manner, which is distinct from other INHATs. In this review, we summarize the biological function of NOC2L in histone acetylation regulation, P53-mediated transcription, ribosome RNA processing, certain development events and carcinogenesis. We propose that NOC2L may be explored as a potential biomarker and a therapeutic target in clinical practice.The online version contains supplementary material available at 10.1186/s12967-023-03877-2. In the nucleus of cell, the acetylation and de-acetylation of histone is in a dynamic balance, which precisely regulates gene transcription and expression , 2. HistNOC2 like nucleolar associated transcriptional repressor (NOC2L) is an inhibitor of histone acetylation –11. SpecAs a novel HDAC-independent INHAT, NOC2L provides a novel insight into the investigation of histone acetylation. In order to delineate the biological function of NOC2L, further studies were conducted to investigate NOC2L-targeted proteins and the underlying regulation mechanisms. NOC2L has been demonstrated to target TP53 , 10, 11,NOC2L  is also known as NIR, NET7, NET15 and PPP1R112. NIR is commonly used as the synonyms of NOC2L. NOC2L was first identified by Hublitz et al. in an attempt to find novel INHAT by conducting low-stringency iterative analyses of the Basic Local Alignment Search Tool (BLAST) to screen databases for proteins containing putative INHAT regions. A cDNA coding for an unknown protein was identified and its human full-length sequence was obtained by PCR from a prostate cDNA library [As a protein coding gene, NOC2L gene is located on chromosome at 1p36.33 and contains 19 exons with an open reading frame including 2757 nucleotides, according to the BLAST analysis.NOC2L protein is 84,919 Da and contains 749 amino acids in human, and its orthologs range from worms to mammals (NCBI Reference Sequence: NP_056473.3). NOC2L is located at the nucleolus primarily, as is indicated in the Human Protein Atlas (HPA) database . HoweverNOC2L is wide and ubiquitous, as indicated in the Genotype-Tissue Expression (GTEx) database  because P21 promoter has a higher P53 occupation level than other P53 target genes, such as NOXA and PIG3 [NOC2L knockdown, increased apoptosis was observed in P53-containing cells but not in P53-deficient cells [Hublitz et. al further investigated the consequence of t on P53 . NOC2L cand PIG3 . This P5nt cells , 10, 11.Before the identification of NOC2L, other INHATs have not been demonstrated to physically interact or functionally associate with P53 , 17, 21,P53 is regulated by multiple proteins, which can influence the function of P53 –26. SincAURKB), a highly expressed serine and threonine protein kinase [During the investigation of NOC2L’s biological function, Aurora kinase B  was found to bind directly to MDM2 in the central acidic and zinc finger domain (amino acids 222-366) and the N terminus (amino acids 1-122) of MDM2 . PreviouThese above findings raised the possibility that NOC2L, P53 and MDM2 may constitute a ternary complex like the previous proposed P53-NOC2L-Aurora B model , 8, 42. As a result of the NOC2L association with MDM2, NOC2L facilitates MDM2-mediated P53 repression by sustaining the stability of MDM2 itself and the MDM2-P53 complex, which further represses the P53-mediated transcriptional activation significantly .As the investigation of NOC2L-mediated P53 regulation continued, scientists turned their attention to the role of NOC2L on other proteins homologous with P53 . P63 resP21 which is targeted by P63 [In addition to the P53-dependent tumor suppression function mentioned above, P63 shows significant ability to regulate epithelial development including cell growth and differentiation . P63 cond by P63 .P21 promoter region, resulting in the transcriptional inhibition of downstream genes in a P53 resembling way [NOC2L was verified to physically interact with TAp63 and affect its transcriptional activity in a negative way . The binling way . Throughling way .In summary, NOC2L can regulate TAp63 activity negatively in a cell-cycle controlled manner . Just asBased on the knowledge of the biological properties and function of NOC2L, further investigation was conducted to examine how NOC2L affects biological events. It has been proposed that NOC2L can participate in some key biological events, including the rRNA biogenesis , embryonAs mentioned previously, NOC2L predominantly exits in the nucleolus , 11. HowNOC2L-knockdown downregulated the levels of these rRNAs and other associated pre-rRNAs [Wu et. al confirmed that NOC2L is indeed involved in the processing of the 40S and 60S subunits of ribosome . They fire-rRNAs . This obre-rRNAs . Meanwhire-rRNAs , leadingre-rRNAs . Upon fure-rRNAs . Since Ure-rRNAs , NOC2L ire-rRNAs , 52, 55.re-rRNAs .As indicated before, NOC2L has been recognized as a transcriptional repressor –11. HereNOC2L-knockout mice all died during embryonic development [NOC2L has been recognized as a P53 suppressor , while Pelopment . Based oWhen cells undergo physiological DNA breaks during V(D)J recombination of lymphocytes, P53 is required to be suppressed temporarily in order to ensure the differentiation of T and B cells . Since NNOC2L generated [NOC2L deficient mice have a remarkable decrease in both double positive (DP) and single positive (SP) T lymphocytes while an increase in double negative (DN) T lymphocytes [P53 knockdown, the defect described above partly but not fully recovered [Mice bearing lymphoid-restricted deletion of enerated . Resultsphocytes . Scientiphocytes . During phocytes . During phocytes . With thphocytes . Upon P5ecovered , suggestecovered , 50.In conclusion, this study indicates that NOC2L participates in early T-lymphocyte development by repressing P53 . In addiAnother important target of NOC2L is P63, as mentioned before . As a clNOC2L conditional knockout (CKO) mice were generated for further investigation [NOC2LCKO mice died at birth [During the development of murine fetus, NOC2L is observed to express from embryonic day12.5 to postnatal day0 in all epidermal cells, suggesting its importance in epidermal development . Followitigation . As a reat birth . The epiat birth . Furtherat birth .NOC2LCKO mice, it is notable that they are similar to the phenotype of P63 deficiency [NOC2LCKO mice may be contributed to abnormal regulation of P63 [GATA3 were both demonstrated to be downregulated during embryogenesis [NOC2L deficiency leads to the incapacity to enter asymmetric cell division and complete stratification process [Taken together the above-mentioned defects observed in ficiency . In thisn of P63 . Upon fuogenesis . Furtherogenesis . Since Pogenesis , downreg process .NOC2LCKO mice, it was further confirmed that the abnormalities in epidermis development were partially contributed to P53 hyper-acetylation [NOC2L-deficient epidermal cells experienced impaired cell proliferation and enhanced apoptosis [Besides the investigation of how NOC2L regulates epidermis development through P63, the role of P53 was also considered. As mentioned before, NOC2L can inhibit the acetylation of P53 , therefotylation . Since bpoptosis .In conclusion, NOC2L plays an essential role in regulating epidermis development by association with both P53 and P63.NOC2L knock-out has been found to significantly repress cell proliferation in the goal of the Dependency Map (DepMap) database [As a negative regulator of tumor suppressor P53 , NOC2L mdatabase , indicatNOC2L between cancer and cancer adjacent tissues [NOC2L increased in most cancer tissues than their counterpart, suggesting that it may be an oncogene  database was exploited to analyze the expression of  tissues . We founene Fig. . FurtherTCGA database and human colorectal cancer (CRC) tissues analysis both indicate that NOC2L is upregulated in CRC. Furthermore, the level of NOC2L is significantly related to the poor outcome of CRC patients. These evidences imply that NOC2L may play an important role in the tumorigenesis and progression of CRC .Retinoblastoma protein (RB) is another key tumor suppressor and its downregulation or inactivation have been demonstrated to facilitate CRC –69. NOC2In both cell and mouse xenograft, NOC2L has been demonstrated to promote tumor growth and cancer cell proliferation through RB , confirmAs a common cancer among females, breast cancer is one of the most studied cancers . MultiplNOC2L knockdown, indicating an important role of NOC2L in breast cancer progression [NOC2L was demonstrated to be upregulated in breast cancer patients . Moreovegression .FOXO3) promoter region [NOC2L was found to be recruited by Enhancer of zeste homolog 2 (EZH2) and cooperate with EZH2 through physical interaction to promote the methylation of H3K27 in the vicinity of forkhead box O3  between NOC2L-high and NOC2L-low patients is statistically significant in several types of cancers, including adrenocortical carcinoma, bladder Urothelial Carcinoma, head and neck squamous cell carcinoma, brain Lower Grade Glioma, liver hepatocellular carcinoma and sarcoma(p<0.05). Other types of cancers show no significant differences of OS.


Cannabis sativa L. has led to a renewal in breeding and resulted in an impressive variability of chemotypical characteristics that required the division of cannabis into different recognized chemotypes. The chemotype V has been overlooked in terms of phytochemical composition due to the almost total absence of cannabinoids, on which biomedical attention is focused. Systematic approaches addressing diverse chemotypes are, however, needed to discriminate and define phytochemical aspects beyond cannabinoids. Such thoroughly characterized chemotypes guarantee blinding in controlled studies by mimicking the sensory properties of hemp and may help to unravel the “entourage effect”. Capitalizing on the ability of cannabis to synthesize a large number of non-cannabinoid phenolic compounds, we here investigated, for the first time, the composition of the Ermo chemotype V and identified new compounds: two dihydrophenanthrenes and the methoxy-dihydrodenbinobin. All three compounds suppress pro-inflammatory leukotriene biosynthesis in activated macrophage subtypes by targeting 5-lipoxygenase, but substantially differ in their capacity to elevate the levels of specialized pro-resolving lipid mediators and their precursors in M2 macrophages. We conclude that the discovered compounds likely contribute to the anti-inflammatory properties of Cannabis sativa L. chemotype V and might promote inflammation resolution by promoting a lipid mediator class switch.The growing general interest surrounding  Cannabis sativa L. was one of the first plants ever domesticated by humans, who spread its cultivation worldwide over the past 10,000 years. Due to its considerable and heterogeneous use, the plant can be considered as a multi-purpose crop, an object of constant breeding and selection +; C17H19O5 requires m/z 303.1227.Compound 5. Red amorphous powder. C3D6O, 400 MHz) and 13C NMR : m/z 301.1066 [M + H]+; C17H17O5 requires m/z 301.1076.Compound 10. Brown amorphous powder. 1H NMR  and 13C NMR : m/z 303.1230 [M + H]+; C17H19O5 requires m/z 303.1227.Compound g, 20 min, RT) using Histopaque®-1077 . PBMC were obtained from the interphase after hypotonic lysis of erythrocytes using water (2–5 mL) and two consecutive washing steps with PBS pH 7.4 (50 mL) .Leukocyte reduction system chamber (LRSC) filters were provided by the Central Institute for Blood Transfusion and Immunological Department of Tirol Kliniken GmbH (Austria) with the informed consent of the volunteers. Only healthy blood donors between 18 and 65 years of age without medication for chronic diseases, fever, or deficiency symptoms and after physical examinations by trained medical personnel were included in the study. Human peripheral blood mononuclear cells (PBMC) were freshly isolated from LRSC filters. After dilution of the cell concentrates in PBS pH 7.4 containing 12.5 mM citrate and 14 mM glucose , immune cells were separated via isopycnic density gradient centrifugation  for 6 days in macrophage medium . These monocyte-derived macrophages were polarized for another 48 h in macrophage medium with 100 ng/mL lipopolysaccharide and 20 ng/mL interferon-γ  to obtain M1 or with 20 ng/mL interleukin-4  to obtain M2 phenotypes ,31.2) was stimulated with Staphylococcus aureus (LS1)-conditioned medium  after 15 min preincubation with DMSO (0.1%) or test compounds and stopped by addition of ice-cold MeOH (2.5 mL) containing deuterium-labeled internal standards (200 pg d8-5S-hydroxyeicosatetraenoic acid (HETE), d4-leukotriene (LT)B4, d5-lipoxin (LX)A4, d5-resolvin (Rv)D2, d4-prostaglandin (PG)E2, and 2000 pg d8-arachidonic acid ) [g, 10 min, 4 °C). The supernatant was mixed with acidified water  and loaded onto solid phase cartridges ), which were conditioned with MeOH (6 mL) and equilibrated with water (2 mL). Columns were washed with water (6 mL) and hexane  before lipid mediators were eluted with methyl formiate (6 mL). The organic phase was evaporated to dryness using a TurboVap LV  and the remaining lipids were dissolved in MeOH/water (1:1), centrifuged twice , and subjected to UPLC-MS/MS analysis. Chromatographic separation of lipid mediators was carried out at 55°C on an Acquity UPLC BEH C-18 column  using an ExionLC AD UHPLC system . The gradient of mobile phase A  and mobile phase B  was ramped at a flow rate of 0.35 mL/min from 35.6% to 84.4% B within 12.5 min followed by 5 min of isocratic elution at 97.8% B. Lipid mediators and fatty acids were analyzed in the negative ion mode by scheduled multiple reaction monitoring (detection window: 120 s) using a QTRAP 6500+ Mass Spectrometer (Sciex), which was equipped with an electrospray ionization source. The curtain gas was set to 40 psi, the collision gas to medium, the ion spray voltage to −4000 V, the heated capillary temperature to 500 °C, and the sheath and auxiliary gas pressure to 40 psi. Transitions selected for quantitation and the corresponding declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) are listed in Lipid mediator biosynthesis in human M1/M2 macrophages ) . Samples2 (2 mM) and arachidonic acid (20 µM) were added [1 (2 ng) as internal standard. Clean-Up C-18 Endcapped SPE cartridges  were conditioned with MeOH  and equilibrated with water (1 mL). After acidification (530 µL PBS plus 60 mM HCl) and centrifugation , supernatants containing lipid mediators were loaded onto the columns, which were first washed with water (1 mL) and then MeOH/water . After elution of lipid mediators with MeOH (300 µL), water (120 µL) was added and samples were centrifuged  and subjected to LC-PDA analysis. 5-LOX products, including all trans-isomers of LTB4 and 5-HETE, were separated at 40 °C and a flow rate of 0.45 mL/min on a Kinetex C-18 LC column  using a Nexera X2 UHPLC system . The step gradient of mobile phase A  and mobile phase B  was kept at 14% B for 2 min, followed by 46% B for 2 min and 90% B for another 2 min. LTB4 isomers and 5-HETE were detected with a photodiode array detector  at 280 nm and 235 nm, respectively. Lipid quantitieswere calculated by internal calibration using PGB1 as reference standard.Human recombinant 5-LOX  was pre-treated with vehicle  or test compounds for 10 min in 1 mL PBS pH 7.4, 1 mM EDTA and 1 mM ATP on ice before CaClre added . After 1p < 0.05) and data were log-transformed for statistical analysis. For multiple comparisons, repeated-measures one-way ANOVA followed by Dunnett´s post hoc test was performed. p values < 0.05 were considered statistically significant. Data were analyzed using Microsoft Excel , and statistics were performed using GraphPad Prism . IC50 values were calculated by non-linear regression (GraphPad Prism 9.3.1).Data are expressed as mean ± s.e.m. and single data from n = 4–8 independent experiments. Significant outliers were removed (Grubbs’test, Cannabis sativa belongs to the chemotype V and is almost completely devoid of cannabinoids, which makes it an excellent candidate to ensure blinding in clinical trials. We investigated, for the first time, the phytochemical profile of this fiber hemp and identified three new compounds: two dihydrophenanthrenes 9, 10 and a quinone 5 closely related to denbinobin 4. Metabololipidomics profiling showed that the three compounds target 5-lipoxygenase and suppress pro-inflammatory LT biosynthesis in activated macrophage subtypes. While each of the compounds might contribute to the resolution of inflammation, their capacity and specificity to elevate the levels of specialized pro-resolving lipid mediators and their precursors significantly differs. Taken together, our results demonstrate the potential of phytochemical and pharmacological characterization of cannabis varieties not only to explain the different biological activities of the chemotypes but also to add to our understanding of the “entourage effect” [The Ermo strain of


Glycine max. In this study, we characterized the seed microbiota of Glycine clandestina, a perennial wild relative of soybean (G. max (L.) Merr.) to expand our understanding about the effect of other storage procedures such as the periodic regeneration of seed stocks to bulk up seed numbers and secure viability on the seed microbiome of said seed. The G. clandestina microbiota was analysed from Generation 1 (G1) and Generation 2 (G2) seed and from mature plant organs grown in two different soil treatments T (treatment [native soil + potting mix]) and C (control [potting mix only]). Our dataset showed that soil microbiota had a strong influence on next generation seed microbiota, with an increased contribution of root microbiota by 90% and seed transmissibility by 36.3% in G2 (T) seed. Interestingly, the G2 seed microbiota primarily consisted of an initially low abundance of taxa present in G1 seed. Overall, our results indicate that seed regeneration can affect the seed microbiome composition and using native soil from the location of the source plant can enhance the conservation of the native seed microbiota. Research into understanding the structure, composition and vertical transmission of crop seed microbiomes has intensified, although there is much less research into the seed microbiomes of crop wild relatives. Our previous study showed that the standard seed storage procedures  can influence the seed microbiome of domesticated  The plant microbiome consists of a multitude of microbes that have important functions in enhancing the health and productivity of the host plant in their natural environments . Plant oPlant seed provides a stable environment for a microbial community protected by the seed coat  and enabPlant domestication and intense agricultural practices have resulted in variations in the composition of the inherent microbiome from wild crop relatives, usually with a loss of microbial diversity in the domesticated crop ,17. ThisSeed Vaults conserve the seed germplasm of numerous crop cultivars and their closest wild relatives for future crop improvement and essential scientific research work following International Genebank guidelines by acting as a secondary backup for every seedbank in the world . There iGlycine clandestina, a perennial wild crop relative of the domesticated soybean, Glycine max (L. Merr)  (G. clandestina plant organs [G1 (T and C) plant organs ]; and G1 seed, [G1 (T and C) plant organs ]  by exporting the grouped rarefied feature table at the genus level to determine the number of shared and unique genera across G1 (T and C) plant organs; and G1 seed and G1 (T and C) plant organs.The sequencing data were processed using Qiime2 as per Chandel et al.  with theC) seed]  and 11722 seed)] . Also, VG. clandestina seed [G1 and G2 (T and C) seed] and 371,487 sequences were assigned to 822 ASVs for microbiome profiling of G. clandestina plant organs [G1 plant organs and G2 (T and C) seed]. After rarefaction and collapsing biological replicates, the ASV table was assigned to 163 genera for microbiome profiling of G. clandestina seed  for microbiome profiling of ina seed  and 412 t organs .G. clandestina seed microbiome, the samples were grouped by treatment, and alpha and beta diversity analyses were performed. Alpha diversity comparison based on observed features showed significant  variations in bacterial diversity. The observed features were significantly (p < 0.05) higher in G2 (T) than G2 (C) seed, while no significant differences were observed between G1 seed and G2 (T), nor G1 and G2 (C) seed (p = 0.001) between G1 and G2 (T and C) seed, the ANOSIM results explained the higher proportion of the variance between microbiome composition of G1 and G2 (T) seed , G1 and G2 (C) seed . When variance was compared between the G2 (T) and G2 (C) seed, the variation  was lessened, but still significant  seed . For β-dnificant .Gammaproteobacteria (93.9%), Actinobacteria (1.8%) and Alphaproteobacteria (1.8%), as opposed to Bacilli (83.5%), Gammaproteobacteria (11.5%) and Alphaproteobacteria (1.61%) in G2 (T), with Bacilli (78.0%), Gammaproteobacteria (19.8%) and Actinobacteria (1.3%) in G2 (C), respectively  seed. For instance, in G1 seed, the three most dominant classes were ectively .Massilia (58.5%), Pseudomonas (30.3%), Uliginosibacterium (2.1%) and Comamonadaceae (1.5%) as opposed to Paenibacillus (71.2%), Cohnella (9.3%), Pantoea (4.4%), Uliginosibacterium (3.3%) and Bacillus (2.9%) in G2 (T) and Paenibacillus (73.6%), Pseudomonas (14.8%), Pantoea (3.9%), Cohnella (3.1%) and Nocardioides (1.2%) in G2 (C) seed, respectively  seed. For instance, in G1 seed, the five most dominant genera were ectively .p < 0.05) higher in G2 (T) seed than in G2 (C) seed, respectively , Comamonadaceae  , Burkholderia , Rhodanobacter , Pedosphaeraceae , Asticcacaulis , Micropepsaceae , Actinoplanes , Pseudolabrys , Hephaestia , Mucilaginibacter  and Thuera  (Paenibacillus increased significantly (p < 0.05) in G2 (T and C) seed at the expense of most dominant genera Massilia (58.5%) which declined significantly (p < 0.05) in G2 (T and C) seed  seed  and G2 (C) seed across two soil treatments were identified using pairwise comparison (Tukey test) of ASVs greater than 1% and less than 1% but greater than 0.1% relative abundance, respectively. In total, there were two (>1%) and 11 ASVs  that were significantly  A, Strept0.01654) B. Intere C) seed A. Additi C) seed B.H = 85, p = 1.30 × 10−15) between plant organs. For G1 (T) plants, the observed features were significantly (p < 0.05) higher in the root than the above-ground organs  seed). No significant differences were observed among the above-ground organs  seed (p < 0.05) higher in the root than in the above-ground organs  seed). In contrast to G2 (T), significant differences were observed between the shoot and G2 (C) seed, and the leaf and G2 (C) seed (p = 0.001) separation for the bacterial composition of plant organs within and across treatments G2 (T and C). However, no significant separations were observed between leaf and shoot within and across the treatments for G2 (T and C)  seed . For G1 (C) seed . When plT and C) .G. clandestina microbiome across G1 plant organs  was found to be influenced by soil type. The most prominent bacterial class in G1 plant organs, except G2 seed in each treatment, was Gammaproteobacteria, accounting for 48.5–92.8% of the ASVs abundance (Bacilli in the G2 seeds of both treatments (G1 (T and C), which accounted for 78–83.3%, whilst in other organs, Bacilli only accounted for 0.2–2.4%. In G1 (T) plants, roots were the most microbially-diverse plant organs, accounting for 44 classes, followed by G2 seed, accounting for 37 classes. Shoot and leaf microbiomes comprised of 29 and 26 classes, respectively. In G1 (C), roots also dominated the microbiome with 43 classes followed by shoots consisting of 38 classes, with leaf and G2 seed accounting for 33 and 25 classes, respectively (Gammaproteobacteria (93%), “Unclassified Bacteria” (1.9%), Alphaproteobacteria (1.4%), Actinobacteria (1.3%) and Verrucomicrobiae (0.7%). The G1 (T) shoots contained Gammaproteobacteria (84.7%), “Unclassified Bacteria” (5.8%), Bacilli (2.3%), Actinobacteria (2.2%) and Alphaproteobacteria (1.9%). Whilst G1 (T) roots were comprised of Gammaproteobacteria (48.5%), Alphaproteobacteria (22.6%), Verrucomicrobiae (6.1%), Actinobacteria (4.7%) and Bacteroidia (3.9%). Comparatively, G2 (T) seed was dominated by Bacilli (83.3%), Gammaproteobacteria (11.2%), Alphaproteobacteria (1.6%), Actinobacteria (1.2%) and Verrucomicrobiae (0.6%) (Gammaproteobacteria (83.2%), Alphaproteobacteria (5.3%), “Unclassified Bacteria” (4.8%), with Bacilli (1.5%) and Actinobacteria (1.5%) making up the balance. The distribution in G1 (C) shoots was Gammaproteobacteria (74.7%), “Unclassified Bacteria” (9.3%), Alphaproteobacteria (4.1%), Actinobacteria (3.8%) and Bacilli (2.5%); while in G1 (C) roots, Gammaproteobacteria (58.6%), Alphaproteobacteria (16.3%), Clostridia (5.7%), Verrucomicrobiae (4.6%) and Actinobacteria (2.9%) were prevalent. As mentioned previously, G2 (C) seed was dominated by Bacilli (78%), with the remainder of the class distribution being Gammaproteobacteria, (19.7%), Actinobacteria (1.3%), “Unclassified Bacteria” (0.3%) and Alphaproteobacteria (0.3%)  . For G1 a (0.3%) .The Venn diagrams displaying ASVs distributed by plant organs demonstrated that more unique genera were associated with the roots of G1 plants for both treatments with 96 genera in G1 (T) and 88 genera in G1 (C) plants. There was a reduced number of organ specific bacterial ASVs for upper plant organs, especially in the leaf (nine genera), shoot (15 genera) and G2 seed (14 genera) for G1 (T), and leaf (10 genera), shoot (27 genera) and G2 seed (five genera) for G1 (C) plants. There were 70 genera shared between all plant organs in both G1 (T) and G1 (C) . In totaThe Venn diagrams displaying G1 ASVs distributed across G1 plant organs demonstrated that both the above-ground and below ground G1 (T and C) organs were colonized by G1 seed microbiota. The G1 seed microbiota contributed largely to the plant microbiota and only six genera in G1 (T) and seven genera in G1 (C) were identified as unique to G1 seed A,B. For G. clandestina seedings in potting mix supplemented with native bulk soil inoculum from the original plant source enhanced the seed bacterial transmissibility by 36.3% in G2 seed, suggesting that this approach can promote the conservation of the native seed microbiome during seed bulk generation. This, in turn, may contribute to the long-term viability of the seed in seed banks and thus increase seed germination and survivability.Seed banks periodically must revive seeds into full plants to harvest their seeds to replenish and increase their own seed stocks to maintain seed viability for long-term sustainable storage, and also to supply accredited seed vaults with viable seeds for curation . To our G. clandestina seed was mainly dominated by the microbial classes Gammaproteobacteria, Alphaproteobacteria, Actinobacteria and Bacilli, which is consistent with previous studies of the seed microbiome of the related domesticated crop Glycine max (soybean) [G. clandestina seed microbiota (G1 and G2) was primarily occupied by bacterial genera Massilia, Pseudomonas, Paenibacillus, Cohnella, Pantoea and Uliginosibacterium. Most of these genera have been reported as being associated with domesticated plant species including maize [Massilia (58.4%) and Pseudomonas (30.3%) dominated G1 seed, whereas the G2 seed microbiome was predominantly Paenibacillus with an average of 71.2% in G2 (T) and 73.6% in G2 (C). It has been postulated that the emergence of specific bacterial taxa, including rare taxa, can provide essential or new functions that can promote plant growth and nutrient cycling and can either provide an alternative or counterbalance functions that were missing in the abundant taxa of seed microbiomes [Paenibacillus were one of the dominating endophytic bacteria found in barley seed [Paenibacillus strains can enhance the seed germination rate due to their ability to produce cytokinins [In general, soybean) ,29. Overng maize , soybeanng maize ,32, wheang maize , cucumbeng maize , ryegrasng maize , rice [3ng maize  as well ng maize . In thisrobiomes ,38,39. Trobiomes  that emeley seed , wheat pley seed ,42. Prevtokinins ,44,45. Itokinins  that a rtokinins .G. clandestina plant microbiota. For instance, the below-ground and above-ground plant organs consisted of about 87–91% and 67–86% of G1-associated microbial communities, respectively. The seed-transmitted microbiota making up the majority of the plant microbiota has been reported on previously in other crop plants [Triticum aestivum) seedlings were mainly derived from seed, although the plant microbiome composition was suggested to vary dependent on soil bacterial community composition. Interestingly, our results strongly indicated the influence of soil type on the assembly of G2 seed microbiota, whereby 25% more G1 seed microbes were vertically transmitted to G2 (T) seed compared to G2 (C) seed. Factors such as soil type, external environment, host genotype, dispersal agents, pollinators, and the floral microbiome have also been identified as potential drivers of the assembly and structure of seed microbiomes [The data from this study indicated that there was clear a difference in seed microbiota composition based on soil type, with an increased number of low abundance taxa in the G2 (T) seed and a significant increase in the abundance of 11 genera. Previous studies demonstrated that the integration of two different ecosystems could result in the emergence of rare taxa as observed. Examples include the uneven mixing of two soil types of different physiochemical and bacterial compositions in soil microcosms , two soip plants ,50. Accop plants . G1 micrrobiomes , and thirobiomes . Notablyrobiomes . A revierobiomes  verifiedrobiomes .Nevertheless, our study also showed that a significant amount of the G1 seed microbiota remains conserved across G2 (T and C) seed. In line with our findings, studies have detected a significant pool of conserved seed bacterial microbiota across plant generations in maize  and ryegArabidopsis [Populus [G. max [G. clandestina roots, and thereby increased the bacterial diversity of the G. clandestina root microbiome. The influence of media type on the composition of root microbiome has been reported for Lolium perenne [4+, NO3− and pH levels) of local soil, which subsequently guided the assembly of the plant microbiota composition, although the initial soil bacterial diversity was identified as the main driver of the seedling microbiota composition [Overall, clear differences were observed between the bacterial profiles of the above-ground plant organs  and roots. These differences were mainly related to the presence or absence of low abundance classes and variations in the abundance of dominant bacterial classes. These results agree with previous studies in soybean , wheat  and Arabbidopsis  which sh[Populus  and G. m [G. max  microbio [G. max  showed t perenne , sugar b perenne , and bar perenne  microbioposition .Our data showed that the largest subset of G2 (T) seed microbiota was recruited from roots compared to leaf and shoot. We found that 88.3% of G2 (T) bacteria originated from the roots, and by inference, the rhizosphere and rhizoplane surrounding the plant. Without native soil supplementation, 87.7% of genera from soil contributed to the G2 (C) microbiome. However, if we disregard the taxa that are common to all three (70 taxa), the proportion of taxa from the soil, through the root system, becomes 26.3% for G2 (C) seeds, and 49.4% with G2 (T) seeds, an almost 90% increase in the contribution of the soil microbiome to the next generation seed with soil treatment. Previous studies have suggested that the composition of the seed microbial communities reflects the microbial communities associated with roots ,63. ThisBacilli, were much less abundant in roots, which were mainly occupied by Gammaproteobacteria. At the genus level, this corresponded with a significant increased abundance of Paenibacillus in G2 seed. Yang et al. [Enterobacteriaceae and Paenibacillaceae become less abundant in roots when plants were grown in soil, suggesting an influence of soil microbial communities on the composition of the root microbiome. Paenibacillus belong to a group of phosphate-solubilizing bacteria [Pseudomonas, Enterobacterales, Bacillus and Paenibacillus in most soil samples. Interestingly, they also reported a lower abundance of Pseudomonas and an increased number of OTUs for Enterobacterales after the addition of N and NP. This is consistent, to some extent, with our results, as the abundance of Pseudomonas significantly declined, especially in G2 (T) seed, while the above-ground organs (shoot and leaf) were dominated by Enterobacterales  seed-borne bacteria compared to seedlings derived from plants solely grown in commercial potting mix. This was largely through vertical transmission of low abundance taxa that were present in the roots of the native soil-treated plants. Given that a mere 10% soil inoculum from the original plant soil did influence the assembly of seed microbiota, we suggest that replanting seed in the glasshouse with a larger ratio of local soil, or using native soil only, may improve the conservation of the seed microbiota from G1 to G2 and subsequent generations. A better understanding about the role of soil nutrient composition in plant microbiome assembly would be extremely helpful in crafting strategies to conserve native seed microbiota.In conclusion, 16S rRNA profiling of


By contrast, the I38T substitution had no impact on MCV19 polymerase activity, and this mutation was genetically stable over four passages. In conclusion, our results show a similar pathway of resistance to BXA in influenza B viruses highlighting the major role of the I38T PA substitution and suggest that I38T may differently impact the fitness of influenza variants depending on the viral type, subtype, or lineage.Baloxavir marboxil (BXM) is an antiviral drug that targets the endonuclease of the influenza polymerase acidic (PA) protein. Antiviral resistance, mainly mediated by the I38T PA substitution, readily occurs in both A(H1N1) and A(H3N2) viruses following a single dose of BXM. Influenza B resistance to BXM remains poorly documented. We aimed to generate baloxavir-resistant contemporary influenza B/Yamagata/16/1988- and B/Victoria/2/1987-like viruses by in vitro passages under baloxavir acid (BXA) pressure to identify resistance mutations and to characterize the fitness of drug-resistant variants. Influenza B/Phuket/3073/2013 recombinant virus  and B/Quebec/MCV-11/2019  were passaged in ST6GalI-MDCK cells in the presence of increasing concentrations of BXA. At defined passages, viral RNA was extracted for sequencing the PA gene. The I38T PA substitution was selected in MCV19 after six passages in presence of BXA whereas no PA change was detected in rg-PKT13. The I38T substitution increased the BXA IC Seasonal influenza is a highly contagious respiratory disease with significant public health and economic impacts. Each year, approximately 1 billion cases of influenza infections are recorded in the world . Such anAntiviral drugs can provide important clinical benefits for severe cases of influenza A and B infections. The neuraminidase (NA) inhibitors have constituted the main class of anti-influenza agents over the last two decades, including oral oseltamivir (the most widely prescribed NAI compound), zanamivir, peramivir, and laninamivir . NAIs haBaloxavir marboxil (BXM) is a novel RNA polymerase inhibitor that has been approved for the treatment of uncomplicated influenza in the USA and Japan in 2018 and in Europe in 2021. Its active compound, baloxavir acid (BXA), targets the cap-dependent endonuclease activity located within the influenza A and B polymerase acidic (PA) proteins, thus inhibiting viral mRNA synthesis and therefore blocking viral replication . BXM affThe major objective of this study was to induce antiviral resistance in IBV for the two influenza B/Victoria/2/1987-like and B/Yamagata/16/1988-like lineages by passaging the viruses under BXA pressure in order to identify which mutation could occur under BXA pressure in influenza B viruses and to predict whether such mutation may alter the in vitro properties of eventual variants.Viruses from the B/Victoria/2/1987 lineage (MCV19 isolate) and B/Yamagata/16/1988 lineage (rg-PKT13) were serially passaged under BXA pressure with increasing drug concentrations. Mutations in the PA gene were investigated at different passages in presence or absence of the drug. The B/Victoria/2/987-like MCV19 isolate showed evident cytopathic effects in presence of increasing concentrations of BXA. The PA-I38T substitution was detected in MCV19 after six serial passages in ST6GalI-MDCK cells corresponding to 200 nM of BXA. No additional substitutions were found in other viral genes . There were no mutations in the PA gene of the control viral sample recovered after six passages without BXA. Contrasting with MCV19, the B/Yamagata/16/1988-like rg-PKT13 virus showed weaker cytopathic effects and some passages needed to be repeated at the same BXA concentration. We lost the PKT13 virus in presence of 100 nM and could not identify new mutations (by Sanger sequencing) at lower BXA concentrations. 50 values for the B/Victoria/2/1987-like MCV19 and rg-B/Washington/02/2019 (a B/Victoria/2/1987-like recombinant virus rescued with reverse genetics using the contemporary vaccine strain) viruses and their PA-I38T variants are reported in 50 values by 13.7-fold and 21.3-fold in MCV19 and rg-B/Washington/02/2019 backgrounds, respectively. For comparison, the PA-I38T substitution was shown to increase the IC50 value of the rg-PKT13 virus by 12.6-fold [The mean BXA IC50/mL, respectively .In replicative capacity experiments performed on ST6GalI-MDCK cells, the viral titers for both MCV19 WT and I38T viruses reached a peak at 48 h p.i. with mean viral titers of 7.8 and 7.2 log TCIDectively . A signiReplication kinetics of MCV19 and its I38T variant in HAE cells revealed reduced viral loads for the mutant compared to the WT at all time points, i.e., 24, 48, 72, 96, and 120 h p.i. A, while The MCV19 WT and I38T viruses were passaged four times in cell culture in the absence of selective pressure with BXA. Viral genes comprising the viral ribonucleoproteins  were then sequenced. The I38T substitution was still present as a pure population (based on sequence chromatograms) with no other mutations. The I38T substitution is thus stable in the B/Victoria/2/1987 lineage background.The minigenome experiment assessing the relative polymerase activity of the MCV19 WT and its PA-I38T variant showed a 10% increase for the I38T variant compared to the WT . HoweverThe emergence of drug-resistant viruses represents a major public health problem that can compromise the clinical outcome of the newly-approved BXM. Understanding the mechanisms of resistance and the evaluation of the fitness of such variants is therefore needed to better understand their potential for dissemination among the population. In that matter, M2 channel inhibitor use is no longer recommended for the treatment of IAV infections, since all circulating IAVs are no longer susceptible to this drug due to the widespread dissemination of the S31N substitution .Mutations of reduced sensitivity to BXA were first detected in phase II and III of BXM clinical trials. This resistance phenotype was explained by substitutions in the sequence of PA protein, particularly at the I38 amino acid position. Such clinical studies involved adults, adolescents, and children infected with A(H1N1) pdm09 and A(H3N2) viruses ,11. VeryIn this study, we passaged IBV in cell culture to induce baloxavir resistance. We and others previously demonstrated that the NA mutations induced by in vitro passages were similar to those observed in clinical cases of resistance to NA inhibitors ,19. Acco50 values than influenza A strains [50 values, respectively) compared to influenza B (<25-fold increase) [We cannot exclude the possibility that the I38T substitution and other important genetic changes might have occurred at a lower frequency so that more sensitive sequencing tools would have been needed. Our work confirms that, as for seasonal influenza A(H1N1) and A(H3N2) viruses, the I38T substitution is the major marker of baloxavir resistance in IBV (B/Victoria/2/1987 lineage), highlighting the importance of such a mutation. Indeed, I38 is highly conserved within the endonuclease domain of IAV and IBV and co-crystal structural analysis showed that the I38T substitution alters the van der Waals contacts between the endonuclease and BXA, resulting in reduced stability of the drug-endonuclease linkage . Notably strains . On the ncrease) ,15. Contncrease) ,22,23.13. Notably, we found that the I38T variant retained ≈ 80% of its polymerase activity and grew at similar viral titers when compared to the WT counterpart. The replication kinetics of rg-PKT13 WT and I38T on HAE also showed no impairment in viral fitness (We previously rescued and characterized the recombinant influenza B/Phuket/3073/2013 (rg-PKT) WT virus and different PA variants (I38T/M and E23K) in vitro and in experimentally infected mice  fitness B. In micHere, we focused on investigating the impact of I38T in the IBV B/Victoria/2/1987-like background using a contemporary clinical isolate (MCV19) that was subjected to passages under in vitro BXA pressure. As we did with the B/Yamagata/16/1988 lineage, we further developed a reverse genetics system for the B/Victoria/2/1987 lineage using the current vaccine strain (B/Washington/02/2019) and confirmed the effect of the I38T PA substitution on the BXA resistance phenotype . Then, wNotably, the impact of PA mutations that confer resistance to BXA on virus transmissibility could not be reliably predicted by in vitro replicative properties. It remains interesting to assess the impact of the I38T PA substitution on the viral fitness using an appropriate animal model. A recent study demonstrated that the PA-I38T substitution did not alter IAV (A(H3N2) and A/H1N) pdm09 strains) and IBV (B/Brisbane/60/2008 and B/Phuket/3073/2013 strains) contact or airborne transmissibility in ferrets despite attenuated replicative capacities in MDCK cells . AnotherIn conclusion, the impact of baloxavir resistance mutations still needs to be further investigated in IBV using different animal studies to assess infectivity (mouse models) and/or transmissibility (guinea pig or ferret models). There is also a need to select and standardize the most efficient in vitro methodologies to predict in vivo viral fitness for PA mutants. It is also of importance to monitor the emergence of influenza viruses with a phenotype of reduced drug susceptibility, especially I38X variants that are considered to be the main baloxavir-resistant marker. In that regard, pyrosequencing is an interesting tool to rapidly detect the presence of this substitution in clinical specimens .Madin–Darby canine kidney (MDCK) cells overexpressing α2,6 sialic acid receptors  were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS)  and 7.5 µg/mL puromycin . MDCK and human embryonic kidney 293T cells (ATCC) were maintained in MEM and Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% FBS, respectively. MucilAir is a reconstituted human airway epithelium (HAE) from human nasal primary cells collected from a pool of 14 donors. HAE was purchased from Epithelix  and cultured at the air–liquid interface with MucilAir culture medium in Costar Transwell inserts  according to the manufacturer’s instructions. Baloxavir acid (BXA) was synthesized at Shionogi & Co., Ltd., Osaka, Japan. Drug dilutions were made in sterile water.Influenza B/Phuket/2073/2013 (PKT13), the current B/Yamagata/16/1988-like vaccine strain, was obtained from the National Institute for Biological Standards and Control  and used for the rescue of the recombinant PKT13 virus (rg-PKT13) by reverse genetics . Influen5 MDCK and 7.5 × 105 293T cells was grown in a 6-well plate with DMEM supplemented with 10% FBS and Hepes at 37 °C for 48 h. One µg of each plasmid and lipofectamine 2000 (3 µL/µg DNA) in 1.2 mL optiMEM  were used to transfect the MDCK/293T co-culture. After 6 h of incubation at 33 °C, the transfection medium was replaced with DMEM with 0.1% bovine serum albumin (BSA), TPCK (3 µg/mL) and Hepes. The co-culture was then incubated at 33 °C for 72 h before collecting the supernatant. Recombinant viruses were then amplified by infecting ST6GalI-MDCK confluent cells. All IBV segments were then sequenced using the Applied Biosystems 3730xl DNA Analyzer .To confirm the role of eventual drug-induced mutations in the influenza B/Victoria/2/1987-like background, we developed a reverse genetics system using influenza B/Washington/02/2019 strain  which is the current vaccine strain. Amino acid sequences of the HA and NA proteins of MCV19  have 98.4% and 99.6% matched identities with those of B/Washington/02/2019. Reverse genetics and PCR-mediated mutagenesis were performed using the bidirectional pBZ plasmids . Co-transfection experiments were performed as previously described . BrieflyTo induce resistance to baloxavir in IBV, rg-PKT13, and MCV19 viruses were submitted to repeated passages in presence of increasing concentrations of BXA [2. The infection medium was then removed and the cells were overlaid with MEM containing 0.8% sea plaque agarose  supplemented with 1 µg/mL TPCK-treated trypsin  and 3-fold serial dilutions of BXA ranging from 0 to 1215 nM. After three days of incubation at 33 °C, the overlay was removed and the cells were fixated with a formalin solution followed by staining with 0.8% crystal violet. Viral plaques were then counted for determination of the drug concentration resulting in the reduction of plaque-forming units (PFU) by 50% .The phenotype of susceptibility to BXA was determined for baseline influenza B viruses and their PA mutants using a plaque reduction assay . Briefly50) assay with the Reed and Muench endpoint method [The in vitro replicative capacity of the MCV19 isolate and its drug-induced I38T variant was determined in ST6GalI-MDCK cells. Twelve-well plates of confluent cells were washed with PBS, then infected with WT influenza B virus and its I38T variant at an MOI of 0.0001. Supernatants were collected at 12-, 24-, 48-, 72-, and 96-h post-inoculation (p.i.) and titrated by tissue culture infectious dose 50% . Reverse transcription-quantitative PCR (qRT-PCR) assay was performed using primers targeting the influenza NS1 gene of IBV (available upon request). This assay was performed with the QuantiTect Virus + ROX Vial Kit  on a LightCycler® 480 system (Roche Molecular System).The apical layers of HAE were gently washed with warm opti-MEM before infection. The epithelium was infected at an MOI of 0.002 with MCV19 or rg-PKT13 and their corresponding PA I38T variants. Viruses were adsorbed at 33 °C under a 5% COThe genetic stability of the WT MCV19 virus and its drug-selected I38T mutant was assessed after 4 serial passages in ST6GalI-MDCK cells without BXA. A confluent monolayer of ST6GalI-MDCK in 12-well plates was infected at an MOI of 0.0001 per well. After an incubation of 1 h at 33 °C, the infection medium was removed and replaced with fresh MEM supplemented with 1 µg/mL TPCK. After 3–4 days of incubation, the supernatant was collected and viral RNA was extracted, reverse transcribed, and PCR-amplified before sequencing the PA, PB1, PB2, and NP genes with an Applied Biosystems 3730xl DNA Analyzer .A minigenome assay was performed to assess and compare the polymerase activities of MCV19 and its I38T PA mutant. Briefly, the Gaucia luciferase reporter plasmid (pHH21-vM-Luc) was co-transfected with pBZ plasmids containing NP, PB1, PB2, and PA genes of MCV19 in 293T cells using Lipofectamine 2000 reagent . After 18 h of incubation at 37 °C, supernatants were harvested and luminescence was measured with a multilabel plate reader , using an acquisition period of 1 s.p-value < 0.05. Student’s unpaired t-tests were used to compare different experimental conditions in replication kinetics experiments and enzymatic assay.Statistical analyses and graphs were conducted with version 9.1.0 of GraphPad Prism. Statistical significance is defined as


However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen.Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G  causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen. Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory disease in children and infants, causing approximately 33 million cases and 118,000 deaths globally every year 13. RSV is4–9–RSV contains two immunogenic envelope glycoproteins that elicit neutralizing antibodies: RSV F and RSV G . The mem+ T cells  , 32, 33.36–45–in vivo in mouse and cotton rat models of RSV infection. The anti-RSV G mouse monoclonal antibodies (MAbs) 131-2G, 5A6, and 3A5 and the human MAbs 3D3 and 3G12 reduce lung viral loads and weight loss in mouse models -positive T cells  were cocultured in the top chamber of a permeable membrane Transwell insert with RSV-infected human airway epithelial cells in the bottom chamber  and tumor necrosis factor alpha (TNF-α) produced by plasmacytoid dendritic cells and monocytes within PBMCs , despite the >30-fold lower abundance of antibodies against RSV G compared to RSV pre-F , vaccine recipients experienced enhanced respiratory disease (ERD) upon natural RSV infection  cells incubated with human PMBCs revealed that RSV CX4C induced higher levels of type I and III interferons compared to wild-type (WT) RSV  was used to develop a live attenuated RSV vaccine (RSV CX4C). Evaluation of RSV CX4C in an (WT) RSV . Additio(WT) RSV . Evaluat in mice , 60, 61.In another approach, single-point mutagenesis of a different CCD residue, serine 177, which is conserved as a small amino acid glycine or serine, was compared to the CX4C mutant in an RSV G subunit vaccine . In thisDespite the promise of RSV G engineering to improve immunogenicity and overcome safety concerns, it has been unclear if the mutations affect antigen folding and display of RSV G conformational epitopes. This is important because structural distortion of RSV G protein could prevent binding of high-affinity antibodies and reduce vaccine efficacy. In this study, we compare RSV G wild-type and CX4C, S177Q, and S177R mutant proteins for their binding affinities to anti-RSV G MAbs 3G12, 3D3, 2D10, and 131-2G. Additionally, we compare RSV G wild-type and mutant proteins for binding to human reference immunoglobulin to RSV. Finally, we present the structure of antibody 3G12 bound to RSV G CCD S177Q to evaluate at the atomic level the mutant CCD folding and display of the 3G12 conformational epitope. These studies validate the structure and antigenicity of the RSV G S177Q mutant and support its further development as an RSV vaccine immunogen.ecto) wild-type (WT) and mutant  proteins (residues 64 to 298) were designed, expressed, and secreted in mammalian cells and purified from the cell medium as described previously  or mouse MAb 131-2G, respectively. Biosensors were submerged into 1:2 serial dilutions of RSV Gecto WT or mutant proteins to measure on-rates and then submerged into buffer to measure off-rates (DK]) for each antibody-antigen interaction was then calculated  were conducted using human reference immune globulin to RSV (human anti-RSV Ig), which has been shown to contain RSV neutralizing antibodies . The data indicate that the RSV Gecto S177Q mutant protein displays immunodominant epitopes for human anti-RSV Ig.ELISA signals and area under the curve (AUC) calculations are shown in To further show at the atomic level that the S177Q mutation does not disrupt CCD folding and display of conformational epitopes, we used X-ray crystallography to solve the structure of RSV G CCD S177Q bound to 3G12 antibody . Recombiin vivo compared to wild-type RSV G protein  amino acids 64 to 298 (UniProtKB entry P03423), in frame with an N-terminal tissue plasminogen activator (TPA) or CD5 signal sequence and tandem C-terminal 6 histidine and Twin-Strep purification tags, was cloned into cytomegalovirus (CMV) promoter-driven expression plasmid pCF or a derivative of pcDNA3.1 (186) to encode CWAIAC (CX4C) instead of the wild-type sequence CWAIC (CX3C). Serine 177 was mutated to glutamine or arginine for the S177Q and S177R mutants, respectively. Recombinant RSV Gecto proteins were expressed by transient transfection in CHO-S cells using electroporation (Maxcyte STX) and purified from the medium by StrepTrap affinity chromatography. All proteins were concentrated to 1 mg/mL and dialyzed into phosphate-buffered saline (PBS). Purity was verified by SDS-PAGE (A synthetic gene encoding RSV (strain A2) G protein ectodomain  targeting the gp120 protein . The plaecto proteins to anti-RSV G MAbs. Anti-human IgG Fc capture AHC biosensors  or anti-mouse IgG Fc capture AMC biosensors (for MAb 131-2G) were submerged in Octet buffer  for 60 s. The sensors were loaded for 120 s with 1 µg/mL of human or mouse anti-RSV G MAb and then submerged into Octet buffer for 120 s. Association of MAbs with wild-type and mutant RSV Gecto was measured for 300 s, and dissociation in Octet buffer was measured for 600 s. RSV Gecto proteins started at 40 nM and were 2-fold serially diluted to 0.63 nM to measure binding to anti-RSV G MAbs. Higher starting concentrations were required to be able to fit curves for RSV Gecto CX4C protein. A global association 1:1 model was used to fit at least three curves to determine the on- and off-rates to calculate the equilibrium dissociation constant (DK). All binding assays were performed in duplicate.An Octet RED384 biolayer interferometry instrument was used to determine binding affinities of wild-type and mutant RSV Gecto proteins at 1 µg/mL in PBS at 4°C overnight. The plates were washed three times with wash buffer . Plates were blocked overnight at 4°C with blocking buffer . The blocking buffer was decanted, and human reference immune globulin to respiratory syncytial virus  was added starting at a 1:100 dilution in blocking buffer and serially diluted 3-fold for 1 h at 37°C. The plates were washed three times with wash buffer. Goat anti-human IgG Fc secondary antibody conjugated to horseradish peroxidase (HRP) (Invitrogen no. A18817) was added at a 1:3,000 dilution for 1 h at 37°C. The plates were washed two times with wash buffer. Plates were developed for 10 min with phosphate citrate buffer, 12.5 µL of 30% hydrogen peroxide, and two o-phenylenediamine dihydrochloride (OPD) tablets. The reaction was stopped with 2 N sulfuric acid, and the plates were measured at the optical density at 490 nm (OD490) using a Molecular Devices Spectramax plate reader. Human anti-RSV G MAb 3G12 was used as a positive control, starting at 1 µg/mL and serially diluted 3-fold. No antigen was used as a negative control. Curves were graphed in GraphPad Prism and are the average of four replicates.Flat-bottom, high-binding Costar 3590 ELISA plates were coated with wild-type and mutant RSV GP03423) was cloned into pRSFDuet-1 with an N-terminal methionine and a C-terminal 6-histidine tag. The S177Q mutation was introduced by Phusion site-directed mutagenesis and verified by Sanger sequencing. Recombinant RSV G CCD S177Q was expressed in T7Express Escherichia coli overnight at 18°C. Cells were lysed by ultrasonication in wash buffer  with 0.1 mM MgCl2, protease inhibitors, and benzonase. RSV G CCD S177Q was purified from clarified E. coli lysates by affinity chromatography using a HisTrap FF crude column and washed with wash buffer containing 6 M urea. Protein was eluted in wash buffer containing 500 mM imidazole.A synthetic gene encoding RSV (strain A2) G CCD amino acids 157 to 197 (UniProtKB entry The Fab fragments of human anti-RSV G MAb 3G12 were produced using the Pierce Fab preparation kit (Thermo Scientific 44985). Briefly, MAb 3G12 was incubated with immobilized papain in PBS for 5 h at 37°C. Fc fragments were removed using immobilized protein A. Fab purity was verified by SDS-PAGE.2. Diffraction data were collected at the Advanced Light Source on beamline 5.0.1. Data were processed using DIALS and scaled using Aimless (within CCP4). Phaser was used for molecular replacement, and Phenix Refinement and Coot were used for structure refinement.To generate the Fab 3G12-RSV G CCD S177Q complex, Fab 3G12 was incubated with a 4-molar excess of RSV G CCD S177Q on ice for 30 min. A Superdex 75 size exclusion chromatography column was used to purify the complex and eluted in 10 mM Tris-HCl (pH 8) and 150 mM NaCl. The complex was concentrated to 8.26 mg/mL. Crystals were grown by hanging drop vapor diffusion in a well solution of 1.8 M ammonium sulfate and 100 mM sodium acetate trihydrate (pH 4.4) at room temperature. Crystals were transferred to a cryoprotectant solution of 2 M ammonium sulfate, 100 mM sodium acetate trihydrate (pH 4.4), 25% glycerol, and 18% of a 1:1:1 ratio of 100% ethylene glycol - dimethyl sulfoxide - glycerol (EDG) and flash frozen in liquid N7T8W. All other data have been made available in this article.Coordinates and structure factors for the Fab 3G12-RSV G CCD S177Q complex structure have been deposited in the Protein Data Bank under accession code