Introduction: Most hepatically cleared drugs are metabolized by cytochromes P450 (CYPs), and Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines provide curated clinical references for CYPs to apply individual genome data for optimized drug therapy. However, incorporating novel pharmacogenetic variants into guidelines takes considerable time.Methods: We comprehensively assessed the drug metabolizing capabilities of CYP2C19 variants discovered through population sequencing of two substrates, S-mephenytoin and omeprazole.Results: Based on established functional assays, 75% (18/24) of the variants not yet described in Pharmacogene Variation (PharmVar) had significantly altered drug metabolizing capabilities. Of them, seven variants with inappreciable protein expression were evaluated as protein damaging by all three in silico prediction algorithms, Sorting intolerant from tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and Combined annotation dependent depletion (CADD). The five variants with decreased metabolic capability (<50%) of wild type for either substrates were evaluated as protein damaging by all three in silico prediction algorithms, except CADD exact score of NM_000769.4:c.593T>C that was 19.68 (<20.0). In the crystal structure of the five polymorphic proteins, each altered residue of all those proteins was observed to affect the key structures of drug binding specificity. We also identified polymorphic proteins indicating different tendencies of metabolic capability between the two substrates (5/24).Discussion: Therefore, we propose a methodology that combines in silico prediction algorithms and functional assays on polymorphic CYPs with multiple substrates to evaluate the changes in the metabolism of all possible genomic variants in CYP genes. The approach would reinforce existing guidelines and provide information for prescribing appropriate medicines for individual patients. Genetic polymorphisms are the primary cause of inter-individual variations in drug metabolism . The majCYP2C19 [MIM: 124020]   are metabolized by CYP enzymes , and cyt 124020] ) is well 124020] . CYP2C19s (PPIs) , mephenys (PPIs) , antideps (PPIs) , benzodis (PPIs) , and ants (PPIs) . Highly variants . The Assvariants . HoweverCYP2C19 from 2,504 representative population subjects in the 1000 Genomes Project, not yet considered in PharmVar  files of 2,504 cohorts in the 1000 Genomes Project phase III database  were dowber 2018 . The dipPharmGKB .Subjects were assigned to the ultra rapid metabolizer (UM), rapid metabolizer (RM), normal metabolizer (NM), intermediate metabolizer (IM), poor metabolizer (PM), possible IM, possible PM, or indeterminate .CYP2C19 region  of 2,504 cohorts in the 1000 Genomes Project phase III database  was synthesized and cloned into the mammalian expression vector pcDNA™3.4 with HindⅢ site. Site-direct mutagenesis was performed based on the WT expression plasmid and verified by Sanger sequencing (The open reading frame (ORF) of wildtype (WT) quencing . WT and The remaining cells were washed twice with .1 M of potassium phosphate buffer (pH 7.4), and resuspended in a lysis buffer containing 100 mM phosphate (pH 7.4), 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, .1 mM phenylmethane sulfonyl fluoride, and 20% glycerol. Microsomes with recombinant CYP2C19 were extracted by centrifugation at 12,000 The microsomal fraction from human embryonic kidney cells was separated on a 10% SDS-PAGE to detect the recombinant CYP2C19 expression. The gels were then transferred to a polyvinylidene difluoride (PVDF) membrane and at 85 V for 90 min by using a semi-dry apparatus . The membrane was immunoblotted with anti-CYP2C19 antibody  and incubated with horseradish peroxidase-conjugated (HRP) anti-rabbit IgG . Protein bands were visualized with ECL Western Blotting Detection System (Santa Cruz Biotechnology) and the gel images were investigated by Chemiluminescence imaging system . Commercially available baculosomes coexpressing CYP2C19 and oxidoreductase  were used as positive control and the empty vector as a negative control, since Expi293F™ cells do not express CYP2C19 .P450 2C19 was monitored by CO-difference spectrum to quantitate specific P450 that functions as the cytochrome P450 family using a UV visible spectrophotometer  . MicrosoSpecific P450 contents were calculated using the following formula:S-mephenytoin and omeprazole, in vitro markers and clinical substrates for CYP2C19-mediated metabolism designated by the United States Food and Drug Administration (U.S. FDA) (S-mephenytoin and pre-incubated at 37°C for 5 min. The reaction was performed with nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system  for 30 min at 37°C. The reaction was stopped on ice by adding acetonitrile containing 2.5 μM of chlorpropamide, and centrifuged at 16,000 ®, Torrance, CA, United States) in mobile phase comprised (A) distilled water containing .1% formic acid and (B) 100% acetonitrile containing .1% formic acid at a flow rate of 200 μL/min for 5 min. The mass spectra of the two substrates and their respective metabolites were recorded by electrospray ionization in the positive ion mode. The turbo-ion spray interface was operated in the positive mode at 5,000 V and 400°C. The optimum collision energies for the ionization of OH-mephenytoin and 5′-hydroxy omeprazole were 17 and 10 eV, respectively. Multiple reaction monitoring modes using specific precursor-to-product ion transitions were applied for quantification. S-mephenytoin was detected at ion transitions of 230 m/z → 150 m/z, whereas omeprazole was detected at the transitions of 362 m/z → 214 m/z. The limit of quantification (LLOQ) of the assay was .02 μM for S-mephenytoin and .01 μM for omeprazole. LC-MS/MS were performed using the and procedures provided by SPMED Co., Ltd., Busan, Republic of Korea. All experiments were done in triplicate and the mean value t-test using R, and p < .05 was considered to indicate statistical significance..S. FDA) , were usThe crystal structure of human CYP2C19 was generated using the atomic coordinate set structure of human microsomal cytochrome P450 2C19 [PBD: 4GQS ] in ProtThe 2,504 cohorts were classified as 810 NMs (32.35%), 73 UMs (2.92%), 412 RMs (16.45%), 814 IMs (32.15%), 178 PMs (7.11%), 11 possible IMs (.44%), one possible PMs (.04%), 106 unknown metabolizers (4.23%), and 99 indeterminable subjects (3.95%) .CYP2C19*9, *3, *38, and *13, respectively, were also selected, with variants was identified in 344 subjects  matched the negative control, and WT matched the positive control  indicated 31.8% of WT metabolism, c.991G>A (CYP2C19*38) revealed 37.8% of WT metabolism, and c.1228C>T (CYP2C19*13) indicated 100.1% of WT metabolism. The assay results identified that the metabolizing capability of c.431G>A (CYP2C19*9) and c.1228C>T (CYP2C19*13) were consistent with their CPIC clinical function, similar to c.636G>A (CYP2C19*3) which classified as no function.The metabolizing enzyme activities of recombinant P450 2C19 in the biotransformation of S-mephenytoin to OH-mephenytoin were measured . P450 2CCYP2C19*9). There were three variants exhibited rapid metabolizing enzyme activity over 150% of WT. Of these, the result of c.1465C>T (174.7%) was nearly twice higher compared to WT. The remaining seven polymorphic P450 2C19 were confirmed to have biotransformation capability close to WT, except for that carrying c.593T>C with 49.6% of WT metabolism.Of the 14 polymorphic CYP2C19, the production of OH-mephenytoin was not observed from the P450 2C19 carrying c.373C>T or c.394C>T. The metabolic capability of P450 2C19 containing c.389C>T or c.1330G>C was 15.5% and 15.9% of WT metabolism, respectively. The results for these four variants were lower than those of c.431G>A (CYP2C19*9), c.991G>A (CYP2C19*38) or c.1228C>T (CYP2C19*13) were observed to have 63.1%, 70.0% or 97.5% of WT metabolism, respectively. The assay results of P450 2C19 carrying c.991G>A (CYP2C19*38) or c.1228C>T (CYP2C19*13) were consistent with their CPIC clinical function, normal function.The metabolizing enzyme activity of P450 2C19 containing each variant was compared with that of the WT by measuring the production of 5-hydroxy omeprazole using omeprazole as a substrate . P450 2CS-mephenytoin. The assay results of ten variants were similar to that of WT metabolism. Among them, two of which had a metabolic capability of less than 50% of WT metabolism (c.389C>T and c.593T>C), and two were over 150% (c.164C>G and c.985C>T) to S-mephenytoin.Remarkably low concentrations of 5-hydroxy omeprazole were detectable for c.394C>T (11.3%) and c.373C>T (22.6%). The P450 2C9 carrying c.1330G>C also revealed a low metabolizing capability, which was 41.9% of WT metabolism. Two polymorphic P450 2C19 indicated a faster metabolic rate over 150% of WT. The P450 2C19 containing c.1465C>T was measured to have a three times higher metabolic capability (269.0%) than WT metabolism. The assay results of this variants was over 150% of WT metabolism in both substrates. The other one (c.326G>C) indicated 152.2% of WT metabolism for omeprazole but approximated WT for p = .0132) was observed in the case of polymorphic P450 2C19 containing c.1465C>T. However, the metabolic capabilities to the two substrates were both more than 150% of WT metabolism . The metabolic capabilities of polymorphic P450 2C19 containing c.1330G>C for the two substrates were both less than 50% of WT metabolism , and polymorphic P450 2C19 containing c.221T>C were both 50%–150% metabolic capability of WT . There were differences in the amount of change in metabolic capability due to these three polymorphic CYP2C19, but the differences in the tendency were not confirmed.We analyzed metabolic capability differences between the two substrates for each polymorphic CYP2C19, and identified eight polymorphic CYP2C19 with a difference of more than 25% . Of thesS-mephenytoin 155.6%, omeprazole 120.7%, p = .0116), c.326G>C , c.389C>T , c.593T>C , c.985C>T .The remaining five polymorphic CYP2C19 indicated different tendencies of metabolic capability in the two substrates; c.164C>G  (The results of the t (SIFT) , Polymort (SIFT) , and Comt (SIFT)   ; Table 3in silico prediction algorithms agreed that the variants caused protein damage except SIFT evaluated c.784G>A as tolerated, despite PolyPhen-2 and CADD evaluating it as possibly damaging, and with an exact score of 24.90.As for the eight variants with low protein expression, all three CYP2C19*9), c.991G>A (CYP2C19*38), and c.1228C>T (CYP2C19*13)), and 15 were not. While P450 2C19 containing c.1228C>T (CYP2C19*13) indicated 50%–150% metabolic capability of WT for both substrates, P450 2C19 carrying each of c.431G>A (CYP2C19*9) and c.991G>A (CYP2C19*38) exhibited differential metabolic capabilities for S-mephenytoin (less than 50% metabolic capability of WT) and omeprazole (50%–150% metabolic capability of WT). All three in silico prediction algorithms predicted c.431G>A (CYP2C19*9) as a protein damage variant and c.991G>A (CYP2C19*38) as a benign variant. Since these in silico prediction algorithms provide one predictive value for a single variant, it was impossible to predict the difference in metabolic capacities by substrate type.Of the 18 genomic variants in sufficiently expressed P450 2C19 polymorphic proteins, three were associated with the star allele  in P450 2C19 polymorphic proteins with 50%–150% metabolic capability of WT for S-mephenytoin were evaluated as benign by all three in silico prediction algorithms, whereas the two (c.326G>C and c.831C>A) were predicted by two as benign and by one as protein damaging.Of the 15 variants not present in the allele definition table, all three polymorphic P450 2C19 carrying each of c.373C>T, c.394C>T, and c.1330G>C indicated decreased metabolic capabilities less than 50% of WT for both substrates. These variants were consistently evaluated as protein-damaging variants by all three in silico prediction algorithms. Six of the remaining eight genomic variants  in polymorphic P450 2C19 proteins with 50%–150% metabolizing capabilities of WT were evaluated as tolerated/benign by all three in silico prediction algorithms. SIFT and CADD evaluated the remaining two genomic variants (c.831C>A and c.985C>T) as benign; PolyPhen-2 evaluated it as possibly damaging.For omeprazole, polymorphic P450 2C19 proteins containing each of the 10 genomic variants not in the allele definition table exhibited 50%–150% metabolic capability of WT. Of these, as described above, two polymorphic P450 2C19 carrying each of c.389C>T and c.593T>C were evaluated as protein damaging by in silico prediction algorithms. Proteins containing the remaining three variants were considered to have more than 150% metabolic capability compared to WT for only one of the two substrates, and in silico predictions were also conflicting.P450 2C19 containing each of the four variants  exhibited metabolic capability of 150% or more compared to WT for either substrates. One genomic variant (c.1465C>T) in P450 2C19 with more than 150% metabolic capability of WT for both substrates was consistently evaluated as damaging by all three Six SRSs and the active site of CYP2C19 were presented on 4GQS  in PDB . The fivCYP2C19  were considered that few mature CYP2C19 were present in the endoplasmic reticulum (ER) membrane . This isCYP2C19 .S-mephenytoin metabolizing enzyme activity assay confirmed that the polymorphic P450 2C19 protein containing four individual variants with reduced metabolic capability compared to c.431G>A (CYP2C19*9), known as the decreased function allele . Based on the electrophoretic measurement of metabolites of omeprazole, P450 2C19 carrying p c.373C>T, c.394C>T, or c.1330G>C had significantly lower enzyme activities than P450 2C19 carrying c.431G>A (CYP2C19*9) (CYP2C19*9) has a metabolic capability of 63.1% of WT for omeprazole, these three variants could also be classified as genotypes with decreased function since the polymorphic proteins encoded by these variants have a metabolic capability of less than 50% of WT. The structural changes observed in the stereoscopic structure of 4GQS containing each variant were located in the decisive structure for drug discrimination, supporting the functional analysis results  . Althoug results . P450 2C results . CYP2C19henytoin . Therefoin silico prediction algorithms have been primarily applied to detect rare Mendelian disease variants, we found that a certain combination of these in silico prediction algorithms can be used to screen candidate variants for substantial changes in drug metabolism, especially for decrease function variants as well as variants with severely lowered expression  showed significant differences in drug metabolism compared to WT ; Table 3pression . Since m in CYPs .in silico prediction algorithms generate results without considering the type of substrate. In our study, polymorphic P450 2C19 proteins indicating different tendencies of metabolic capability in the two substrates were detected , which was found to be lower than that of CYP2C19*9 to S-mephenytoin, but similar to WT to omeprazole    . The repe 86.8%)   . The str= .0017) . P450 fa= .0017) . Therefoin silico protein damage prediction algorithms generate results that do not consider the type of substrate. High-throughput expression screening assays might also have advantages over conventional functional analysis in that they reduce the intensity of time and labor; however, further advancement is still needed to investigate drug metabolism (CYP2C19 has only one isoform, our results for each genomic variant are suitable for providing functional information supporting the existing guidelines.A limitation of this study is that the metabolizing enzyme activity assay for this approach is a one-at-a-time functional assay requiring a relatively long time and is labor-intense. In particular, it is impossible to determine the drug metabolic capability to vary by substrate because tabolism . It is aCYP2C19 genomic variants with in vitro and in silico assessments using S-mephenytoin and omeprazole. All spontaneously present variants were synthesized using HEK293 cells, which allowed us to use the human cell system without reducing the contribution of the polymorphic CYP2C19. The results verified that there are polymorphic proteins containing genomic variants with significantly changed metabolizing capabilities compared to that of the WT, although they have yet to be considered in existing pharmacogenetic guidelines. It was also confirmed that there are polymorphic proteins with different disposition of metabolizing enzyme activity depending on substrates. These genomic variants causing changes in metabolic capabilities analyzed in this study will be reported to the PharmVar Database to be evaluated in the updating CPIC guidelines and proposed as a priority consideration for clinical testing. Therefore, we propose a methodology that combines analysis methods using functional assays on CYP protein correlated with binding to multiple substrates and prediction methods using in silico prediction algorithms to evaluate the changes in the metabolism of all possible genomic variants in CYPs. Further research on both functional analysis methods and in silico prediction algorithms is needed to improve measurement accuracy, and the classification criteria re-established through the process would reinforce existing guidelines and provide information for prescribing appropriate medicines for individual patients.In summary, we characterized the metabolic enzyme activity of


Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro protein expression system.The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based The online version contains supplementary material available at 10.1186/s13036-023-00323-1. The T7 promoter for the RNA polymerase of bacteriophage T7 consists of 18 base pairs of sequence (5’ – TAATACGACTCACTATAG – 3’ ) –4.in vivo and in a cell-free translation system (CFTS). For example, bacterial cell-free translation systems commonly use the T7 RNA polymerase alongside the endogenous sigma 70 system [in vitro translation systems.Due to the T7 system’s versatility, the T7 system can be used both 0 system . Further0 system , mammali0 system  in vitroWe investigated whether translation in a cell free transcription – translation system (TxTl) can be increased by improving the strength of the promoter. It has been shown that increasing plasmid concentration directly correlates with increased translation yields in bacterial TxTl . We reasDue to the robustness and high popularity of T7 RNA polymerase, there has been a lot of effort in engineering T7 RNA polymerase promoter sequences , 10. We Each transcription reaction was analyzed on a urea PAGE gel with the product stained with DFHBI1T, the ligand for the aptamer. The resulting image shows only correctly folded full length aptamer products , or MyTXTL, a commercial E. coli TxTl extract from Arbor Biosciences , we analyzed reactions at 25°C and 37°C  than classic T7 templates.E. c oli TxTl in POPC / cholesterol liposomes [Cell-free translation systems are key components of most synthetic minimal cell designs . We testiposomes . We prepin vitro translation systems. Other in vitro translation systems are used for different applications [E. coli translation machinery purified individually [Leishmania tarentolae extract [Spodoptera frugiperda Sf21 cell line extract [Next, we asked how will T7Max compare to classic T7 in other ications , 31, incvidually , wheat gvidually , Leishma extract , insect  extract , and rab extract . All of We created templates for eGFP expression in each of those cell-free systems, with the only difference between templates being the T7 RNA polymerase promoter: either T7Max or classic T7.E. coli in this case was 9384, while T7Max value was 15671; normalizing T7 to 100 gives T7Max value of 167  were different in each extract, we normalized the results: the classic T7 promoter is assigned value 100, and the T7Max template fluorescence is proportionally scaled for each sample. For example, the raw fluorescence value for classic T7 promoter Finally, we looked to other applications for T7Max. Robust, sensitive, and transportable disease detection systems are in great need, and many rely on the amplification of nucleic acids . Apta-NAWe designed Apta-NASBA primers to detect the aggR gene associated with enteroaggregative E. Coli, using T7Max, which contains features known to be of value in NASBA primers, such as no pyrimidine residues early in the transcript , 14, as While T7Max consistently outperforms T7 promoter in expression from both linear and circular plasmids, as indicated by both protein activity or abundance (Western blot or activity assays), we observed significant increase in translation yields in the case of linear DNA templates with RecBCD nuclease inhibitor protein GamS. The T7Max shows remarkable improvement in translation yields under those single specific conditions. In expression from circular plasmids, T7Max consistently outperforms T7 (both in protein abundance and in qPCR measurements) by 20% to 100%; in GamS experiments T7Max outperforms T7 by more than 300%. The T7 advantage in expression from plasmids remains consistent for all proteins tested in this work, including data from all species of TxTl system we tested.in vitro translation reactions , most often with the use of a bacterial translation system and T7 RNA polymerase [Cell free expression platforms find increasingly versatile applications in many areas of bioengineering, synthetic biology, and metabolic engineering –41. Addilymerase , 43. HerThis system utilizes all existing elements of T7 RNA polymerase-driven transcription without changes and only requires replacement of the promoter sequence in the construct.We have demonstrated versatile utility of the T7Max promoter in multiple different cell-free protein expression systems and for proteins over a wide range of sizes and types, as well as significantly increased yields of protein synthesis from linear DNA templates.in vitro transcription and translation. Our hope is this technology will enable further improvements in both transcription and in vitro protein expression to result in better biomedical, biotechnological, and synthetic cell engineering applications [While the sequence of the T7Max promoter has been known , the perications , 45.A series of T7 promoters described previously , 11, 12,The sequences used in transcription screening in this work differed from the original Uhlenbeck paper sequences testing the strength of various promoter variants. The relative order of promoter strength in our experiments is different than the earlier reported relative transcription yields. We speculate this might be due to the use of different sequences of the transcript. In our experiments, the transcript contained G quadruplex in Broccoli aptamer sequence. It is possible that there is, yet to be explored, variability in promoter strength vs transcribed sequence. Similarly, the promoter best in transcription experiments did not produce the absolute strongest translation result – providing more evidence to speculate about possible transcript sequence dependency.The templates were then used as -is in a transcription assay. All reagents, tubes, and plates were pre-chilled on ice. A master mix of transcription reagents was prepared on ice, and 9 μL of the master mix and 1 μL of the 10X templates were combined in a 200 μ L PCR tube, flicked, spun down, and then transferred to a cold, clear bottom 384-well plate. The transcriptions  were incubated for 6 hours at 37°C in a SpectraMax Gemini XS microplate fluorimeter and data collected every 5 minutes . An endpoint measurement was taken and the transcriptions stored at -80°C.The fluorescent data was correlated by resolving the transcriptions in a denaturing polyacrylamide gel. An 8M urea, 10% (19:1) PAGE was prerun for 30 minutes at 100V in a Mini PROTEAN tank (Bio-Rad) electrophoresis chamber using 1X TBE . Transcriptions were diluted 1:1 with 2x TBE Loading Buffer  and the entire 20 μL sample was resolved for 1 hour at 125V. The gel was then equilibrated in 50 mL 1X folding buffer  for 45 minutes. The buffer was then decanted, exchanged with 50 mL 1X folding buffer supplemented with 10 μ M DFHBI-1T, and incubated for 15 minutes at room temperature . The broccoli band was imaged on an Aplegen Omega Lum G using a SYBR Safe filter. The buffer was decanted as before and replaced with 1X Folding Buffer supplemented with 1X SYBR Gold . After a 15 minute incubation at room temperature , the total RNA was imaged using the aforementioned filter.Low range ssRNA Ladder  and 10 pmol of broccoli were run alongside the transcriptions as controls. The RNA produced for both stain s was quantified using GelQuant.NET.https://www.geneious.com/) and purchased from IDT. For the promoter insert primers, the forward primer sequence was 5’-/5Phos/GATCTAATTCTAATACGACTCACTATAGGGAAATAATTTTGTTTAACTTTAAGAA-3’ and the reverse primer sequence was 5’-/5Phos/CCGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCCCTATAGTGAGTCGTA-3’. The T7 promoter sequence was excised from the original plasmid backbone, UTR1-T7RNAP-T500 , via restriction digestion with AgeI and BglII. The T7Max promoter was cloned into backbones containing the genes for eGFP, fluorescein arsenical hairpin (FlAsH) peptide, and Omphalotin A (OphA) by following NEB’s restriction digest protocol (NEB #R0744), 5’ dephosphorylation protocol (NEB #M0289) and T4 DNA ligase protocol (NEB #M0202). Ligated constructs were transformed into the E. c oli strain BL21(DE3) and plated on LB agar plates containing 100 μg/ml carbenicillin. Colony constructs were verified by sequencing.Double stranded T7Max promoter insert was formed from a pair of annealed 5’-phosphorylated primers. Primers were designed with 4 bp 5’ overhangs just upstream of a restriction enzyme digestion site, the forward primer containing the AgeI restriction site and the reverse primer containing the BglII restriction site, using Geneious 7.1.9 . Mixtures of loading buffer and sample were boiled at 95°C for 5 minutes in a Bio-Rad T100 thermo cycler. Boiled samples were fractionated on a 37.5:1 Acrylamide:Bis-Acrylamide SDS-Page gel and then transferred to a 0.2 μ m nitrocellulose membrane using a Mini-PROTEAN tank (Bio-Rad) according to the manufacturer’s protocol. Gels were run for 60 minutes at 100V in 800 mL of 1X SDS running buffer . Gels were transferred for 60 minutes at 100V in 1L of 1X transfer buffer . Electrical current was provided by Bio-Rad Power Pac 3000. Membrane was incubated with 5% nonfat milk in TBST  for 60 minutes on a horizontal rocker (Benchmark) before mouse IgG1 anti-his primary antibodies (1:5000), purchased from Biolegend, were added to the solution. The 5% nonfat milk TBST and mouse IgG1 mixture incubated with the membrane for 60 minutes on a horizontal rocker. After incubation with primary antibodies, the membrane was rinsed three times with TBST followed by three 10 min washes in TBST. The membrane was next added to 5% nonfat milk in TBST containing horseradish peroxidase-conjugate goat anti-mouse IgG1 secondary antibodies (Biolegend 405306) diluted at 1:5000 and incubated on a horizontal rocker for 60 minutes. After incubation with secondary antibodies, the membrane was rinsed three times with TBST followed by three 10 minute washes in TBST. Blots were developed with SuperSignal (Thermo Scientific) immunoblotting detection system according to manufacturer’s protocols. Blots were imaged using the ChemiDoc MP Imaging System (Bio-Rad) running Image Lab version 5.2.1.C-terminus 6xHis-tagged proteins were expressed d before . ConstruE. coli cell extract and TXTL master mix, we followed the protocol outlined by Sun et al. [To prepare the n et al. . For extThe TxTl was prepared from a strain that contains genomic copy of T7 RNA polymerase under acUV5 promoter. We did not induce T7 expression growing the cells for TxTl prep, therefore we assume there was no basal T7 RNA polymerase in the TxTl prep. We added T7 RNA polymerase to the reactions from 25μ stock in 50% glycerol, purified according to the previously described protocol .oC for 8 hours . Post-incubation, protein expression was determined through measurement of fluorescence (eGFP and FlAsH) or Western Blot (OphA). eGFP fluorescence was standardized to 1μ M fluorescein.The eGFP, fluorescein arsenical hairpin (FlAsH) peptide, or Omphalotin A (OphA) genes with C-terminal His-tags were cloned into the UTR1-T7RNAP-T500 plasmid backbone . The T7 Max promoter was further cloned into these plasmids for downstream experiments. The linear version of the eGFP plasmid was created through restriction enzyme digestion of the circular plasmid with BamHI. To measure the differences in protein expression between the two promoters, 10nM of templates with each promoter type were added to TXTL reactions and incubated at 30N-morpholino) ethanesulfonic acid (MES) buffer and were standardized to samples without the peptide. The excitation and emission spectra of FlAsH intersects with that of Chai Green Dye 20X  in the subsequent quantitative polymerase chain reaction experiments, so 10 μL of the peptide’s TXTL reactions were saved for transcript quantification prior to determining expression levels.FlAsH peptide expression was determined through the addition of 5 μ M FlAsH dye and 20mM 2- at 75oC for 10 minutes . The denatured proteins were pelleted through centrifugation at 3,200g for 2 minutes.Template DNA in 10 μ L of the TXTL reaction was degraded by adding 0.5 μ L of TURBO DNase . The mixture was incubated at 37Forward and reverse primers (Integrated DNA Technologies), for each protein sample were created for downstream reverse transcription and qPCR experiments. The primers were designed based on our GFP sequences, using the IDT Oligo analyzer tool to match the melting temperatures of primer pair. Each primer pair was compatible for transcripts produced from the old promoter and T7 Max. For eGFP, the forward primer was 5’-AAGTTCATCTGCACCACC-3’ and the reverse primer was 5’-TTGAAGTCGATGCCCTTC-3’. For the FlAsH peptide, the forward primer was 5’-TATACCGGTATGTGGGACTG-3’ and the reverse primer was 5’-GATGGTGATGATGGTGATGG-3’. For OphA, the forward primer was 5’-ACGACAATGGCAAGTCCA-3’ and the reverse primer was 5’-GGAAATCCGATGCCTCGT-3’.oC for 1 hour and the reverse transcriptase was inactivated at 65 oC for 20 minutes.To prepare the reverse transcription reaction, 2 μL of the DNase-treated sample was mixed with 2 μL of 10 μM reverse primer, 4 μL of 5X Protoscript II Reverse Transcriptase Buffer, 1 μL of Protoscript II Reverse Transcriptase , 2 μL of 0.1M dithiothreitol (DTT), 1 μL of 10mM dNTP, 0.2 μL of RNase Inhibitor , and 8 μL of nuclease-free water. The reverse transcription reaction was incubated at 42oC, 30 cycles of 15 second denaturation at 95oC, 15 second annealing at 50oC, 1 minute extension at 68oC, and 1 cycle of 5-minute final extension at 68oC. The amplification curves plotted through the Open qPCR software to determine Cq values and averages across 3 replicates of each promoter type were calculated separately.The quantitative PCR reaction mix was prepared by mixing 2 μL of complementary DNA from the reverse transcription with 2 μL of 10 μM forward and reverse primers, 11.25 μL OneTaq Hot Start 2X Master Mix with Standard Buffer , 1.25 μL Chai Green Dye 20X , and 7.5 μL of nuclease-free water. The qPCR was completed using Open qPCR (Chai Biotechnologies) with the following thermocycling program: 1 cycle of 30 second denaturation at 95oC for 8 hours. Every 2 hours, including at the start of the incubation, 10 μ L samples were removed to measure protein expression and quantify transcription.For experiments involving the kinetic determination of protein expression and transcript comparison, 50 μ L of TXTL reactions with 10 nM DNA templates were incubated at 30In any qPCR reaction, the lower Cq value indicates higher abundance of the analyzed sequence. While sometimes qPCR data is presented as relative RNA abundance after performing calibration curves, often the Cq values are presented directly. The latter approach is chosen in this work, to avoid any possible error introduced by the calibration curves and to show the data in the least processed form.Leishmania tarentolae extract (Jena Bioscience), insect Spodoptera frugiperda Sf21 cell line extract (Promega), and rabbit reticulocyte extract (Promega). All of those extracts were used according to the manufacturer’s protocols.To compare T7Max performance, we used commercially available PURE system (NEB), wheat germ extract (Promega), To maintain directly comparable values, all translations were performed in uncoupled mode: the mRNA template was transcribed separately. To preserve the original transcription yields, the transcription reaction was not purified, concentrated, or altered in any way before adding equal volume aliquots of each T7 and T7Max transcription to the corresponding translation reactions.Leishmania tarentolae extract expression cassette was designed with UTR sequences based on Jena pLEXSY_invitro-2 vector. Spodoptera frugiperda Sf21 cell line extract expression cassette was designed with UTR sequences based on Promega pF25K ICE T7 Flexi vector. All expression cassette sequences are in Table SThe GFP template for E coli and PURE reactions was the same, PCR product from the T7 and T7Max GFP plasmids used in other experiments in this paper. We did not codon-optimize GFP for eukaryotic translation, because in those experiments we wished only to compare yield of GFP between T7 and T7Max. With both sequences in each comparison pair being non-codon optimized, the difference in expression can still be directly compared even though the absolute yields are lower than the yields from a codon-optimized gene. Wheat germ extract expression cassette was designed with UTR sequences based on Promega pF3 WG (BYDV) Flexi vector. For the reported normalized fluorescence values, each three WT T7 expression values were averaged, and error was calculated. Then, this expression value was assigned arbitrary value of 100, and error was scaled accordingly. For example if the raw averaged value was 400 with error value of 40, after normalizing raw value of 400 to 100 the error would be normalized by the same factor, in this case divided by 4, to produce error bar with value 10.GAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCAGCGATACATTAAGACGCCTAAAG-3’ classic T7 primer  5’-TAATACGACTCACTATAGCGTCAGCATCAGCTACAATTATTCC-3’ T7Max primer  5'-AATTCTAATACGACTCACTATAGGGAGACGTCAGCATCAGCTACAATTATTCC-3’Apta-NASBA reactions were performed as previously described, with 10nM input RNA . Primershttps://www.addgene.org/browse/article/28223150/. Maps of plasmids are available in Supporting Figure SWe deposited plasmids with T7 max promoter used in this work on Addgene, Additional file 1: Figure S1. The original uncropped gels for data presented on Fig. Additional file 2: Figure S2. The original uncropped gels for data presented on Fig. Additional file 3: Figure S3. Map of plasmid used in the TxTl experiments, pCI-T7Max-UTR1-deGFP-8xHis-T500.Additional file 4: Figure S4. Map of plasmid used in the TxTl experiments, pCI-T7Max-UTR1-NTerminus8xHis-T500.Additional file 5: Figure S5. Map of plasmid used in the TxTl experiments, pCI-T7Max-UTR1-CTerminus8xHis-T500.Additional file 6: Figure S6. The full uncropped Western Blot image of GFP expression comparison between T7 and T7Max. p1686 is T7 and p2008 is T7Max promoter.Additional file 7: Figure S7. Western blots used to quantify protein expression for different proteins using T7 vs T7Max promoter. The ladder is BLUEstain Protein Ladder (Goldbio).Additional file 8: Figure S8. GFP fluorescence data for expression of proteins in different cell-free extracts, before normalizing T7 value to 100 (as shown on Fig. Additional file 9: Table S1. Complete sequences used in the promoter testing experiments. The aptamer transcription templates had no terminators, so all transcriptions were run-off terminated. Promoter sequence is underlined, the aptamer sequence is in italics and protein coding sequence is in bold.


Lung cancer is the leading cause of cancer death in the world. Identifying prognostic factors is crucial to improve the survival time of those with lung cancer. Our previous studies have reported that human papillomavirus (HPV) infections and epidermal growth factor receptor (EGFR) expression are associated with a better survival prognosis in lung adenocarcinoma. The purpose of this study was to detect the molecular evidence of HPV oncoproteins interfering with EGFR nuclear trafficking related to better prognosis in lung cancer. Based on the study results for a better response to cisplatin in transfected HPV 16E5/16E6/16E7 H292 xenograft animal models, as well as better survival in lung adenocarcinoma patients with either 16E6/18E6 or EGFR expression, we suggest that clinicians should adjust the treatment protocol according to HPV 16E6/18E6 expression and EGFR expression to increase the overall survival time in lung cancer.High-risk human papillomavirus (HPV) infections and epidermal growth factor receptor (EGFR) expression have been reported to be associated with more favorable survival outcomes in lung adenocarcinoma patients. In this study, we utilized transfected HPV 16E5/16E6/16E7 H292 cells to investigate the mechanism of HPV oncoproteins interfering with EGFR nuclear trafficking related to a better response to cisplatin. Furthermore, we correlated HPV 16E6/18E6 expression and differentially localized EGFR expression with the clinical association and survival impact in lung adenocarcinoma patients. Our results found significantly higher phosphorylated nuclear EGFR expression upon epidermal growth factor stimulus and better responses to cisplatin in transfected HPV 16E5/16E6/16E7 NCI-H292 cells and xenograft animal models. Our data were compatible with clinical results of a high correlation of HPV 16E6/18E6 and EGFR expression in non-small cell lung cancer tissues and the synergistic effects of both with the best survival prognosis in a lung adenocarcinoma cohort, especially in patients with older age, no brain metastasis, smoking history, and wild-type EGFR status. Cumulatively, our study supports HPV 16E5/16E6/16E7 oncoproteins interfering with EGFR nuclear trafficking, resulting in increased sensitivity to cisplatin. HPV 16E6/18E6 and EGFR expression serve as good prognostic factors in lung adenocarcinoma patients. Lung cancer remains the leading cause of cancer death worldwide according to 2020 global cancer statistics . TherefoThe epidermal growth factor receptor (EGFR) signaling pathway plays a critical role in tumorigenesis and tumor progression . EGFR ovThe HPV E5 oncoprotein has been shown to induce keratinocyte proliferation through the upregulation of the EGFR signaling pathway . The actSince cervical cancer cells are more sensitive to cisplatin and high-risk HPV infections cause more than 90% of cervical cancer ,18, HPV 2. MSCV-N 16E5 (HPV 16E5), MSCV-N 16E6 (HPV 16E6), and MSCV-N 16E7 (HPV 16E7) plasmids were obtained from Addegene , the nonprofit plasmid repository. The fusion protein constructs were then transfected into the NCI-H292 cell line, as per the instructions, through electroporation . Puromycin  was used to select single cell lines . The transfection efficiency was determined using the tagged protein, hemagglutinin .The human lung mucoepidermoid carcinoma cell line H292 , purchased from the Bioresource Collection and Research Center , was maintained in the Roswell Park Memorial Institute medium  supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. Cells were grown at 37 °C in a humidified atmosphere containing 5% CO6 cells, centrifuged them at 1000 rpm for 4 min, and removed the supernatant. We then added 100 μL of phosphate-buffered saline  per tube containing 3% bovine serum albumin and added 5 μL of antibody  to each tube. Cells were incubated for 30 min on ice in the dark, washed three times, centrifuged at 400 g for 5 min, resuspended in ice-cold PBS, and put on ice in the dark during transport. They were incubated for 30 min on ice and analyzed in a flow cytometer .We collected 1 × 10H292, H292-HPV16E5, H292-HPV16E6, and H292-HPV16E7 cells were stimulated with 100 ng/mL epidermal growth factor . They were incubated at 37 °C for 30 min. Then, nuclear and non-nuclear fractions were isolated and subjected to Western blotting with anti-EGFR  and anti-pEGFR  antibodies on nitrocellulose membranes. The same membranes were also probed with histone 3  or tubulin , for the non-nuclear fraction) as a loading control.3 cells; 100 μL) were seeded into the wells of a 96-well plate containing various concentrations of cisplatin . After incubation for 72 h at 37 °C, 10 μL of the MTT solution was added to each well, and the plates were incubated for a further 3 h at 37 °C. Absorbance was measured at 550 nm using a 96-well reader (Gen-5). Each experiment was performed in triplicate wells for each drug concentration.Due to the clinical evidence that HPV-positive lung cancer patients respond to platinum-based chemotherapy, the 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability after cisplatin administration. Adhesion cells (5 × 105) were resuspended in a 1:1 mixture of PBS and GFR-Matrigel and injected subcutaneously into the right flanks of mice using a 27-gauge needle. After 7 days, these mice were intraperitoneally administered 10 mg/kg cisplatin or saline once a week for 3 weeks. To determine the drug effects, the body weight of mice and tumor volume (width2 × length/2) were monitored every 3 days. After 28 days, the tumor weight and final body weight were recorded after sacrifice. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Taipei Medical University.Finally, we established the xenograft animal models using the H292, H292-HPV16E5, H292-HPV16E6, and H292-HPV16E7 cells to determine the treatment responses after cisplatin administration. BALB/c nude mice were purchased from BioLASCO Taiwan Co., Ltd. . All mice were maintained in laminar-flow cabinets under specific pathogen-free conditions at room temperature in a 24 h night–day cycle. To establish the xenograft models, 5-week-old BALB/c nude mice were anesthetized with pentobarbital; then, H292-HPV16E5, H292-HPV16E6, and H292-HPV16E7 cells , membranous EGFR (mEGFR), and HPV 16E6/18E6 immunostaining were as mentioned in our previous studies  = 0.39 [0.16–0.94], p = 0.034). However, other categorical variables, such as age, gender, smoking history, and EGFR mutations, were not associated with HPV 16E6/18E6 expression or EGFR expressions. Regarding the different biology of lung squamous and adenocarcinoma, we focused the analyses on the clinical characteristics of 173 lung adenocarcinoma patients. We found a significantly lower prevalence of any expression , we found that lung adenocarcinoma patients with +tEGFR+E6 had the longest survival time . Specifically, the clinical significance of +tEGFR+E6 expression was identified in lung adenocarcinoma patients with older age, no brain metastasis, smoking history, and wild-type EGFR status. Next, we analyzed the clinical effects of the combination of HPV 16E6/18 E6 expression and EGFR expressions in 173 lung adenocarcinoma patients. In analyses stratified by HPV 16E6/18E6 and EGFR expression  animal models. The results provided molecular evidence in our prior studies showing better responses to platinum-based chemotherapy in lung adenocarcinoma patients with HPV infections and EGFR expression . With thnEGFR/mEGFR expression in NSCLC tissues with a preference in early-stage lung adenocarcinoma. The relationship between both biomarkers in lung cancer has been less reported. Nevertheless, inconsistent findings in head and neck cancers have been obtained regarding the correlation, with a strong association found between HPV infections and EGFR expression in laryngeal squamous cell carcinoma [Consistent with the results of basic experiments with HPV oncoproteins enhancing EGFR nuclear translocation, we found a strong correlation between HPV 16E6/18E6 expression and arcinoma , and a sarcinoma . The proarcinoma ,34, and arcinoma . SimilarHowever, this study has several limitations. First, we limited our analyses to apoptosis alternation in transfected HPV 16E5/E6/E7 H292 cells compared with H292 cells after cisplatin administration. Second, this is a single-institution retrospective study with small sample size. Third, we did not establish a patient-derived (with HPV infections) xenograft model due to limited tissue availability to investigate the molecular mechanism for better treatment responses to platinum-based chemotherapy.In the future, we will develop patient-derived (using fresh lung adenocarcinoma cancer tissues with or without HPV infections) xenograft models to test the treatment response after cisplatin administration. The tumor mass in the xenograft models will be examined for apoptosis alternation such as caspase enzyme activity or DNA repair enzymes (such as Uracil-DNA glycosylase) and tested for the interaction of HPV oncoprotein and EGFR nuclear trafficking-related factors such as cyclin D1. The detailed molecular mechanism of lung adenocarcinoma patients with HPV infections will be determined.Through a series of basic experiments, we have provided evidence of HPV oncoproteins interfering with the EGFR nuclear trafficking pathway related to better treatment response to cisplatin. Therefore, this study suggests that personalized medicine should be included in the treatment protocol for lung cancer patients according to the status of HPV oncoproteins and EGFR expression.


Some of the H6N6 viruses could infect mice without mammalian adaptation, and even caused death in this species. Therefore, our study demonstrated that the H6 AIVs posed a potential threat to human health and highlighted the urgent need for continued surveillance and evaluation of the H6 influenza viruses circulating in the field.H6 avian influenza virus (AIV) is one of the most prevalent AIV subtypes in birds globally. To investigate the current situation and characteristics of H6 AIVs circulating in China, we analysed the epidemiology, genetic evolution and pathogenic features of this subtype. During 2000-2021, H6 subtype AIVs spread widely through Southern China and presented high host diversity. On analysing 171 H6 viruses isolated during 2009-2021, dynamic reassortments were observed among H6 and other co-circulating AIV subtypes, and these generated a total of 16 different genotypes. A few H6N6 strains possessed L226 and S228 mutations of hemagglutinin (H3 numbering), which may enhance the affinity of H6 viruses to human receptors. H6N6 viruses also exhibited divergent pathogenicity and growth profiles  During the last century, four influenza pandemics were documented: H1N1 in 1918, H2N2 in 1957, H3N2 in 1968 and H1N1 influenza in 2009 [The genome segments of pandemic viruses were partially derived from avian influenza viruses (AIVs) . Over thSubtype H6 low pathogenic avian influenza viruses are currently circulating widely throughout the globe. In 1965, H6 AIVs were isolated from a turkey in Massachusetts in the United States . The isoTo monitor AIV genetic evolution and evaluate AIV pandemic potential in a timely manner, a sustained surveillance of live poultry markets (LPMs) has been conducted in mainland China since 2009. In this study, we investigated characteristics of the epidemiology, genetic evolution and pathogenicity of H6 AIVs in China.Mouse experiments were carried out with the approval of the Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC (20211119087). All experiments associated with live H6 viruses were conducted in biosafety level 2 (BSL2) laboratories.50) with the Reed and Muench method [In this study, the H6 AIVs were isolated from environmental samples from LPMs or poultry farms according to the guideline on AIV surveillance of Chinese Centre for Disease Control and Prevention (China CDC). Samples were collected from avian-linked environments once per-month across 31 provinces, autonomous regions, and municipalities in mainland China. Viruses were isolated in specific pathogen free (SPF) embryonated chicken eggs at 37°C for 48 hours. A hemagglutinin (HA) assay was run with 1% turkey red blood cells (TRBCs) to confirm the presence of influenza viruses in the allantoic fluid of the eggs. The HA subtypes of all viruses were identified by real-time reverse transcription-PCR . Virus titrations were conducted in 9-day-old SPF embryonated eggs to determine the 50% embryo infectious dose  were isolated from live poultry related environmental samples by the Chinese National Influenza Centre (CNIC). In order to study the epidemiology of H6 subtype viruses in China, a total of 1524 H6 sequences, generated by the viruses from 2000 to 2021, were downloaded from the public databases, including the Global Initiative of Sharing All Influenza Database (GISAID), GenBank , and CNIC. Information on the subtypes, hosts and geographic distributions of these 1524 H6Nx subtype viruses was collected for further spatiotemporal analysis.https://guix.gnu.org/en/packages/velvet-1.2.10External Link) and Newbler Assembler version 2.5 were used to trim low-quality reads, and sample and de novo assemble the filtered reads. Then, all contigs were blasted against a database generated by CD-HIT which clustered all FluA sequences collected from the NCBI Influenza Virus Database and GISAID EpiFlu database . After that, sequences with the highest similarity were selected as references and bowtie2 version 2.1.0  was used for read mapping. Lastly, FluA genome sequences were obtained by extracting the consensus sequences from the mapping results, with a coverage depth of at least 30× at each site on the eight segments.An RNeasy Mini kit  was used to extract virus RNA from the allantoic fluid of infected SPF embryonated eggs. The complete genome of H6 AIV was characterized by Illumina next-generation sequencing as follows. The extracted RNA was subjected to reverse transcription and amplification. Then whole-genome sequencing of FluA on the Miniseq high-throughput sequencing platform was implemented . Data anhttps://beast.community/2018-11-14.10.4_released.html), all sequences were aligned using MAFFT . By utilizing the model of nucleotide substitution selected in Model Finder of IQ-TREE , phylogenetic trees were generated with a Bayesian Markov Chain Monte Carlo method. Best-fit clock model (the uncorrelated relaxed clock) was selected using marginal likelihoods. The Markov Chain Monte Carlo (MCMC) analyses were run for 100 million generations, with samples drawn every 10,000 generations. Lastly, phylogenetic trees were visualized in iTOL (https://itol.embl.de/) and Figtree .To build the system phylogeny tree by BEAST v1.10.4 (50) was calculated using the Reed–Muench formula [Madin–Darby canine kidney cells (MDCK) were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% foetal bovine serum , HEPES , penicillin , and streptomycin . Confluent MDCK cells were infected with selected viruses at the multiplicity of infection (MOI) of 0.001. After 1 hour of adsorption, the viral inoculums were removed, then the inoculated cells were incubated at 37°C with DMEM containing 2 μg/mL TPCK-treated trypsin . Supernatants were collected at 0-, 6-, 12-, 24-, 36-, 48-, 60- and 72- hours post infection (hpi), respectively, and were stored at-80°C before viral titration in MDCK cells. The tissue culture infectious dose affecting 50% of the cells , and interpretation of IC50 values was performed using the WHO-AVWG  criteria. The criteria for influenza A viruses were as follows: compared to the median IC50 value of all tested viruses by (sub)type and drug, normal inhibition was considered <10-fold, reduced inhibition was 10-to 100-fold, and highly reduced inhibition was > 100-fold (https://www.who.int/entity/wer/2012/wer8739.pdf).Oseltamivir carboxylate  was prepared in sterile distilled water and stored at -20 °C. Neuraminidase (NA) inhibitor (NAI) susceptibility was assessed by a fluorescence-based NA enzyme inhibition assay based on the NA-Fluor™ kit . The IC50).Eight-week-old SPF female C57BL/6 mice . The mock group was inoculated intranasally with 50 μL of PBS. Body weights and the survival of mice were monitored daily, and mice with a 25% weight loss were euthanized for ethical reasons. At 14 dpi, serum samples were collected from the surviving mice. After being centrifuged and separated, the serum samples were treated with receptor-destroying enzyme (Denka Seiken) for 18 hours at 37°C and heat-inactivated at 56°C for 30 minutes. Then the HA inhibiting (HI) antibody titres of the samples were tested with 0.5% (vol/vol) turkey erythrocytes. Serum samples with HI titres of > 10 were classified as antibody positive. The Karber method was performed to calculate the 50% mouse infectious dose  or 107 EID50 (50 µL) of the indicated viruses. Three mice in each group were euthanized at 1, 3 and 5 dpi. Nasal turbinate, tracheal tissue, and lungs were collected to detect the virus titres using a TCID50 assay.To further compare the replication abilities of the H6 viruses in infected mice, nine mice per group were inoculated intranasally with 10The data are displayed as means ± SD. Statistical significance was determined using Student’s t-test and the GraphPad Prism 9.0 software package (GraphPad Software Inc.). A P-value < 0.05 indicated statistical significance.Among all the H6 viruses, H6N6 strains accounted for the largest proportion (49%), followed by H6N2 (34%) and H6N1(14%). By contrast, the ratios of other H6 subtype viruses, including H6N8, H6N5, H6N3, H6N4 and H6N9, were low A. BeforeGeographically, an increase in the number of H6 AIVs was observed from northern to southern China . We clasTo clarify the host distributions of H6 subtype viruses, we analysed the proportion of H6 in each avian species. As shown in Figure S1A, the proportion of H6 AIVs was highest in ducks (53%), followed by environmental samples and wild fowl (14% in each), chickens (11%), geese (8%), and swine and humans (both < 1%). In terms of each H6 subtype, H6N6 exhibited the highest host diversity, being distributed in all species except for humans, followed by H6N2, H6N8 and H6N1. Chickens played a major role as H6N1 hosts, instead of ducks. Although only three H6N9 isolates were detected in China during this period, this subtype was still isolated from two host species. In addition, there was one report of H6N3 and one of H6N4 in samples from duck and wild fowls . These results indicate that ducks were the most important reservoir for H6 viruses, and H6N6 viruses had the most diverse host species among all the H6Nx viruses.To determine the genetic evolution of H6 viruses in China, phylogenetic analysis of all eight gene segments were conducted. As shown in Phylogenetic analysis of the seven genes of H6 viruses, including the NA, basic polymerase 2 (PB2), basic polymerase 1 (PB1), polymerase (PA), nucleoprotein (NP), matrix (M), and non-structural protein (NS) genes, demonstrated distinct diversity , G228 (170/171), E190 (171/171) and G225(171/171)(H3 numbering) at the receptor-binding sites, suggesting that these viruses have maintained a high affinity for the avian-type receptor (α2-3-SA) . HoweverIn the NA stalk region, two H6N6 isolates (JX24/09 and HN01/09) possessed a 27-nucleotide deletion, resulting in the loss of nine amino acids (Table S1) that might be associated with increased virulence in mammals . MoreoveAmino acid changes in PB2, including E627 K and D701N, contribute to the increased virulence and transmission of influenza viruses in mammals . In our Substitutions which were reported to be associated with drug resistance to NAIs  were not6 EID50 in 50 μl PBS. Mice were inoculated with PBS as the control. The survival condition and bodyweight loss of these mice were monitored for 14 days. None of the mice died, and the body weight loss was <20% for those inoculated with all selected viruses at 106 EID50 . However, the bodyweight of mice infected with JX24/09 and HN/28868 decreased significantly , suggesting that these H6N6 AIVs may present different pathogenic phenotypes in mice.Based on the genotype classification, a total of 29 representative viruses were selected for the pathogenicity characterization study. Each virus was inoculated into 8-week-old female C57B/L6 mice intranasally at 107 EID50. The survival and bodyweight loss of the animals were monitored until 14 dpi. As shown in We further infected mice intranasally with JX24/09 and HN01/09 as controls at the dose of 10Tissues, including lung, tracheal tissue, nasal turbinate, intestine tract, brain, and eyeballs, were collected from infected mice at 5 dpi. Viruses were detected only in the respiratory tissues, including lung, tracheal and nasal turbinate tissues. These results indicate that H6N6 viruses preferentially infected the respiratory system, rather than extra-pulmonary tissues.Although viral titres in the tracheal tissue and nasal turbinate tissues infected with JX24/09 virus were similar to those of HN01/09, the viral titre in lung tissue infected with JX24/09 was significantly higher than that of HN01/09 (p < 0. 01) C. These in vivo and in vitro. We also included a control H6N6 virus (GD30383) that does not have NA stalk deletions to study its virulence, replication, and infectivity compared with JX24/09 and HN01/09. As shown in 1–105 EID50 of JX24/09 or HN01/09 virus, no mice died, and the bodyweight loss was < 20%  value of JX24/09 (106.1EID50) was 25 times lower than that of HN01/09 (≥107.5EID50) and GD30383 (≥107.5 EID50), which was expected (To investigate the pathogenicity of the two representative H6N6 viruses (JX24/09 and HN01/09) in detail, their virulence, replication and infectivity were evaluated as < 20% A. Howeveexpected B.Figure50 value of JX24/09 was 103.3EID50, which was almost 63-fold lower than that of HN01/09 (105.1EID50) and 16-fold lower than that of GD30383(104.5EID50)  . These d5 EID50 of virus, the titres of the HN01/09 and GD30383 viruses were significantly lower than those of JX24/09 virus in the lung tissues of mice at 1, 3 and 5 dpi and tracheal tissues of mice at 5 dpi (p < 0.01) . Although the HN01/09 virus exhibited a trachea viral titre slightly higher than that of JX24/09 at 5 dpi at the dose of 107 EID50, the nasal turbinate viral titre of JX24/09 was higher than that of HN01/09 (p < 0.01) . Furthermore, at 1 dpi, the viral titres of JX24/09 were higher than those of HN01/09 in lung and nasal turbinate (p < 0.01) . These data suggested that JX24/09 replicated more efficiently in the upper airway and lungs of mice than HN01/09 and GD30383.When inoculated with 10The growth-kinetic properties of the three viruses in MDCK cells were also compared. Data showed  that the two selected viruses had different replication abilities at 37°C. JX24/09 replicated efficiently and resulted in a higher viral titre than HN01/09, which had the lowest titre at most timepoints (6-36 hpi) (P < 0.05). In contrast, GD30383 replicated poorly at all timepoints. In conclusion, JX24/09 replicated rapidly in MDCK cells and maintained higher viral titres than that of HN01/09 and GD30383, which was consistent with the tissue tropism results.H6 AIVs have been sporadically reported to infect different species of wild birds and domestic poultry during their cocirculation around the world. They have caused several pandemic threats, including H7N9, H9N2 and H5N1 virus outbreaks, for many years ,24–27, pIn this study, we conducted a comprehensive prevalence analysis of H6 AIVs for the first time, using 1524 sequences obtained from 2000 to 2021. H6 subtype AIVs were widely detected throughout 21 provincial regions in China , and theBased on the results of a study that showed ducks were the main hosts of H6 viruses, and the previous conclusion that wild waterfowl were the natural reservoirs that contributed to the geographical spread of AIVs via long-distance migration , we specIn our study, the genotypic diversity of H6 viruses and 16 genotypes was revealed in the genome analysis, providing an indication of the intricate reassortment that occurs among these viruses during their active circulation with other viruses. The same characteristics were seen for the highly pathogenic avian influenza (HPAI) H5N1 virus and the H7N9 virus. For instance, during the 1997 outbreak in Hong Kong, H5N1 influenza viruses identified in this region were considered to be complicated reassortants that acquired the ability to infect and kill mammals gradually . PreviouH6 AIVs had been found to replicate in mice without preadaptation, but with less weight loss and an absence of death . In thisIn summary, given that H6 viruses have acquired the ability to replicate in mammalian hosts, we cannot rule out the possibility that they will eventually evolve into viruses with efficient transmissibility in mammals and even human populations via the accumulation of reassortments or mutations. Therefore, long-term surveillance of H6 AIVs in China is urgently needed.Click here for additional data file.


Angiostrongylus cantonensis (AC) is well-documented that parasitizes the host brain and causes eosinophilic meningitis. The migration route of AC in permissive hosts is well demonstrated, while in nonpermissive hosts, it remains to be fully defined. In the present study, we exploited live imaging technology, morphological and pathological configuration analysis, and molecular biological technologies to explore the migration route of AC and the accompanying tissue damage in nonpermissive and permissive hosts. Our data indicated that, in nonpermissive host mouse, AC larvae migrated from intestinal wall to liver at 2 hours post-infection (hpi), from liver to lung at 4 hpi and then from lung to brain at 8 hpi. AC larval migration caused fatal lung injury (pneumonia) during acute and early infection phases, along with significant activation of Stat3/IL-6 signaling. In addition, AC induce sustained interstitial pneumonia in mouse and rat and pulmonary fibrosis only in rat during late infection phase. Moreover, during the early and late infection phases, Th2 cytokine expression and Stat3 and IL-6 signaling were persistently enhanced and myeloid macrophage cells were notably enriched in host lung, and administration of Stat3 and IL-6 inhibitors (C188-9 and LMT-28) attenuated AC infection-induced acute pneumonia in mice. Overall, we are the first to provide direct and systemic laboratory evidence of AC migration route in a nonpermissive host and report that infection with a high dose of AC larvae could result in acute and fatal pneumonia through Stat3/IL-6 signaling in mice. These findings may present a feasible to rational strategy to minimize the pathogenesis induced by AC. Angiostrongylus cantonensis is a well-known nematode parasitizing the host brain and is the leading cause of eosinophilic meningitis worldwide. Our study reports the migration route of Angiostrongylus cantonensis larvae in non-permissive host mouse and discovers that the larvae could induce fatal pneumonia in mouse lung during acute and early infection phase characterized by activation of Stat3/IL-6 signaling. Moreover, administration of inhibitors targeting Stat3/IL-6 signaling could significantly attenuate the AC-induced pneumonia of non-permissive host mouse, providing an effective candidate target for intervention of this severe parasitic pneumonia. These findings expand our understanding of Angiostrongyliasis cantonensis and indicate that more attention to AC-induced pneumonia is required when treatment for Angiostrongyliasis cantonensis in the future. Angiostrongylus cantonensis (AC) is a food-borne nematode that infects hosts via ingestion of foods containing infective third-stage larvae of AC (AC L3) [Parasitosis is attributed to infection by parasites, which can cause host disease directly by migration from one organ to another in the body ,2 or ind (AC L3) . Based o (AC L3) . In perm (AC L3)  and fina (AC L3)  where ad (AC L3) . During  (AC L3) , eosinop (AC L3)  at 21 dp (AC L3) . However (AC L3) . In anot (AC L3) , indicatAscaris, hookworms, Strongyloides, Paragonimus filariasis, and Toxocara [Strongyloides, penetrate the host lung, while others, such as Trichuris, do not. Generally, parasites can cause pneumonia through direct stimulation or cytokine-induced systemic inflammation [Ascaris could cause a severe eosinophilic reaction, which may lead to eosinophilic pneumonia [Conventional pneumonia is characterized by fluid or pus filling the air sacs and is commonly attributed to pathogen infections, including infections caused by bacteria, viruses, fungi and parasites . InterstToxocara . Some neammation . It has neumonia . As AC i, and hosts infected with AC often exhibit a shift from a Th1 to Th2 response [Many studies have reported that parasite infection triggers the host immune response, mainly the Th1 and Th2 responses . The Th1response . FurtherIn the present study, we used live imaging technology to explore the migration route of AC in nonpermissive host mice, revealing that after infection, AC L3 migrated from the intestinal wall to the liver at 2 hpi, then to the lung at 4 hpi and the brain at 8 hpi. Surprisingly, we observed fatal pneumonia in the mouse lung during the acute and early phases of AC infection. In addition, AC could lead to sustained slight interstitial pneumonia during the late infection phase. Moreover, the immune response of the host to AC exhibited a Th2 bias characterized by activation of Stat3/IL-6 signaling and enrichment of innate immune myeloid cells. Inhibitors targeting Stat3 or IL-6 could significantly attenuate the pneumonia induced by AC, expanding our understanding of angiostrongyliasis cantonensis and providing effective candidate targets for intervention in AC-induced pneumonia.All research and animal care procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University.Biomphalaria glabrata (B. glabrata), as described previously [B. glabrata was homogenized and digested for 30 minutes at 37°C with pepsin solution. After that, AC L3 were counted under a stereomicroscope  and placed in a 1.5 mL centrifuge tube with 100 μL distilled water prior to animal infection. To trace the migration route of AC in mice, AC L3 were washed with saline followed by incubation with the green fluorescent dye Nuclear Green LCS1  for 1 hour at room temperature. Then, AC L3 were washed three times with saline before fluorescence imaging. The labeling efficacy was assessed by imaging under a fluorescence microscope.Third-stage larvae of AC (AC L3) were obtained from positive eviously . BrieflyThe mice were infected with 100 to 300 AC L3 labeled with green fluorescence by oral gavage and sacrificed at 0-, 2-, 4-, 8- and 12-hours post-infection followed by removal of the liver, lung and brain. Live imaging was used to test the existence of AC L3 in specific tissues under a living imager . After that, the liver, lung and brain were excised and homogenized to determine AC L3 counts under a stereomicroscope .Female six-week-old C57BL/6 mice and Sprague–Dawley (SD) rats were purchased from Charles River Laboratories  and randomly divided into the mentioned groups, and 5 animals each group. Each mouse and rat in the following study was infected with 30 and 100 AC L3, respectively, by oral gavage. At the experimental endpoint, mice and rats were anesthetized and sacrificed for the following experiments. All animals were housed in a specific pathogen-free, temperature-controlled environment with a 12-hour light/dark cycle. All animal procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.To examine the histomorphological configuration alterations caused by AC L3 migration in mice, the livers, lungs and brains were removed after AC infection, fixed in formalin , and embedded in paraffin. Tissues were sliced into 4-μm serial sections and stained with hematoxylin and eosin (H&E) or Masson trichrome according to reported previously methods [-ΔΔCt method. The primers used in this study are listed in Total RNA from the lung or brain was extracted with the use of Trizol Reagent  following the manufacturer’s instructions. The concentration and purity of RNA were determined with a NanoDrop One . Complementary DNA (cDNA) was synthesized by using a Revert Aid First Strand cDNA Kit  according to the manufacturer’s guidelines. RT–qPCR was carried out using SYBR Green  on a LightCycler 480 Real-Time PCR System . RT-qPCR was performed for 10 minutes at 95°C followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Relative mRNA levels were normalized to the housekeeping gene β-actin and analyzed by the 2Tissue samples were homogenized and lysed with RIPA lysis buffer  containing protease and phosphatase inhibitor cocktail  on ice for 5 minutes, followed by centrifugation at 12000 rpm for 15 minutes. The supernatant was collected and subjected to protein quantification with a bicinchoninic acid (BCA) kit . A total of 30 μg protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride  membranes , which were blocked with 5% nonfat skim milk for 2 hours at room temperature before overnight incubation with primary antibodies  at 4°C. The lung tissues were fixed for 24 hours, embedded in paraffin and cut into 4-μm serial sections. For immunofluorescence, the sections were deparaffinized and subjected to antigen retrieval at 100°C for 15 minutes in citrate buffer . Next, the sections were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature prior to primary antibody incubation at 4°C overnight. Subsequently, sections were incubated for 1 hour at room temperature with fluorescence-labeled secondary antibodies as listed in To evaluate the apoptosis of lung cells caused by AC L3 migration, flow cytometry was carried out. The lung tissues were homogenized on ice and filtered with a 40-μm cell filter to prepare a single-cell suspension. The prepared cells were washed twice with cold PBS, followed by staining with APC-labeled annexin V and propidium iodide (PI) for 30 minutes at 4°C in the dark. Then, the cells were washed twice and further analyzed on a CytoFLEX flow cytometer .2O2 prior to incubation with an HRP-labeled secondary antibody and diaminobenzidine (DAB) for color development. Next, hematoxylin was used to counterstain the cell nucleus, and the sections were dehydrated, mounted with neutral gum and photographed with the use of an inverted microscope .Consecutive formalin-fixed and paraffin-embedded lung tissue sections were subjected to immunohistochemical staining as previously described. In brief, the sections were blocked with 10% fetal bovine serum for 1 hour, followed by incubation with primary antibody overnight at 4°C. After washing with PBS three times, sections were incubated with 3% Ht test or one-way analysis of variance .Statistical analyses were performed with GraphPad Prism 7.00 . All data are displayed as the mean ± standard derivation (SD) of no less than three independent replicates. Significance was determined with a two-tailed Student’s Parasites usually parasitize the host in a tissue-specific manner. It is well known that AC will eventually parasitize the brain of nonpermissive hosts but the lungs of permissive hosts after infection . During The parasite migration process in the host leads to pathological injury, including hemorrhage and inflammatory responses . We hencAfter the acute infection phase (within 12 hours), AC L3 left the mouse liver and lung, and the infection process entered the early phase (from 1 to 7 days post-infection) when AC L3 completely migrated to the mouse brain. To explore how AC worms influenced the mouse brain and lung during this phase, we first examined the morphological alterations at the indicated numbers of days post-infection (collectively dpi) and found no obvious bleeding spots or thickening of meninges in the mouse brain Figs  and S2A.Next, we aimed to pinpoint how AC affected host pneumonia during the early infection phase. To this end, we detected the expression of Th1  and Th2 cytokines  that were reported to be involved in parasitic immunity in mouse and rat lungs. The results in mouse lungs indicated that the levels of Th1 cytokines  were significantly elevated by approximately 3-fold at 1 dpi compared with 0 dpi but gradually decreased to normal at 2–4 dpi and slightly increased again from 5–7 dpi  upper. IAs AC induced a notable inflammatory response and injury in the host lung during the acute and early infection phases, we next asked whether this alteration would cause host death. For this purpose, mice were separately infected with different doses of AC L3 , and survival curves were generated during the early infection phase (1–7 dpi). The results showed that no mouse died after infection with 30, 60 and 120 AC L3, while 10% of mice infected with 300 AC L3 died at 1 dpi, 70% died at 2 dpi and all died at 3 dpi . To pinpBased on laboratory observations, nonpermissive host mice infected with 30 AC L3 exhibited severe eosinophilic meningitis at 21 dpi and died before 28 dpi. In contrast, permissive host rats infected with 100 AC L3 survived more than 42 days. To further explore how AC exerted influences on the host lung and brain during the late infection phase (7 to 21 dpi for mice and 7–42 dpi for rats), mice and rats were infected with 30 and 100 AC L3, respectively, followed by morphological and pathological observation of the brain and lung on the indicated numbers of days post-infection. We found that AC caused notable hemorrhage and thickening meninges in the mouse brain surface at 21 dpi Figs  but no vNext, we aimed to detect the expression of Th1 and Th2 cytokines in mouse and rat lungs during the late infection phase. Among Th1 cytokines in the mouse lung, the level of TNF-α was significantly elevated by less than 2-fold  and that+ and IBA1+ innate immune cells exhibited sustained enrichment during the late infection phase (+ adaptive immune cells (T cells) were significantly aggregated at 7 and 14 dpi but returned to basal levels (+ antigen-presenting cells (dendritic cells) were notably enriched only at 14 dpi  exhibited no change . FurtherPersistent inflammation can trigger collagen deposition, which leads to fibrosis . To furtOur data indicated that AC infection could induce fatal pneumonia in nonpermissive host mice during the acute and early infection phases . Fatal pAs it is well known that humans and mice infected by AC develop eosinophilic meningitis, an increasing number of studies in recent decades have focused on characterizing and searching for therapeutic strategies for AC-induced meningitis . We shouStrongyloides, Paragonimus, and Toxocara [AC was first discovered in rat lungs by Chen in 1935 , and theToxocara , and theToxocara . TogetheParasitic infection triggers host immune resistance, mainly via Th1- and Th2-type responses . GeneralIt has been reported that chronic helminth infection induces a host Th2-type response bias and that STAT6 signaling is central for this phenotype . SimilarIn summary, in this study, we provided the first laboratory evidence to confirm the migration route of AC L3 in nonpermissive host mice and discovered that AC larvae could induce fatal pneumonia in mice during the acute and early infection phase while interstitial pneumonia during the late infection phase; this condition was characterized by activation of Stat3/IL-6 signaling. Moreover, administration of inhibitors targeting Stat3/IL-6 could attenuate AC-induced pneumonia in nonpermissive host mice, corroborating that Stat3/IL-6 signaling mediates the AC-induced pneumonia and providing an effective candidate target for intervention of parasitic pneumonia.S1 Video(MP4)Click here for additional data file.S2 Video(MP4)Click here for additional data file.S3 Video(MP4)Click here for additional data file.S1 Fig(A) The gross morphology of mouse livers at 2, 4, 8, 12 hours post infection of AC (n = 3). (B) The pathological configuration of mouse livers at 2, 4, 8, 12 hours post infection of AC was displayed with H&E staining (n = 3). Magnifications: 40×, 100× and 200×.(TIF)Click here for additional data file.S2 Fig(A-B) The pathological configuration of mouse (A) and rat (B) brain at 1, 2, 3, 4, 5, 6, 7 days post infection of AC was displayed with H&E staining (n = 3). Magnifications: 40×, 100× and 200×.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(A) The representative image of mouse brain at 7, 14, 21 days post infection as shown by H&E staining (n = 3). (B) The relative mRNA level of IL-4, IL-6, IL-10 and IL-13 in mouse brain at 0, 7, 14, 21 dpi of AC (n = 4). (C) The representative image of rat brain at 7, 14, 21, 28, 35, 42 days post infection as shown by H&E staining (n = 3). (D) The relative mRNA level of IL-4, IL-6, IL-10 and IL-13 of rat brain at 0, 7, 14, 21 dpi of AC (n = 4).(TIF)Click here for additional data file.S5 Fig(A-B) The relative mRNA levels of Th1 cytokines  in mouse  and rat  lungs were determined by RT–qPCR (n = 4). *p < 0.05, **p < 0.01, ****p < 0.0001 compared to 0 dpi.(TIF)Click here for additional data file.S6 Fig(A-B) Densitometric analysis of western blot in mouse(A) and rat(B) lungs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to 0 dpi.(TIF)Click here for additional data file.S7 Fig(A-B) The relative mRNA level of CD11b, IBA1, CD3 and CD103 in mouse  and rat  lungs (n = 4). (C) The relative immunofluorescence density of CD11b, IBA1, CD3 and CD103 in rat lungs at 0, 7, 14, 21, 28, 35 and 42 dpi. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to 0 dpi.(TIF)Click here for additional data file.S8 Fig(A-D) Relative mRNA level of RIP3, RIP1, Caspase-3 and Caspase-8 in mouse (A) and rat (C) lungs at the indicated dpi of AC (n = 4). Cell death of mouse (B) and rat (D) pulmonary cells at different time point of AC infection was tested by flow cytometry analysis (n = 4).(TIF)Click here for additional data file.S9 Fig(A) Masson staining of mouse lungs at different dpi of AC (n = 3). (B) Quantitative analysis of masson staining in A. (C) The relative mRNA level of Col1a1, Col3a1 and α-SMA in mouse lungs after AC infection (n = 4). (D) Protein level of TGF-β, Col1a1, Col3a1 and α-SMA in mouse lungs were exhibited by immunoblotting (n = 3). (E) Relative density of the indicated proteins in D. **p < 0.01, ***p < 0.001 compared to 0 dpi.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Our study showed that the Mexican lineage H5N2 AIV originated from the North American wild bird gene pool viruses around 1990 and is currently circulating in poultry populations of Mexico, the Dominican Republic, and Taiwan. Since the implementation of vaccination in 1995, the highly pathogenic AIV (HPAIV) H5N2 virus was eradicated from Mexican poultry in mid-1995. However, the low pathogenic AIV (LPAIV) H5N2 virus has continued to circulate in domestic poultry populations in Mexico, eventually evolving into five distinct clades. In the current study, we demonstrate that the evolution of Mexican lineage H5N2 AIVs involves gene reassortments and mutations gained over time. The current circulating Mexican lineage H5N2 AIVs are classified as LPAIV based on the amino acid sequences of the hemagglutinin (HA) protein cleavage site motif as well as the results of the intravenous pathogenicity index (IVPI). The immune pressure from vaccinations most likely has played a significant role in the positive selection of antigenic drift mutants within the Mexican H5N2 AIVs. Most of the identified substitutions in these viruses are located on the critical antigenic residues of the HA protein and as a result, might have contributed to vaccine failures. This study highlights and stresses the need for vaccine updates while emphasizing the importance of continued molecular monitoring of the HA protein for its antigenic changes compared to the vaccines used.We have demonstrated for the first time a comprehensive evolutionary analysis of the Mexican lineage H5N2 avian influenza virus (AIV) using complete genome sequences ( Orthomyxoviridae, genus Influenzavirus A, and have negative-sense RNA genomes consisting of eight segments coding for at least 10 proteins [Avian influenza viruses (AIVs) are classified in the family proteins . To dateproteins . Accordiproteins . Wild aqproteins . Infectiproteins .The first evidence of the presence of H5N2 AIV in Mexican poultry was demonstrated serologically in October 1993 , and then = 83) and 106 new complete full genomes generated in this study using both maximum likelihood (ML) and Bayesian approaches. Furthermore, to determine H5 HA genetic diversity, cleavage site motif, and antigenic evolution, we performed the analysis of 284 complete HA genes related to the Mexican lineage H5N2 virus.Despite vaccination efforts, repeated AIV surveillance, and biosecurity measures, H5N2 LPAIVs remain enzootic in poultry populations in Mexico. This LP virus has spread to neighboring countries of Guatemala in 1998 and EI Salvador in 2001 , 2005 inhttp://fusiongenomics.com, accessed on 7 May 2021) and the DNAstar SeqMan NGen software .A total of 106 samples sequenced in this study were derived from H5N2 AIVs that were isolated from Mexican poultry . These sn = 334), PB1 (n = 335), PA (n = 324), HA (n = 384), NP (n = 324), NA (n = 305), M (n = 269), and NS (n = 294). All publicly available complete genome sequences and complete HA gene sequences of Mexican lineage H5N2 AIV were downloaded from the Influenza Research Database , accessed on 29 November 2021 and GenBank (https://www.ncbi.nlm.nih.gov/genbank/), accessed on 29 November 2021. This leads to datasets of 189 complete full genome sequences and 284 complete H5 gene sequences that were used in this study.Various sequence datasets were used throughout this study. For the origins of Mexican lineage H5N2 AIV gene segments, the sequences of initial Mexican H5N2 AIV strain A/chicken/Mexico/31381–7/1993 isolated in October of 1993 were used for BLAST with the output of 500 sequences. After removing identical sequences and sequences that did not contain full-length coding regions, the numbers of sequences when combined with recent Mexican lineage H5N2 AIV are as follows: PB2 (The nucleotides in the coding regions of segments 1 (PB2), 2 (PB1), 4 (HA), 5 (NP), and 6 (NA) were aligned using Muscle in MEGA-X . The fulhttp://tree.bio.ed.ac.uk/software/tracer, accessed on 1 December 2021). All parameter estimates for each run showed ESS values >200. The nonparametric Coalescent Bayesian Skyline Plot (BSP) model [A Bayesian approach was implemented to explore additional evolutionary information. TempEst v1.5.3 was used to examine temporal signals in each dataset , taking P) model  was usedTo estimate reassortment events, a coalescent-based phylogenetic network model was employed to allow the reassortant network and the embedding of each segment tree within the network to be jointly inferred with CoalRe .All animal experiments were carried out according to the Mexican Regulations for Animal Welfare. Based on H5 HA cleavage site sequences, a total of 16 viruses with the amino acid motif of PQKRKR/G at the cleavage site were selected for the intravenous pathogenicity index (IVPI) test. To determine the IVPI of field isolates, 10 specific pathogen-free (SPF) chickens (4–6 weeks old) were inoculated intravenously with 0.1 mL of a 1:10 dilution of virus in sterile isotonic saline solution according to the OIE standard protocol [p-value to classify a site as positively or negatively selected was set at 0.1 for SLAC and 0.01 for the MEME method. The cut-off value for the posterior probability in the FUBAR method was set at 0.9 to reflect a positive or negative selection at a given site.Site-specific selection pressures for all segments of the Mexican H5N2 AIVs were measured as nonsynonymous (dN)–synonymous (dS) nucleotide substitutions per site. The differences were estimated using the SLAC (Single-Likelihood Ancestor Counting) , MEME   from A/VThe origins of each of the eight genomic segments of Mexican H5N2 AIV were determined from sequence datasets that comprised AIV sequences related to the initial Mexican H5N2 virus (A/chicken/Mexico/31381–7/1993) by BLAST and sequences generated in this study. As shown in The ML phylogenetic trees showed that the Mexican H5N2 AIV had spread to the neighboring countries of Guatemala, EI Salvador, and the Dominican Republic, and had reached Japan and Taiwan in East Asia since its first isolation in October of 1993 . Consist−3 sub/site/year  for the HA gene and 5.49 × 10−3 sub/site/year  for the NA gene. The estimated rates for the other genome segments ranged from 3.59 × 10−3 to 4.43 × 10−3 sub/site/year. The mean tMRCA estimations showed that Mexican lineage H5N2 AIV originated around 1990  were estimated using a Bayesian coalescent approach for each genome segment separately. The means and 95% highest posterior density (95% HPD) intervals of all genomic segments are shown in Our analysis reveals that the HA, NA, M2, NS1, and NS2 genes have accumulated more nonsynonymous substitutions per site compared to the genes encoding polymerase complex , PA-X, and NP, which exhibited lower nonsynonymous substitutions per site n = 49), 87 aa (n = 87), 79 aa (n = 2), and 52 aa (n = 27). Notably, PB1-F2 with 87 aa was only observed in 2016–2019 viruses. Out of 189 viruses, twenty-four viruses showed a non-functional PB1-F2 protein due to a mutation that resulted in an early stop codon in the PB1-F2 open reading frame.Various amino acid (aa) lengths of PB1-F2 were present within the dataset, including 90 aa . The analysis revealed that the vast majority of codons were subject to purifying selection. Positive selections were detected in PB2 (1 residue), PB1 (3 residues), PA (2 residues), PA-X (4 residues), HA (12 residues), NP (1 residue), NA (4 residues), M2 (4 residues), NS1 (8 residues), and NS2 (2 residues) . The bion = 83). In addition, we generated 106 full genome sequences of Mexican lineage H5N2 AIVs collected between 2016 and 2019. Phylogenetic trees were constructed using full genome sequences of all Mexican lineage H5N2 AIVs available in public databases such as GenBank (n = 284). 2 = 0.90). The HA sequence displayed a strong clock signal. The epidemic history of the Mexican lineage H5N2 AIV from 1994 to 2019 was analyzed using the Bayesian skyline plot (BSP) that depicts changes in the estimated effective population size through time. The BSP showed the enzootic circulation of the viruses in Mexico despite the vaccination program launched in 1995. The effective population size peaked in 2005 and was maintained at a high level until 2015  and 1995 Queretaro isolates (PQRKRKTR/G and PRKRKRKTR/G) in Mexico and one 2014 isolate (PKREKREKR/G) in Taiwan. The basic amino acids at the cleavage site were counted including the Arg residue (−1 position) immediately preceding the junction between HA1 and HA2 subunits but excluding the −4 position found in the LPAI cleavage site consensus motif. Within the examined dataset, a cleavage site containing one basic amino acid was observed in the early isolates of Mexico (1994–2007), Japan (2005–2006), EI Salvador/Guatemala (2000–2003), and the Dominican Republic (2007). A cleavage site containing two to three basic amino acids was observed in recent isolates of Mexico (2016–2019) and the Dominican Republic (2017–2019), while it showed in all isolates of Taiwan (2003–2019).Since the first isolation of a low pathogenic H5N2 virus in chicken in 1993 in Mexico, H5N2 AIV has become enzootic in poultry despite an extensive vaccination program launched in 1995. To investigate the antigenic evolution of Mexican H5N2 AIVs, the full-genome sequences of 106 Mexican H5N2 AIVs were generated. These viruses are distributed throughout 15 states in Mexico and detected by the AIV surveillance system between 2016 and 2019 . PhylogeForty-five critical residues in the H5 HA protein have been determined by monoclonal antibody escape mutant studies ,37,38,39http://www.cbs.dtu.dk/services/NetNGlyc, accessed on 2 January 2022). Analysis of 210 complete Mexican H5 HA sequences showed 18 potential N-glycosylated sites among HA proteins of 1994 to 2019 isolates  shows typical LP polymorphism of two basic amino acids: PQRKTR/G (n = 87), PQRETR/G (n = 1), and PQKGKR/G (n = 1), and three basic amino acids: PQKRKR/G (n = 16) and SQKRKR/G (n = 1). However, examination of viral pathogenicity by intravenous pathogenicity indexing (IVPI) in four- to eight-week-old chickens of selected Mexican H5N2 isolates with cleavage site sequence of PQKRKR/G demonstrated low pathogenicity of the virus (Our data showed that Mexican lineage H5N2 AIVs are enzootic in Mexico, the Dominican Republic, and Taiwan. This H5N2 virus has undergone rapid genetic diversification. It is five times more diversified in recent isolates than the initial isolates in 1994. Notably, Mexican H5N2 AIVs have diversified into at least five clades as of 2019. Analysis of the H5 HA gene showed a low pathogenic sequence motif at the cleavage site for Mexican lineage H5N2 AIVs except for some early 1994–1995 isolates in Mexico and one 2014 isolate in Taiwan. The H5 HA cleavage site sequence of 2016–2019 Mexican H5N2 viruses (he virus . Since HThe implementation of a vaccination program in 1995 within Mexican poultry coupled with improved biosecurity measures and active surveillance has eliminated the occurrence of high pathogenic H5N2 viruses. However, the low pathogenic H5N2 viruses continue to persist in Mexican poultry, as demonstrated in this study. The HA protein is the major antigenic component of the virus, and currently, forty-five amino acids are characterized as critical residues for antibody–antigen binding by escape mutant studies ,37,38,39In summary, we have used complete genome sequence data to perform a comprehensive analysis of the evolution of the Mexican lineage H5N2 AIVs since its emergence in 1993/1994. The evolutionary properties of the major antigenic determinants of the HA gene were analyzed against the two currently used vaccines in 2016–2019 Mexican H5N2 AIVs. Findings from this study suggest the need for vaccine updates and emphasize the importance of continuing molecular characterization of the antigenic determinant for its antigenic and phenotypic changes.