The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018–2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations. The highly pathogenic (HPAI) avian influenza AH5N1) A/goose/Guangdong/1/1996 (Gs/Gd) virus emerged in China in 1996 and subsequently evolved into multiple genetic clades  A/goose/,3,4. FroPrevious studies have shown that live bird markets (LBMs) and poultry trade are major contributors to the persistence and spatial distribution of avian influenza viruses (AIVs), particularly A(H5N1), A(H5N6), A(H7N9) and A(H9N2) subtype viruses ,15,16,17Surveillance studies in Pakistan have frequently described the prevalence and evolutionary relationships of avian A(H9N2) viruses since 1990s, as the virus is endemically circulating in the poultry population ,29,30,31n = 5) were cultured in 9-day-old embryonated chicken eggs for 48 h and amniotic–allantoic fluids were tested for influenza A virus by hemagglutination assay (HA) using 1% chicken red blood cells [During September 2018–March 2019, avian influenza virus (AIV) surveillance was conducted in six LBMs in three districts  of Punjab Province, Pakistan . These Lod cells . Individod cells . HemagglTotal viral RNA was extracted from the cultured isolates using the QIAamp Viral RNA Mini Kit  following the manufacturer’s instructions. Full-genome sequencing of influenza A virus was performed by next-generation sequencing. Briefly, cDNA synthesis and RT-PCR amplification were performed using the SuperScript III Platinum One-step qRT-PCR kit  using the following three primers as previously described : Opti1-FAll available influenza A(H5Nx) sequences were downloaded from NCBI and GISAID databases (as of 4 March 2021). Large dataset phylogenies were reconstructed using FastTree in Geneious v7.1.9  and the datasets were then sub-sampled to include clade 2.3.4.4a–h viruses. For each gene segment, dated phylogenies and changes in relative genetic diversity were estimated using the uncorrelated lognormal relaxed clock with the Gaussian Markov random field (GMRF) Bayesian skyride model and the SRD06 codon position model in BEAST v1.10.4 . At leasDuring 2018–2019, we detected 77 (17.1%) of 449 samples from chickens and quails as being positive for influenza A virus based on hemagglutination inhibition assay and NGS sequencing , includiFrom the 77 Pakistani influenza-A-positive samples, we recovered A(H5N8) virus genomes from 2 chickens, 1 quail and 1 environmental (chopping board) sample collected from Lahore LBMs . PhylogeThe mean time of the most recent common ancestor (TMRCA) of all Pakistan H5-HA viruses is estimated as August 2018  viruses possessed the polybasic amino acid cleavage site (PLREKRRKR/GLF), identical to other clade 2.3.4.4b viruses. Pakistan A(H5N8) viruses also have a D155N HA mutation (H5 numbering), with additional G46E and E251K mutations in 1 quail and 1 environmental sample. Mutations S155N and E251K in A(H5N1) virus are known to increase HA affinity to human α-2,6 receptors . The N8-The internal gene constellations of Pakistan A(H5N8) viruses are unequivocally derived from the same Saudi Arabia A(H5N8) lineage . The meaEarly clade 2.3.4.4b viruses were detected in geese and ducks in China during 2013–2014. The mean TMRCA estimates of the HA and NA genes of clade 2.3.4.4b are August 2012 (95% HPD: October 2011–April 2013) and April 2013 (95% HPD: November 2012–August 2013), respectively. We also observed several key mutations of clade 2.3.4.4b that occurred on the trunk of phylogenetic trees . Some ofSkyride analysis of all eight gene segments of clade 2.3.4.4b viruses exhibited an increase in genetic diversity that peaked in early 2017  infection in Russia in late December 2020, clustering in subgroup 8 that contains clade 2.3.4.4b A(H5N8) viruses which circulated in domestic and wild birds across Europe during May 2020–February 2021 c. The meLive bird markets (LBMs) are associated with avian influenza transmission and outbreaks, providing environmental sources for virus persistence  and amplBetween 2018 and 2019, we detected and identified four avian HPAI A(H5N8) viruses in chickens and quails from LBMs in Lahore. We also detected environmental contamination with A(H5N8) from the same markets. Our phylogenetic analyses of Pakistan A(H5N8) viruses showed close genetic and phylogenetic similarities, all belonging to clade 2.3.4.4b viruses, showing that LBMs play a significant role in transmission of the virus among domestic birds. We showed that all eight gene segments of Pakistan HPAI A(H5N8) viruses were most closely related to viruses from Saudi Arabia, suggesting they were most likely introduced to Pakistan through the movement of live poultry from neighboring regions, while the similar TMRCA dates across all eight genes indicate that the viruses are likely derived from a single avian source that was introduced approximately three months before virus detection. The presence of viruses on chopping boards suggests that environmental contamination may occur through slaughtering or processing of infected live poultry. A previous study in China indicated that the highest H7 positivity rates came from chopping boards . The preThe emergence of HPAI A(H5N8) clade 2.3.4.4b viruses in Pakistan in chickens is likely associated with poultry production. In Pakistan, poultry production has been growing significantly since the 1960s, with an increase of 126% in total meat production and an increase of 71% in total egg production during 2000–2010, with the main chicken exports being made to neighboring regions, including Afghanistan, Iran and Turkey . A studyIn this study, we also showed that clade 2.3.4.4b viruses exhibited markedly rapid diversification that peaked in 2017, with the acquisition of non-synonymous amino acid mutations on most viral genes. Some of these mutations are associated with mammalian adaptation, suggesting domestic poultry and wild birds are playing a significant role in virus evolution and potential cross-species transmission. Environmental contamination in LBMs also provides a persistent source for poultry infection, and effective control strategies to mitigate environmental contamination with AIV in LBMs are needed. The recent infections of poultry workers from Russia and of gray seals  indicate


Human type A influenza viruses A(H1N1)pdm09 have caused seasonal epidemics of influenza since the 2009–2010 pandemic. A(H1N1)pdm09 viruses had a leading role in the severe epidemic season of 2015/16 in the Northern Hemisphere and caused a high incidence of acute respiratory infection (ARI) in Ukraine. Serious complications of influenza-associated severe ARI (SARI) were observed in the very young and individuals at increased risk, and 391 fatal cases occurred in the 2015/16 epidemic season. We analyzed the genetic changes in the genomes of A(H1N1)pdm09 influenza viruses isolated from SARI cases in Ukraine during the 2015/16 season. The viral hemagglutinin (HA) fell in H1 group 6B.1 for all but four isolates, with known mutations affecting glycosylation, the Sa antigenic site , or virulence (D222G/N in two isolates). Other mutations occurred in antigenic site Ca (A141P and S236P), and a subgroup of four strains were in group 6B.2, with potential alterations to antigenicity in A(H1N1)pdm09 viruses circulating in 2015/16 in Ukraine. A cluster of Ukrainian isolates exhibited novel D2E and N48S mutations in the RNA binding domain, and E125D in the effector domain, of immune evasion nonstructural protein 1 (NS1). The diverse spectrum of amino-acid substitutions in HA, NS1, and other viral proteins including nucleoprotein (NP) and the polymerase complex suggested the concurrent circulation of multiple lineages of A(H1N1)pdm09 influenza viruses in the human population in Ukraine, a country with low vaccination coverage, complicating public health measures against influenza. Human influenza A viruses (IAV) are a major threat to public health and the global economy, causing annual seasonal epidemics and occasional novel pandemics. Influenza viruses, including the H1N1 swine-origin 2009 pandemic lineage viruses, H3N2 viruses, and influenza B viruses, infect 5–10% of adults and 20–30% of children annually [When effective, influenza A and B vaccines protect the general population and particularly people from high-risk groups  against severe disease, or influenza-like illness (ILI). However, the vaccine composition must be annually changed because of antigenic drift among influenza viruses in the human population.The development of recommendations for vaccine composition is based on phylogenetic and immunological analyses of influenza viruses in the previous epidemic season. The phylogenetic analysis is, however, usually limited just to the analysis of nucleotide gene sequences of viral hemagglutinin (HA) and neuraminidase (NA) genes. Even using state-of-the-art analysis methods, including antigenic cartography built from hemagglutination inhibition (HI) assay data , the preType A influenza viruses, particularly pdmH1N1 viruses and their genetic descendants that are the cause of seasonal H1N1 epidemics, continually evolve in the human population. H1N1 viruses have accumulated mutations that are associated with higher replication (fitness), increased binding to α-2,6-sialic acid receptors on respiratory epithelial cells, and escape from innate and adaptive immune responses . In the In Ukraine during the 2015/16 influenza epidemic season, it was hypothesized that the A(H1N1)pdm09 influenza strains that were circulating in the Northern Hemisphere were contributing to markedly increased disease incidence and serious complications in patients. The A(H1N1)pdm09 influenza subtype dominated the epidemic curve in the 2015/16 season . While 3Influenza vaccination rates in Ukraine during 2015/16 were only 0.3% in comparison to those in the European Union (EU), due to a lack of systematic national or regional-level vaccination campaigns . HoweverIn order to improve our understanding of the Ukrainian 2015/16 epidemic, we analyzed the genetic changes that occurred in the dominant influenza subtype, A(H1N1)pdm09, during this period. While in virus genetics studies of IAV, the focus is typically directed to the surface proteins (HA and NA) that are the key targets for neutralizing antibodies, changes in the virulence and pathogenicity of influenza viruses can also be harbored mutations in the polymerase, matrix and nucleoprotein, and non-structural proteins . Thus, wViruses isolated in Ukraine in the 2015/16 season belong to the 6B genetic group in which two new subgroups, namely 6B.1 and 6B.2, emerged in that season . These sThe phylogenetic analysis of HA revealed a number of amino-acid substitutions in A(H1N1)pdm09 influenza viruses from Ukraine isolated during the 2015/16 epidemic. HA P83S, I321V, and S203T substitutions existed in Ukrainian isolates . These sIn the large Ukrainian 6B clade branching off the A/Norway/3023/2014 strain, the K163Q, A256T, K283E substitutions also arose. Such mutations are characteristic of the 6B genetic group, which is divided into two subgroups, 6B.1 and 6B.2 . Most ofFour Ukrainian isolates from Odessa were in the 6B.2 subgroup, having acquired HA substitutions R113K, D127E , E164G (HA2), and the E47Q (HA2). Similar to the A/England/377/2015 strain, they also harbored substitutions characteristic of the 6B.2 genetic group at V152T and V173I. The rare V152T aminoacyl substitution has occurred in 18 isolates in nature, and in cell culture, and affects antigenic properties [Variation of potential glycosylation sites in HA. The acquisition or loss of glycosylation sites leads to changes in the topology of the hemagglutinin (HA) protein structure. Acquiring a site for glycosylation may be beneficial for the virus, as, due to this, the masking of antigenic sites that are recognized by the antibodies of the human organism occurs. Thus, the viruses can potentially regulate their antigenicity [genicity . For A sequences, we found that all A(H1N1)pdm09 isolates sequenced from the 2015/16 season in Ukraine formed a subclade of the A/SouthAfrica/3626/2013 reference strain [All Ukrainian isolates in the clades phylogenetically descending from the common ancestor of A/St.Petersburg/27/2011 and A/SouthAfrica/3626/2013 acquired the amino-acid substitutions V241I and N369K. The A/SouthAfrica/3626/2013 subclade, that included all Ukrainian isolates, harbored I34V, N44S, N200S, I321V, and K432E, substitutions that were absent in most of the reference strains . The majMost of the Ukrainian isolates belonged to the 6B.1group and acquired amino-acid substitutions such as V13I, I34V, V264I, N270K, and I314M . The A/KMatrix proteins. Two matrix proteins, M1 and M2, are encoded in the MP gene (segment 7) of influenza A viruses. For phylogenetic analysis, isolates were taken from the 2014/15 and 2015/16 epidemic seasons. All isolates including the A/California/07/2009 vaccine strain had the D21G mutation in the M2 protein pdm09 influenza virus NP for the 2015/16 season in Ukraine. However, isolates differed from the vaccine strain with the A22T, V100T, L122Q, S498N substitutions  such as PKR and OAS, modulating pro-inflammatory gene expression, and inducing host protein shutoff by disrupting mRNA processing and export .Ukrainian isolates differed from the vaccine strain with NS1 substitutions E55K, L90I, I123V, K131E, and N205S, which were common in other viruses in the 2014/15 and 2015/16 epidemic years . Recent In the 2015/16 season, a series of new amino-acid substitutions was found in the NS1 protein in the major subgroup of strains in Ukraine, as follows: D2E, N48S, and E125D . All isoIt was suggested that D2E and N48S in the RNA binding domain, and E125D in the NS1 effector domain, are involved in host adaptation . E125D iSome isolates had other substitutions in NS1 such as N48S and M124T, which were identified in 12.5% of the studied viruses; three isolates had the L95I replacement. A second subgroup of seven isolates lacked the mutations of the major subgroup but instead included I18V, or V129I and I182V, respectively .NS2. In the NS2 protein, three amino-acid substitutions, N29S, T49N, and M83I, were found compared to the vaccine strain. However, how these mutations might affect viral virulence is unknown.The RNA-dependent RNA polymerase complex of influenza viruses is a heterotrimer that consists of three proteins, encoded by three separate gene segments: PA, PB1, and PB2. Besides viral transcription and replication, the polymerase complex is increasingly recognized as an essential host factor for host adaptation .PA polymerase protein. The PA gene remained similar to the vaccine strain, with a few notable substitutions. All Ukrainian isolates acquired V100I, P224S, N321K, I330V, and R362K pdm09 viruses with an increased prevalence in comparison to the two previous influenza seasons . To undeAs contemporaneous vaccinations in Ukraine are typically <5% , the potThe World Health Organization (WHO) encourages National Influenza Centers (NICs) to conduct ongoing influenza virologic surveillance, to monitor the spread of viruses and their continuous evolution. Combining data from phylogenetic and molecular analyses of influenza viruses is essential to detect virus variants that have undergone antigenic drift, variants with enhanced virulence, or variants with reduced sensitivity to antivirals. Combined genetic, antigenic, and phylogenetic analyses provide improvements in the process of vaccine virus selection and inform patient treatment regimens ,32,52.As expected, most of the diversity occurred in the HA and NA genes , with thVariations in other viral proteins were also observed. All 6B-lineage NS1 proteins contained a spectrum of variations  associated with host interferon inhibition and increased virulence in the A(H1N1)pdm09 virus in human and animal models ,36,37. IOther substitutions were found in NA, NP, M1, M2, NS2, and the polymerase complex among Ukrainian strains, but their effect(s) on the viral life cycle and clinical significance requires further study ,48,49,50The observed rapid spread of influenza A(H1N1)pdm09 viruses may be in part explained by antigenic changes in HA, possibly reflecting an escape from immune responses, increased transmissibility, or a change in virulence. Several mutations observed sporadically in Ukrainian A(H1N1)pdm09 influenza viruses in the 2015/16 season became much more prevalent in subsequent years in other countries, suggesting a fitness advantage and ongoing evolutionary pressure . For exa2) [Nasal swabs and sputum samples were collected during the routine diagnosis of influenza-like illness (ILI) in patients from ten sentinel hospitals and eight outpatient clinics in four cities of Ukraine . We randomly selected 1246 specimens for PCR diagnostics and sequencing analyses from 4964 hospitalized patients identified by the WHO SARI case definition guidance, including all conditions: acute respiratory infection (ARI) with an onset during the previous 7 days requiring overnight hospitalization; history of fever >38 °C; cough; and shortness of breath or difficulty breathing. All clinical samples were collected under bioethical guidelines for human subjects research under Institutional Review Board approval #281 (11/01/2002), approving authority: Ministry of Health of Ukraine. Clinical samples were analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the influenza A matrix gene (MP) segment and for the influenza type  and subtype  . Clinica2) . Viruseshttp://platform.gisaid.org/) database. Reference strains included vaccine strain A/California/07/2009 for A(H1N1)pdm09, and isolates from around the world that were temporally and geographically most similar to viruses isolated in Ukraine were included in the phylogenetic analysis. The nucleotide sequences were translated into protein sequences. In the 2015/16 season in Ukraine, we isolated the A(H1N1)pdm09 influenza viruses. The analysis of genetic changes of pandemic viruses was carried out for all of the genes encoding the two surface proteins; hemagglutinin (HA) and neuraminidase (NA); polymerase complex proteins ; nucleoprotein (NP); M1 and M2 matrix proteins; and non-structural proteins such as NS1 and NS2/NEP (nuclear export protein). The phylogenetic analysis and analysis of mutations were performed using the MEGA7 program by the NJ (neighbor-joining) method and Kimura 2-parameter model, with 1000 bootstrap iterations [For the phylogenetic analysis, sequences of entire genomes of pandemic A(H1N1)pdm09 influenza viruses were obtained from the Global Initiative on Sharing Avian Influenza Data pdm09 influenza virus that was obtained from the Protein Data Bank. The Chimera 1.11.2rc software was used to visualize mutations .


Vitamin D deficiency has been associated with type 2 diabetes (T2D) and metabolic syndrome (MS) and its components. However, it is unclear whether a low concentration of vitamin D is the cause or consequence of these health conditions. Thus, this study aimed to evaluate the association of vitamin D concentrations and its genetic risk scores (GRSs) with MS and its component diseases, such as T2D, in middle-aged and elderly participants from rural eastern China.A subset of 2393 middle-aged and elderly individuals were selected from 70,458 participants of the Nantong Chronic Diseases Study of 2017–2018 in China. We used two 25-hydroxyvitamin D (25[OH]D) synthesis single-nucleotide polymorphisms (SNPs) (DHCR7-rs12785878 and CYP2R1-rs10741657) and two 25(OH) D metabolism SNPs (GC-rs2282679 and CYP24A1-rs6013897) for creating GRSs, which were used as instrumental variables to assess the effect of genetically lowered 25(OH) D concentrations on MS and T2D based on the Wald ratio. F statistics were used to validate that the four SNPs genetically determined 25(OH) D concentrations.Compared to vitamin D sufficient individuals, individuals with vitamin D insufficiency had an odds ratio  of MS of 1.30 (1.06–1.61) and of T2D of 1.32 (1.08–1.64), individuals with vitamin D deficiency had an ORs (95% CI) of MS of 1.50 (1.24–1.79) and of T2D of 1.47 (1.12–1.80), and those with vitamin D severe deficiency had an ORs (95% CI) of MS of 1.52 (1.29–1.85) and of T2D of 1.54 (1.27–1.85). Mendelian randomization analysis showed a 25-nmol/L decrease in genetically instrumented serum 25(OH) D concentrations using the two synthesis SNPs (DHCR7 and CYP2R1 genes) associated with the risk of T2D and abnormal diastolic blood pressure (DBP) with ORs of 1.10 (95%CI: 1.02–1.45) for T2D and 1.14 (95%CI: 1.03–1.43) for DBP.This one sample Mendelian randomization analysis shows genetic evidence for a causal role of lower 25(OH) D concentrations in promoting of T2D and abnormal DBP in middle-aged and elderly participants from rural China.The online version contains supplementary material available at 10.1186/s12877-021-02307-6. Metabolic syndrome (MS) encompasses a cluster of conditions such as abdominal obesity, hypertension, dyslipidemia, and hyperglycemia , that coVitamin D deficiency is common in European, Indian, South American, and Chinese populations and is particularly notable in middle-aged and elderly Chinese populations , 11. VitDHCR7/NADSYN1 (7-dehydrocholesterol reductase) and CYP2R1 (25-hydroxylase) affect the synthesis of 25(OH)D; the transport gene GC (group-specific component) encodes the vitamin D-binding protein, and the catabolism gene CYP24A (24-hydroxylase) is involved in the clearance of 25(OH) D ) and analyzed using the Wilcoxon rank sum test. Categorical variables are expressed as percentages; the Pearson’s chi-square test was used for comparison between MS/T2D cases and MS/T2D non-cases. Furthermore, clinical characteristics  were compared between the quintiles of 25(OH) D concentration groups using ANOVA for continuous variables with normal distribution, the Kruskal-Wallis test for continuous variables with non-normal distribution, and Pearson’s chi-square test for categorical variables.βZX)), assuming linear effects of each vitamin-D related SNP per additional allele on 25(OH)D. The Cragg-Donald F-statistic βZY)). Genetically determined A(βZX) and C(βZY) were calculated using the Wald ratio estimator (βIV = βZY/βZX) : 1.10 [1.02–1.45]) and abnormal DBP (1.14 [1.03–1.43]). Moreover, we did not find any association between genetically instrumented serum 25(OH) D concentration of two metabolism and all four SNPs with T2D. Null results were obtained between genetically instrumented serum 25(OH) D concentrations using any single SNP and MS or T2D in the middle-aged and elderly participants from eastern rural China (data not shown).Table IV per 25-nmol/L increase in 25(OH) D concentrations has been shown in Additional file synthesis and T2D and abnormal DBP (OR [95%CI]: 1.08[1.03–1.38] and 1.09[1.02–1.37] per 25-nmol/L 25(OH) D decrease).The results of the sensitivity analyses of another two-stage regression estimator for calculating the ORVitamin D levels are known to influence the development of MS and component diseases, but the causality or direction of the association has been uncertain. This study showed that genetically determined 25(OH) D levels are causally associated with T2D and abnormal DBP in middle-aged and elderly rural participants from east rural China.Many epidemiological studies have found inverse associations between serum 25(OH) D levels and MS and its component diseases , 37, 38.P = 0.0290) [P = 0.0003) [DHCR7/NADSYN1 and CYP2R1, transport gene GC, and catabolism gene CYP24A1 contribute to variability in circulating biomarkers of vitamin D levels [MR studies have provided no evidence to suggest that genetically increased serum 25(OH) D concentrations are associated with a lower risk of MS, T2D, or hypertension , 27, 45. 0.0290) . A previ 0.0003) . The preD levels , 47. IntD levels . AnalysiD levels , 49. In D levels .Using a genetic variant as a proxy for vitamin D levels has been considered to provide better causal inferences for several reasons. First, unlike vitamin D levels, genetic variants are generally not associated with behavioral, social, and physiological factors that confound the association between vitamin D and MS and its associated diseases. Second, genetic variants associated with vitamin D levels are not influenced by other diseases, and the estimates are therefore less biased. Third, a genetic variant will often reflect exposure throughout the life course and will not change with disease status –53. FinaA limitation of the present study was the single measurement of vitamin D levels. The lack of 25(OH) D GWAS in Asian populations that would allow building of an ethnic-specific genetic score limits the use of MR. Furthermore, the four SNPs explain only approximately 1–4% of the variance in the 25(OH) D phenotype . A GRS cThe serum 25(OH) D concentration was inversely associated with MS and T2D risk in the rural middle-aged and elderly participants. However, MR analysis demonstrated concordance between genetically determined 25(OH) D using two synthesis SNPs and the risk of T2D and abnormal DBP in middle-aged and elderly participants from eastern rural China. This indicates a risk effect of lower serum 25(OH) D concentrations on the development of T2D and abnormal DBP. Conversely, genetically determined vitamin D was not significantly associated with the development of MS; lower vitamin D concentrations are therefore unlikely to have a causal role in the development of MS. Therefore, further trials will be required on vitamin D supplementation before advocating the use of vitamin D supplements or food fortification for the prevention of MS and T2D.Additional file 1. Causal coefficients from MR analysis.


Background: The association between the CYP17A1 and ATP2B1 SNPs and essential hypertension (referred to as hypertension) is far from being consistent. In addition to the heterogeneity of hypertension resulting in inconsistent results, gene–gene and gene–environment interactions may play a major role in the pathogenesis of hypertension rather than a single gene or environmental factor.Methods: A case–control study consisting of 1,652 individuals  was conducted in Maonan ethnic minority of China. Genotyping of the four SNPs was performed by the next-generation sequencing technology.Results: The frequencies of minor alleles and genotypes of four SNPs were different between the two groups (p < 0.001). According to genetic dominance model analysis, three  SNPs and two haplotypes (CYP17A1 rs1004467G-rs11191548C and ATP2B1 rs1401982G-rs17249754A) were negatively correlated, whereas rs1401982 SNP and the other two haplotypes (CYP17A1 rs1004467A-rs11191548T and ATP2B1 rs1401982A-rs17249754G) were positively associated with hypertension risk . Two best significant two-locus models were screened out by GMDR software involving SNP–environment (rs11191548 and BMI ≥ 24 kg/m2) and haplotype–environment (CYP17A1 rs1004467G-rs11191548C and BMI ≥ 24 kg/m2) interactions (p ≤ 0.01). The subjects carrying some genotypes increased the hypertension risk.Conclusions: Our outcomes implied that the rs1004467, rs11191548, and rs17249754 SNPs and CYP17A1 rs1004467G-rs11191548C and ATP2B1 rs1401982G-rs17249754A haplotypes have protective effects, whereas the rs1401982 SNP and CYP17A1 rs1004467A-rs11191548T and ATP2B1 rs1401982A-rs17249754G haplotypes showed adverse effect on the prevalence of hypertension. Several SNP–environment interactions were also detected. Essential hypertension (referred to as hypertension) is a regular multifactorial disease affecting about one-fourth of adults worldwide . Convers2+ transporting, plasma membrane 1 gene (ATP2B1) and cytochrome P450, family 17, subfamily A, and polypeptide 1 gene (CYP17A1)  can screen and analyze hypertension risk genes . For insCYP17A1) , 7. The c groups –12. In phe GWASs . HoweverCYP17A1 encodes the P450c17 protein, a member of the cytochrome P450 superfamily of enzymes speeding up plenty of chemical synthesis processes involving steroids, cholesterin, and other blood fats  , the frequency of rs1004467GG genotype in the Chinese Han population was 0.364, which was slightly higher than the genotype frequency of 0.322 in the Japanese population, but both were significantly higher than that (0.104) in the European population. These differences may be caused by evolutionary divergence, or it may be the result of negative selection of rs1004467 risk alleles in European populations. Therefore, we should continue (1) to evaluate the differences in genotypes and allele frequencies in other populations of different ancestry; (2) to screen larger cohorts with clinical BP abnormalities; and (3) to evaluate gene–gene (G × G) and gene–environment (G × E) interactions on BP and hypertension, which are very meaningful and necessary (The above studies have shown significant association between the  factors : (1) ignecessary .p < 0.001) (ATP2B1 (rs1401982 and rs17249754) and CYP17A1 (rs1004467 and rs11191548) SNPs, and their haplotypes, G × G and G × E interactions, with hypertension in the Maonan population.Maonan is one of the mountain ethnic groups with a small population in China . Its liv< 0.001) . HoweverCYP17A1 and ATP2B1: (1) SNPs belonging to tagging SNPs were detected by Haploview . (2) CYP17A1 (rs1004467 and rs11191548) and ATP2B1 (rs1401982 and rs17249754) SNPs were then chosen by SHEsis Main . (3) The minor allele frequency (MAF) of the SNPs was more than 1%. (4) SNPs may be associated with hypertension according to the previous investigations. (5) SNP-related information was acquired from NCBI dbSNP Build 132 (http://www.ncbi.nlm.nih.gov/SNP/).There were five steps for screening four SNPs of A total of 1,652 Maonan subjects were randomly extracted from previously stratified random samples to conduct a cross-sectional study of hypertensive molecular epidemiology . The par2) was calculated as weight/(height2). Sitting BP was determined three times after taking a rest at least 5 min using a manual sphygmomanometer, and the average of three readings was used for BP analysis , triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were tested by commercially available enzyme assays , and allThe genome DNA was isolated from venous blood white cells with phenol-chloroform . All DNA2 was defined as normal weight, overweight, and obesity, respectively (Hypertension was defined as an average systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg, or using drugs for treating high BP . Hyperliectively .t-test, Wilcoxon–Mann–Whitney test, and chi-square test, respectively. The analyses of Hardy–Weinberg equilibrium (HWE), genotype and allele frequencies, pairwise LD, and haplotype frequencies were mainly performed by the SHEsis online genetics software      , 39. Logsis.php) , 36. A pn (GMDR) , 40–42. n (GMDR) . Finally-values) . G × G a-values) . Besides(0.05/3) .p < 0.001). However, there was no difference in age, sex ratio, smoking, and drinking between the control and case groups .The demographic parameters of 1,652 subjects are shown in p < 0.001). p > 0.05 for all). Simultaneously, the rs1401982 SNP enhanced the risk of hypertension, whereas the rs1004467, rs11191548, and rs17249754 SNPs decreased the susceptibility of hypertension in the dominant model .As shown in D' = 0.950) or between the rs1401982 and rs17249754 SNPs (D' = 0.951) was strong in both control and hypertension groups (CYP17A1 rs1004467A-rs11191548T and ATP2B1 rs1401982A-rs17249754G (≥ 67% of the samples). The frequencies of CYP17A1 rs1004467A-rs11191548T, CYP17A1 rs1004467G-rs11191548C, ATP2B1 rs1401982A-rs17249754G, and ATP2B1 rs1401982G-rs17249754A haplotypes were significantly different between the control and case groups. Meanwhile, the haplotypes of CYP17A1 rs1004467A-rs11191548T, CYP17A1 rs1004467G-rs11191548C, and ATP2B1 rs1401982G-rs17249754A had a protective effect for hypertension, whereas the haplotype of ATP2B1 rs1401982A-rs17249754G revealed an increased susceptibility of disease (p < 0.001).LD analysis showed that the four SNPs did not have statistical independence in the control or case group. However, the LD between the rs1004467 and rs11191548 (n groups . As show2 (p = 0.01), with a cross-validation consistency (7/10) and a testing accuracy of 62.7%. Another significant two-locus model  indicated a potential haplotype–environment interaction, with a cross-validation consistency (9/10) and a testing accuracy of 63.5%.The GMDR model was utilized to analyze the interaction of G × G and G × E among SNPs, haplotypes, BMI, age, gender, alcohol, and/or cigarette usage on the risk of hypertension. 2), haplotype–haplotype interaction (CYP17A1 rs1004467A-rs11191548T and CYP17A1 rs1004467G-rs11191548C), and haplotype–environment interaction (CYP17A1 rs1004467G-rs11191548C and age), respectively. In order to obtain the OR and 95% CI for the joint effects, we implemented an interaction study by logistic regression analyses  compared to the individuals with rs11191548 TT and BMI ≥ 24 kg/m2.Entropy-based interaction dendrograms obtained from MDR analysis are shown in analyses . When thATP2B1 and CYP17A1 SNPs, and their haplotypes, G × G and G × E interactions, with hypertension in the Maonan population was observed for the first time. The main findings are as follows: (1) The genotype and allele frequencies of the CYP17A1 rs1004467, CYP17A1 rs11191548, ATP2B1 rs1401982, and ATP2B1 rs17249754 SNPs were significantly different between the control and hypertension groups. (2) The ATP2B1 rs1401982 SNP enhanced the risk of hypertension, whereas the CYP17A1 rs1004467, CYP17A1 rs11191548, and ATP2B1 rs17249754 SNPs decreased the prevalence of hypertension in the dominant models. (3) The frequencies of CYP17A1 rs1004467A-rs11191548T, CYP17A1 rs1004467G-rs11191548C, ATP2B1 rs1401982A-rs17249754G, and ATP2B1 rs1401982G-rs17249754A haplotypes were significantly different between the control and case groups. (4) The CYP17A1 rs1004467A-rs11191548T, CYP17A1 rs1004467G-rs11191548C, and ATP2B1 rs1401982G-rs17249754A haplotypes had a protective effect for hypertension, whereas the ATP2B1 rs1401982A-rs17249754G haplotype increased the risk of hypertension. (5) Several interactions including rs11191548-BMI ≥ 24 kg/m2 (SNP–environment) and rs1004467G-rs11191548C-BMI ≥ 24 kg/m2 (haplotype–environment) on the risk of hypertension were also observed. (6) The individuals with rs11191548 TC/CC genotypes and BMI ≥ 24 kg/m2 raised the risk of hypertension.In this cross-sectional study of hypertensive molecular epidemiology, the association of the ATP2B1 and CYP17A1 have correlation with BP and/or hypertension . The CYP17A1 rs1004467G allele frequency in Chinese Dai in Xishuangbanna, China (CDX), Han Chinese in Beijing, China (CHB), and Southern Han Chinese (CHS) was 28.49, 36.41, and 35.71%, respectively. The CYP17A1 rs11191548C allele frequency in CDX, CHB, and CHS was 25.27, 29.61, and 28.10%, respectively. The ATP2B1 rs1401982G allele frequency in CDX, CHB, and CHS was 27.96, 34.95, and 39.05%, respectively. The ATP2B1 rs17249754A allele frequency in CDX, CHB, and CHS was 18.82, 32.04, and 36.67%, respectively. In the present study, we found that the MAF of these SNPs was lower than other Chinese, especially in the hypertension group, but it was higher in our study populations than in Europeans or Africans. These results might also be a reasonable explanation for the distinct prevalence of hypertension between Chinese and Europeans or Africans.In the past 10 years, according to the results of GWAS scans, both rtension , 7. Howeractions . The genMaonan people not only like to eat beef, pork, and animal viscera, all of which are rich in saturated fatty acid, but also like sour marinated meat, snails, and sour pickles that contain a lot of salt . High-faThere was no statistical significance in alcohol and cigarette consumption rates between control and hypertension groups in our research. The effects of drinking and smoking on hypertension have been reported by previous articles. The extent to which alcohol is associated with hypertension may be partly related to the amount of alcohol consumed –51. Low p < 0.001). These results showed that CYP17A1 and ATP2B1 SNPs were correlated with hypertension and genetic factors might play a part in susceptibility to hypertension. Furthermore, according to genetic dominance model analysis, three SNPs  and two haplotypes (CYP17A1 rs1004467G-rs11191548C and ATP2B1 rs1401982G-rs17249754A) were negatively correlated with hypertension risk, while the rs1401982 SNP and the other two haplotypes (CYP17A1 rs1004467A-rs11191548T and ATP2B1 rs1401982A-rs17249754G) were positively associated with hypertension risk (p ≤ 0.002). Meanwhile, GMDR analysis showed no statistical difference between the interaction of CYP17A1 and ATP2B1 on hypertension. However, two best significant two-locus models were screened out involving SNP–environment (rs11191548 and BMI ≥ 24 kg/m2) and haplotype–environment (CYP17A1 rs1004467G-rs11191548C and BMI ≥ 24 kg/m2) interactions (p ≤ 0.01). The participants with the rs11191548 TT genotype and BMI ≥ 24 kg/m2 had higher risk of hypertension than the individuals with the rs11191548 TC/CC genotypes and BMI ≥ 24 kg/m2. G × E interaction on the development of hypertension was also observed in this cross-sectional study.In the current study, minor allele and genotype frequencies of all four SNPs had a difference in control and case groups (ATP2B1 encoding plasma membrane Ca2+ ATPase 1 (PMCA1) is strongly associated with BP and hypertension. Several studies have confirmed that PMCA1 plays a physiological role in regulating BP and resistance artery function. PMCA1 may be a potential target for the treatment of essential hypertension . The patients/participants provided their written informed consent to participate in this study.B-LW conceived the research, took part in design, performed genotyping and statistical analysis, and drafted the manuscripts. R-XY conceived the research, took part in the design, conducted the epidemiological investigation, collected the samples, and helped to draft the manuscript. C-XL collaborated to the genotyping. G-XD, Y-ZG, and P-FZ conducted the epidemiological investigation and helped to collect the samples. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 81460169). There was no role of the funding body in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Influenza A virus infections occur in different species, causing mild-to-severe symptoms that lead to a heavy disease burden. H1N1, H1N2 and H3N2 are major subtypes of swine influenza A viruses in pigs and occasionally infect humans.A case infected by novel influenza virus was found through laboratory surveillance system for influenza viruses. Clinical specimens were tested by virus culture and/or real-time RT–PCR. The virus was identified and characterized by gene sequencing and phylogenetic analysis.In 2021, for the first time in Taiwan, an influenza A(H1N2)v virus was isolated from a 5-year old girl who was suffering from fever, runny nose and cough. The isolated virus was designated A/Taiwan/1/2021(H1N2)v. Full-genome sequencing and phylogenetic analyses revealed that A/Taiwan/1/2021(H1N2)v is a novel reassortant virus containing hemagglutinin (HA) and neuraminidase (NA) gene segments derived from swine influenza A(H1N2) viruses that may have been circulating in Taiwan for decades, and the other 6 internal genes  are from human A(H1N1)pdm09 viruses.Notably, the HA and NA genes of A/Taiwan/1/2021(H1N2)v separately belong to specific clades that are unique for Taiwanese swine and were proposed to be introduced from humans in different time periods. Bidirectional transmission between humans and swine contributes to influenza virus diversity and poses the next pandemic threat.The online version contains supplementary material available at 10.1186/s12985-022-01794-2. Influenza A viruses infect a variety of host species, including avian, swine, and humans, and cause mild-to-severe symptoms that lead to a heavy disease burden . InfluenIn Taiwan, there is a well-established laboratory surveillance system for influenza viruses circulating in the community. Clinical specimens are routinely collected from outpatients with influenza-like illnesses and transported to commissioned laboratories for influenza testing by virus culture and/or real-time RT-PCR . All thehttp://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic trees were constructed using the neighbor-joining method, and 1000 bootstrap replications were performed to evaluate the robustness.To investigate the molecular phylogenies of the influenza AH1N2)v virus identified in this study, full-genome sequences of the previously identified representative influenza A(H1)v viruses during the period from 2015 to 2021 in the USA ((H1N1)v and (H1N2)v), Germany ((H1N1)v), the Netherlands ((H1N1)v), Denmark ((H1N1)v), Canada ((H1N2)v) and Brazil ((H1N2)v) as well as human seasonal influenza A(H1N1), A(H1N1)pdm09 and A(H3N2) viruses isolated between 1968 and 2019, several swine and avian influenza A viruses were selected as reference sequences and included in detailed analyses v virus were analyzed to investigate the phylogenetic and genetic characteristics of this virus (GISAID accession numbers EPI1918923-EPI1918930).The influenza A(H1N2)v virus was isolated from a 5-year-old child who lives in the county of central Taiwan through the nationwide laboratory surveillance network. She developed symptoms of cough and fever on March 12 and 13, 2021, respectively, and visited the emergency department of a local hospital on March 14. Throat swab specimens were collected at initial admission, tested positive for influenza A by rapid immunochromatography and were further subjected to influenza virus isolation. Oseltamivir was given empirically on the day of her admission without hospitalization. A total of 6 family members, including her great-grandmother, grandfather, grandmother, father, mother and younger brother, were notified, four of whom  were pig farm workers. Notably, the father, mother and younger brother had exhibited mild respiratory symptoms (cough or runny nose) during March 15–22; however, no respiratory specimens had been collected or tested for influenza. The case patient and her younger brother sometimes went to the pig farm on holidays with their family, they did not directly contact with pigs based on the epidemiological investigation.The virus was successfully propagated in MDCK cells. The initial identification of the virus by one local laboratory of the network influenza surveillance using real-time RT–PCR revealed an unsubtyped influenza A virus since only a matrix protein (M) gene assay of influenza A was positive, whereas subtyping assays targeting HA genes of human A(H1)pdm09 and A(H3) viruses were both negative. The virus isolate was then sent to the Taiwan CDC and further characterized as a subtype of A(H1N2)v on the basis of the full-length sequences of the viral HA and NA genes (designated A/Taiwan/1/2021(H1N2)v, TW01/21 in short). Furthermore, throat swab specimens were also obtained from all 6 family members on April 8, 2021, and all tested negative for influenza virus with real-time RT–PCR assays by the Taiwan CDC.The full-length complete genomic sequences of the TW01/21 virus were successfully determined. Regarding the evolutionary relationship of TW01/21 with other globally identified influenza A(H1)v viruses, phylogenetic analyses of the eight viral gene segments were conducted. Based on the phylogeny of viral HA gene sequences v viruses of 2020–2021 based on the complete genome phylogenies is summarized in Table Because of geographical segregation of pig populations and multiple introductions of viruses from either humans or birds, swine influenza viruses differ genetically between continents and regions . In TaiwIn this study, we identified a human case infected by a novel reassortant virus containing HA and NA gene segments derived from swine influenza A(H1N2) viruses that may have been circulating in Taiwan for decades, and the other 6 internal genes  are from human A(H1N1)pdm09 viruses. The phylogenetic analysis revealed that HA and NA genes separately belong to specific clades that are unique for Taiwanese swine and were proposed to be introduced from humans in different time periods. Bidirectional transmission between humans and swine contributes to influenza virus diversity and poses the next pandemic threat.Additional file 1. Supplementary Table 1. The names and accession numbers of viruses were analyzed in this study.Additional file 2. Supplementary Fig. 1. Phylogenetic relationships of representative internal protein-encoding genes for (A) PB2, (B) PB1, (C) PA, (D) NP, (E)M and (F)NS of the A/Taiwan/1/2021(H1N2)v virus. Classification of the specific evolutionary clades is indicated. The recently identified influenza A(H1)v viruses in Canada, Denmark, Brazil, Germany and the Netherlands are included as reference sequences shown in bold. Branch values of more than 70 are indicated.


Both H5N1 and H7N9 subtype avian influenza viruses cause enormous economic losses and pose considerable threats to public health. Bivalent vaccines against both two subtypes are more effective in control of H5N1 and H7N9 viruses in poultry and novel egg-independent vaccines are needed. Herein, H5 and H7 virus like particle (VLP) were generated in a baculovirus expression system and a bivalent H5+H7 VLP vaccine candidate was prepared by combining these two antigens. Single immunization of the bivalent VLP or commercial inactivated vaccines elicited effective antibody immune responses, including hemagglutination inhibition, virus neutralizing and HA-specific IgG antibodies. All vaccinated birds survived lethal challenge with highly pathogenic H5N1 and H7N9 viruses. Furthermore, the bivalent VLP significantly reduced viral shedding and virus replication in chickens, which was comparable to that observed for the commercial inactivated vaccine. However, the bivalent VLP was better than the commercial vaccine in terms of alleviating pulmonary lesions caused by H7N9 virus infection in chickens. Therefore, our study suggests that the bivalent H5+H7 VLP vaccine candidate can serve as a critical alternative for the traditional egg-based inactivated vaccines against H5N1 and H7N9 avian influenza virus infection in poultry. Other than H5N1 AIV, H7N9 AIV also has a significant impact on poultry and human health. In April 2013, three residents in Shanghai and Anhui Province, China, showed rapidly developing lower respiratory infections and were found to be infected with a new re-assortment of AIV known as low pathogenic H7N9 AIV (http://www.fao.org/ag/againfo/programmes/en/empres/H7N9/situation_update.html). Therefore, considering the dual threat to poultry and public health posed by H5N1 and H7N9 AIVs, a bivalent influenza vaccine is needed to prevent a potential pandemic caused by these two subtypes.H5N1 avian influenza virus (AIV) has been widely circulating in China and spread to more than 60 countries, and has caused huge economic losses to the poultry industry worldwide . In addiH7N9 AIV –6. As ofMassive vaccination of the H5+H7 trivalent  vaccine in China contributes substantially to the successful control of influenza virus outbreaks . HoweverEscherichia coli (Non-infectious virus-like particle (VLP) represents one of the most promising alternatives for the traditional inactivated vaccines, owing to its ease of production and scalability, intrinsic safety, and stimulation of strong and persistent immunological response –16. Furthia coli . HoweverIn this study, a bivalent H5+H7 VLP vaccine was prepared by combining H5 and H7 VLP assembled in a baculovirus expression system. The bivalent H5+H7 VLP vaccine provided good protection against HP H5N1 and H7N9 AIV, significantly inhibited viral shedding as well as virus replication in immunized chickens. In addition, the bivalent VLP vaccine was better than the commercial vaccine regarding protection from lung injury caused by HP H7N9 virus infection. Therefore, the bivalent H5+H7 VLP vaccine may serve as an important alternative for the egg-based inactivated vaccines for controlling these two subtypes in poultry.This study was carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The protocols for animal experiments were approved by the Jiangsu Administrative Committee for Laboratory Animals  and complied with the guidelines of Jiangsu Laboratory Animal Welfare and Ethics of Jiangsu Administrative Committee of Laboratory Animals. All experiments involving live viruses and animals were housed in negative-pressure isolators with HEPA filters in biosafety level 3 (BSL3) animal facilities at Yangzhou University in accordance with the institutional bio-safety manual.2. HP H7N9 virus A/chicken/Guangdong/GD15/2016 (GD15) and H5N1 virus A/chicken/Shandong/TT3/2016 (TT3) were isolated and identified previously (Spodoptera frugiperda Sf9 cells (ATCC # CRL-1711) were maintained in SF900III insect serum-free medium (SFM)  supplemented with 5% fetal calf serum (FCS)  at 27°C. Sf9 suspension cells were routinely cultured in insect serum-free SF900II SFM (ThermoFisher Scientific) at 27°C in spinner flasks at a speed of 120 rpm. Chicken embryo fibroblasts (CEF) were cultured in M199 medium supplemented with 5% FCS and maintained at 37°C with 5% COeviously . The virAutographa californica multiple nucleopolyhedro virus (AcMNPV). After 72 h post-transfection, indirect immunofluorescence assay (IFA) was carried out to verify the rescue of these rBVs. The rescued rBVs were then plaque-purified for three rounds in Sf9 cells and were serially propagated in Sf9 cell suspension culture for several passages. The rBVs were designated as rBac-H7, rBac-H5, rBac-NA, and rBac-M1, respectively.Four recombinant pVL1393 transfer plasmids encoding GD15 HA (pVL1393-H7), TT3 HA (pVL1393-H5), GD15 NA (pVL1393-NA), or GD15 M1 (pVL1393-M1) gene was generated as described previously . To gene50) of the rBVs, serial dilutions of the indicated rBVs were added to Sf9 cells in 96-well plates. After 96 h p.i., baculovirus infection was determined by detecting expression of baculovirus envelope gp64 protein using the mouse mAb against gp64  with IFA.To detect viral protein expression by the rBVs, Sf9 cells were infected with the rBVs at a multiplicity of infection (MOI) of 1. At Day 4 post-infection (p.i.), the cells were processed for IFA as described previously . Briefly6 cells/mL) were co-infected with the rBac-H7, rBac-NA, and rBac-M1 at a MOI ratio of 1:1:1. The H5 VLP was produced according to the same procedure. Culture supernatants were harvested at Day 3 p.i. and clarified by low-speed centrifugation  followed by ultracentrifugation at 10,000 g for 2 h at 4°C. The obtained VLP suspension was then placed on formvar-coated (copper 300 mash) grids, negatively stained with 1% uranyl acetate, and dried by aspiration. The VLP particles were then examined under a transmission electron microscope as described previously   were divided into two group (n = 32). Birds were intramuscularly (i.m.) immunized with 15 μg of the bivalent H5+ H7 VLP, or 0.3 mL of the commercial trivalent  vaccine  according to the label. At Week 3 post-vaccination (p.v.), the vaccinated birds of each group were randomized into two subgroups and moved into the biosafety level-3 animal facility for virus challenge. One group (n = 16) was challenged with 106.0 50% embryo infectious dose (EID50) (in 100 μL) of the H5N1 TT3 virus, another group (n = 16) was challenged with 106.0 EID50 the HP H7N9 GD15 virus.To investigate the immunogenicity and efficacy of the bivalent H5+H7 VLP in chickens, 64 4-week-old SPF White Leghorn chickens (n = 11) was challenged with the highly pathogenic H5N1 TT3 virus, another group (n = 11) was challenged with the highly pathogenic H7N9 GD15 virus.Moreover, another group of 22 birds was inoculated with 0.3 mL of PBS as the un-immunized control. At Week 3 p.v., birds were randomized into two subgroups and moved into the biosafety level-3 animal facility for virus challenge. One group . The mortality was recorded daily. Those severely ill or moribund birds were euthanized by inhalation of carbon dioxide and counted in mortality the following day. To monitor viral shedding, oropharyngeal and cloacal swabs were collected on Day 2, 4, and 6 p.c. for virus isolation in ECEs. To determine viral replication in birds, on Day 2 and 4 p.c., three birds of each group were euthanized, and the heart, cecum, lung, and spleen were collected for virus titration in ECEs. The remaining birds of each group were observed for clinical symptoms and mortality.n = 3) was collected on Day 2 and 4 p.c. and fixed with 10% neutral formaldehyde and embedded in paraffin. Tissue sections were then cut 5 μm thick and stained by hematoxylin and eosin (H & E). Histological changes were then examined by a double-blind method and captured with a light microscope  as described previously  assay was performed using 1% chicken erythrocytes with 4 HA unit (HAU) of the homologous viruses using the standard method . Briefly50) or H5N1 TT3 virus (100 TCID50). After incubation at 37°C for 1 h, the virus-sera mixture was then transferred to CEF cells in 96-well tissue culture plates and cultured for 4 days. VN antibody titers were determined as the reciprocal of the highest serum dilution that completely inhibited the cytopathic effect (CPE) caused by virus infection.Virus neutralizing (VN) antibody titers of the serum samples were determined as described previously . Briefly2SO4 and the optical density (OD) was read at 450 nm using a spectrophotometer.HA-specific IgG antibody titers in the sera collected at Week 3 p.v. were determined by enzyme-linked immunosorbent assay (ELISA) as described previously . In briep-values < 0.05 .Statistical analyses were performed by the unpaired t tests using GraphPad Prism . Data are expressed as the mean ± standard deviation (SD). Statistical significance was designated for differences with The HA genes of the H5N1 or H7N9 subtypes and the NA and M1 genes of the H7N9 subtype were amplified and ligated into the transfer plasmid pVL1393. The recombinant baculoviruses expressing the HA, NA, and M1 genes were generated through co-transfection of the transfer vectors with the linear genomic DNA of AcMNPV. The expression of the HA, NA, and M1 proteins were detected in the transfected Sf9 cells using IFA, indicating successful rescue of the recombinant baculoviruses . Subsequ2, significantly higher than that induced by the commercial vaccine at Week 2 p.v. . In addition, a different profile was seen for H7-specific HI antibody response  and challenged with AIV of the H5 or H7 subtypes, respectively. After lethal virus challenge, the unvaccinated chickens rapidly died with typical clinical signs caused by HP influenza virus infection, e.g., skin cyanosis, depression, facial edema, hemorrhage of foot scale, and central nervous system (CNS) signs. In contrast, all the vaccinated chickens survived, without showing any clinical signs during a 14-day observation period , and complied with the guidelines of Jiangsu Laboratory Animal Welfare and Ethics of Jiangsu Administrative Committee of Laboratory Animals.XL and JH conceived and designed the experiments, analyzed the data, and wrote the manuscript. JH, PP, JL, QZ, and RL performed the experiments and analyzed the data. XW, MG, ZH, SH, XL, XJ, and DP provided good suggestions for the experiments and helped to analyze the data. All authors contributed to the article and approved the submitted version.This work was supported by Jiangsu Province Agricultural Science and Technology Independent Innovation Funds [CX (21) 3141], the National Natural Science Foundation of China (32072832), the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX21_3277), the Earmarked Fund for China Agriculture Research System (CARS-40), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The sphingolipid sphingosine-1-phosphate (S1P) promotes tumor development through a variety of mechanisms including promoting proliferation, survival, and migration of cancer cells. Moreover, S1P emerged as an important regulator of tumor microenvironmental cell function by modulating, among other mechanisms, tumor angiogenesis. Therefore, S1P was proposed as a target for anti-tumor therapy. The clinical success of current cancer immunotherapy suggests that future anti-tumor therapy needs to consider its impact on the tumor-associated immune system. Hereby, S1P may have divergent effects. On the one hand, S1P gradients control leukocyte trafficking throughout the body, which is clinically exploited to suppress auto-immune reactions. On the other hand, S1P promotes pro-tumor activation of a diverse range of immune cells. In this review, we summarize the current literature describing the role of S1P in tumor-associated immunity, and we discuss strategies for how to target S1P for anti-tumor therapy without causing immune paralysis. Cancer is a collective term for more than 100 diseases that are defined by the uncontrolled multiplication of cells and the invasion of these transformed cells into other parts of their tissue of origin, or into other tissues. Despite considerable progress in cancer detection, management, and therapy in the last decades, cancer remains the second leading cause of non-natural deaths worldwide ,3. WhileImportantly, tumor initiation and development are multi-step processes that can require several decades in humans. They do not only depend on the tumor cells themselves, but they are shaped by the interaction of transformed cells with their local microenvironment. The tumor microenvironment (TME) is composed of cells such as vascular and lymphatic endothelial cells, pericytes, fibroblasts, and immune cells. An altered extracellular matrix and various gradients of nutrients and gaseous molecules, including oxygen, also contribute to the diversity of the TME . The TMEKRAS mutations. On the downside, epithelial memory together with KRAS mutations are not only protected from future inflammation-related injury but are also predisposed to the development of cancer [It is now firmly established that the immune system shapes tumor development at each stage . Howeverf cancer . Somaticf cancer ,16,17. Af cancer .Once cancerous growth is initiated, altered self-patterns, including neoepitopes and stress-related cell surface architecture, can be recognized by the anti-tumor arm of the immune system. This process presumably occurs regularly in humans, leading to eradication of initial cancerous lesions or at least immune control (immune equilibrium). The theory of cancer immunoediting predicts that if eradication fails, the constant interaction between immunity and the malignant cells during the equilibrium phase may result in the development of a highly immunosuppressive tumor phenotype that allows immune escape and, consequently, tumor growth ,21,22,23+ T cells are often linked to poor prognosis [https://clinicaltrials.gov, accessed on 19 January 2022. From the resulting studies, we prioritized those that directly addressed the role of S1P in tumor immunity and inflammation. Due to this strategy, publications describing the role of S1P metabolism and signaling in cancer unrelated to cancer-associated immunity or inflammation are underrepresented. Another limitation of our approach is that studies exploring S1P metabolism and signaling in an immune context unrelated to cancer are not prominently discussed. Thus, potentially interesting hypotheses on how S1P affects tumor immunity based on extrapolation from other immune contexts are largely absent in the text below.While the immune system is actively engaged in protection against transformed cells in humans, this protection obviously has failed in clinically detectable tumors. Loss of immune protection may involve shaping the mutational landscape ,25, but rognosis ,40,41. Trognosis . A similrognosis ,44,45. Brognosis . This isrognosis ,48. In tThe sphingolipid S1P is a bioactive signaling molecule with powerful impact in physiological as well as pathophysiological settings, which is why its levels are tightly regulated by a number of mechanisms ,52,53,54These enzymes differ in subcellular localization, with SPHK1 being predominantly found in the cytosol and being able to translocate to the plasma membrane. SPHK2 is found at a number of intracellular membranes, such as the endoplasmic reticulum and the mitochondria, and is able to shuttle into and out of the nucleus . S1P, whBesides its intracellular actions, S1P can be exported from cells to serve as a ligand for five G-protein coupled receptors (S1PR1-5), triggering autocrine or paracrine signaling . S1P traSPNS2-deficient mice displayed reduced circulating S1P levels coupled with lymphopenia [A major physiological role of S1P, the regulation of immune cell trafficking  Figure B, was rephopenia . As a cophopenia . The impphopenia . This ocphopenia ,99. S1PRphopenia . This laphopenia , while mphopenia  or S1PR5phopenia . B cell phopenia ,103. Howphopenia . Mature phopenia . In macrphopenia ,107. Thephopenia .The S1P/S1PR axis that maintains lymphocyte trafficking can be disrupted during inflammation, as indicated above for T cells. This occurs not only through regulating S1PR expression but also via an increase in S1P production . When prS1PR1 in endothelial cells, while overexpression of S1PR1 in endothelial cells normalized tumor vessels and improved the response to tumor therapy [As indicated above, S1P signaling regulates the migration, survival, and proliferation of cells. S1P levels in tumors are often elevated either due to increased expression of SPHKs, inflammatory signaling in the TME, tumor enriched environmental cues such as hypoxia, or tumor cell death ,111,112. therapy . Other S therapy ,119. How therapy , which mSPHK1 expression was increased in human colon cancer samples compared to normal colon mucosa, which was further increased in metastatic cancer. Moreover, azoxymethane (AOM)-driven colon cancer in mice increased blood levels of S1P, and SPHK1-deficient mice had reduced colon cancer development [SPHK1 upon SPHK2 ablation in mice were also observed in the AOM/DSS model of colitis-associated cancer (CAC). S1P triggered activation of resident myeloid cells via an NF-κB/IL-6/signal transducer and activator and transcription 3 (STAT3) loop that involved S1PR1 [SPHK2 in tumor cells reduced pro-tumor features of infiltrating macrophages [SPHK1 gene expression. In turn, expression of SPHK1 was associated with an increased expression of immune-related genes such as CD68, CD163, CD4, and forkhead box P3 (FOXP3), indicating increased infiltration of macrophages and Treg [SPHK1 in melanoma cells was associated with shorter survival in metastatic melanoma patients treated with anti-PD-1 ICB. SPHK1 targeting augmented the response to ICB in murine models of melanoma, breast, and colon cancer and limited Treg infiltration [SPHK1 in melanoma cells lead to a shift of macrophages with tumor-promoting to macrophages with tumor-suppressive phenotype markers, which was coupled to T cell recruitment and activation [SPHK1 ablation in Mantle cell lymphoma cells. An induction of natural killer T (NKT) cell activation was observed under these conditions accompanied by an increase in the NKT cell lipid antigen cardiolipin upon SPHK1 ablation [SPHK1-deficient T cells showed a sustained memory response and reduced differentiation to Tregs independent of S1PR signaling, which resulted in activity of T cells against murine melanoma cells and synergy with ICB [As opposed to global scavenging of S1P, inhibition or deletion of either of the two sphingosine kinases is not expected to result in lymphopenia due to the compensatory effect of the remaining kinase. Indeed, both kinases need to be targeted to reach meaningful changes in plasma S1P levels ,120. Theelopment . Increased S1PR1 . Indeed,ed S1PR1 . Interesed S1PR1 . Interesed S1PR1 . This firophages . SPHK1 wand Treg . Both imand Treg ,129. In ltration . Moreovetivation . Unconveablation . Togethewith ICB . In concSGPL1 was genetically ablated in mouse CAC models. Mice lacking SGPL1 in intestinal epithelial cells showed enhanced S1P levels and tumor growth, accompanied by increased STAT3 activation and inflammatory cytokine levels, which were inhibited when colonic SGPL1 levels were increased in WT mice [SGPL1 in myeloid cells also increased S1P levels. Hereby, immune cell specific SGPL1 ablation caused massive immune cell infiltration, delayed tumor formation, and a mix between the previously observed STAT3 pattern and immunosuppressive marker expression. In contrast, SGPL1 ablation in non-myeloid cells elicited rapid formation of colon tumors accompanied by a tumor-favoring microenvironment. Thus, the cellular source of S1P is decisive for triggering inflammation-induced cancer or cancer-induced inflammation, respectively [SGPP1 and PLPP3 expression with relapse-free survival in triple-negative breast cancer patients. Expression of both enzymes correlated with tumor-infiltrating DCs, CD4+ and CD8+ T cells, neutrophils, and macrophages [SGPP1 and PLPP3 was associated with overall survival in lung adenocarcinoma and non-small-cell lung carcinoma (NSCLC) patients, where expression of PLPP3 correlated with tumor-infiltrating immune cells in NSCLC patients [Given the evidence that increasing S1P by SPHKs may limit anti-tumor immune responses and support tumor-promoting inflammation, one may assume that preventing S1P degradation has similar effects. So far, data supporting this notion have emerged mainly from studies where  WT mice . Interesectively . In controphages . Moreovepatients . Such paSPHK1 and the S1P exporter ABCC1 were expressed at higher levels in aggressive HCC when compared with normal liver or cirrhotic tissue. High expression of these genes, which may indicate S1P export, correlated among others with immune signatures related to TNFα and IL6 signaling but also to allograft rejection and IFN-γ, which has anti-tumor properties. Moreover, an association with mixed anti- and pro-tumor immune cell infiltrates was found. Nevertheless, high virtual S1P export was associated with worse disease-specific survival and overall survival [SPNS2-deficient mice exhibit peripheral lymphopenia and defects in B cell homing to secondary lymphoid organs, which results in impaired humoral immunity upon immunization with E. coli and suggests SPNS2 as a target for immunosuppressive therapy [SPNS2 ablation reduced the formation of experimental metastases in mice, while increasing NK cells and CD8+ T cells at the metastatic sites [SPNS2 ablation. However, these effects were immune cell independent, even though depleting SPNS2 again increased NK cells and effector T cells in the lungs [SPNS2 ablation, despite its overall immunosuppressive effect during immunization, warrant further investigation. Interestingly, S1P independent features of SPNS2 have been proposed as well. SPNS2 expression was upregulated in colon adenoma and CRC compared to normal tissues. However, low SPNS2 expression correlated with poor prognosis in CRC and ectopic expression of SPNS2 inhibited cell proliferation, migration, epithelial-mesenchymal transition, invasion, and metastasis in CRC cell lines. This appeared to be S1P independent, rather acting directly via inactivation of AKT signaling [Besides production and degradation, S1P export decisively contributes to its signaling properties. Data from different human hepatocellular carcinoma (HCC) cohorts showed that both survival . Again,  therapy . Despiteic sites . These ric sites . In an ihe lungs . These lignaling . These dGiven the above-mentioned, sometimes antithetic properties of S1P signaling through its individual receptors, targeting S1P metabolizing enzymes, may be expected to result in reduced efficacy compared to targeting S1PRs selectively. Thus, S1PR-specific effects on anti-tumor immunity need to be elucidated.S1PR1 indicate that S1PR1 on CD4+ T cells regulates intratumoral Treg expansion leading to CD8+ T cell suppression and tumor progression through STAT3-dependent activation of Tregs [NLRP3) expression leading to enhanced IL-1β production via the inflammasome, which resulted in enhanced lymphangiogenesis and tumor metastasis in a murine breast cancer model [S1PR1 was the first S1PR to be discovered and is certainly the best explored receptor through which S1P exerts its immunomodulatory functions. Its importance in regulating T cell trafficking via the circulation  would leof Tregs . The rolof Tregs . Consequof Tregs . Besidesof Tregs . Reductiof Tregs ,149. Furof Tregs . This waof Tregs . Howeverer model . Taken ter model ,153,154,er model . While tS1PR2-deficient mice led to accelerated tumor growth, in part due to enhanced infiltration of CD11b+ myeloid cells, which stimulated new vessel formation through secretion of proangiogenic mediators such as vascular endothelial growth factor A (VEGF-A) [S1PR2 gene, and S1PR2-deficient mice develop B cell lymphomas at an advanced age [Similar to S1PR1, S1PR2 is ubiquitously expressed . In the (VEGF-A) . Furthernced age . In summS1PR3 also shows a rather broad tissue expression profile similar to S1PR1 and 2. However, S1PR3 appears to be rather selectively expressed in monocytes within human blood peripheral blood mononuclear cells . SimilarS1PR4 ablation resulted in significantly reduced tumor growth through enhanced CD8+ T cell proliferation. Unexpectedly, this appeared to be partly independent of myeloid cells since enhanced cytotoxic T cell proliferation was triggered via a T cell-intrinsic mechanism [S1PR4 on murine T cells also favored memory formation [S1PR4 was expressed by nutrient-deprived CD8low T cells and increased CXCR4 expression [S1PR4 ablation also enhanced IFN-α/-β production in murine breast tumors, which may aid in increasing intratumoral CD8+ T cell numbers and activity [S1PR4 was absent [S1PR4-deficient DCs, indicating a role for S1PR4 in the induction of TH17 cell differentiation [S1PR4 expression with tumor patient prognosis seem rather counter-intuitive. S1PR4 expression strongly correlated with improved prognosis of triple-negative breast cancer and non-small-cell lung cancer patients, and it was furthermore positively associated with lymphocyte infiltration [S1PR4 on lymphocytes compared to other cells, these data likely do not reflect a functional role of S1PR4, but rather indicate that S1PR4 expression in human tumors can be taken as a marker for tumor-infiltrating lymphocytes. Taken together, these studies underscore the potential of S1PR4 as a possible drug target in immune oncology.In contrast to S1PR1-3, S1PR4 shows a rather specific expression pattern restricted in hematopoietic tissue and endothelium and a lower expression within the gastrointestinal tract, the lung, and the brain ,168,169.echanism . Importaormation . There ipression . CXCR4 ipression ,178. Howactivity ,175. Bess absent ,180. CD4ltration ,137. HowS1PR5 on NK cells [S1PR5 on Trm or effector memory T cells (Tem) with cancer prognosis. Single cell RNA sequencing (scRNA-Seq) of T cells from 12 patients with CRC suggested that Tem highly expressed S1PR5 [S1PR5 expression associated with rapid proliferation and prolonged persistence at the tumor site [S1PR5 seems to be a potent T cell marker for either effector function or retention as well as T cell homing in tumors. The latter was demonstrated by mass cytometry and scRNA-Seq analysis of hepatitis B virus (HBV)-specific T cells isolated from tumors of HCC patients. There, clusters of HBV-specific Trm T cells were identified that showed reduced expression of S1PR5 and whose presence correlated with long-term relapse-free survival of cancer patients [S1PR5 was also associated with improved patient survival, which may fit to observations that glial tumors are enriched in Trm cells [S1PR5 is not only downregulated in Trm, but that its downregulation is a requirement for Trm differentiation [Similar to S1PR4, S1PR5 has a rather restricted expression pattern mostly being expressed by NK cells, T cells, monocytes, and oligodendrocytes ,181. TheNK cells ,182 and NK cells . Interesed S1PR5 . Furthermor site . Thus, Spatients . In patirm cells ,187. In ntiation . Thus, SIn our opinion, the data discussed above clearly indicate that targeting components of the S1P signaling machinery may be beneficial not only to alter tumor cell properties, but to create a favorable immune environment. S1P appears to affect virtually all steps of the cancer-immunity cycle  Figure .Tumor cell death by apoptosis and the resulting interaction of apoptotic cells with antigen-presenting cells (APC) promotes immune evasion, tissue remodeling, and an invasive phenotype in tumors . S1P ser


In April 2016, an indigenous monovalent rotavirus vaccine (Rotavac) was introduced to the National Immunization Program in India. Hospital-based surveillance for acute gastroenteritis was conducted in five sentinel sites from 2012 to 2020 to monitor the vaccine impact on various genotypes and the reduction in rotavirus positivity at each site. Stool samples collected from children under 5 years of age hospitalized with diarrhea were tested for group A rotavirus using a commercial enzyme immunoassay, and rotavirus strains were characterized by RT-PCR. The proportion of diarrhea hospitalizations attributable to rotavirus at the five sites declined from a range of 56–29.4% in pre-vaccine years to 34–12% in post-vaccine years. G1P[8] was the predominant strain in the pre-vaccination period, and G3P[8] was the most common in the post-vaccination period. Circulating patterns varied throughout the study period, and increased proportions of mixed genotypes were detected in the post-vaccination phase. Continuous long-term surveillance is essential to understand the diversity and immuno-epidemiological effects of rotavirus vaccination. Rotavirus is the leading etiology of acute gastroenteritis in children under 5 years old worldwide, causing high mortality, especially in middle- and low-income countries. India accounts for 22% of the total global rotavirus mortality . In IndiThe genome of group A rotavirus is composed of 11 double-stranded RNA segments, of which the VP7 and VP4 genes coding for the outer capsid proteins are used for the classification of the virus into G and P types, respectively. Studies have been conducted across the globe to understand the natural evolution of rotavirus and its relevance in the context of vaccine introduction. Globally, G1P[8], G2P[4], G3P[8], and G9P[8] are the most common genotypes associated with rotavirus diarrhea  was the most common strain (49.5%) in the pre-vaccine period. The other common genotypes were G2P[4] (8%), G9P[4] (7.5%), G9P[8] (4.5%), and G12P[6] (3.8%). Conversely, G3P[8] (44.3%) was the most common genotype in the post-vaccine period, with G1P[8] (15.4%), G2P[4] (7.4%), G9P[4] (4.9%), and G1P[6] (3.7%) being the next most common genotypes . Marked The genotype distribution also varied across the sentinel sites in North India (Tanda and Rohtak) and South India . G1P[6] was seen predominantly in northern sites, while G9P[8] and G12P[6] were seen in southern sites during the pre-vaccination period. In the post-vaccination period, the major circulating strains remained the same in northern sites, with G3P[8] topping the list. G3P[8] emerged in the southern sites as well, with a decline in G9P[8] and G12P[6] .An increased prevalence of G3P[8] and decreased prevalence of G1P[8] were noted in the post-vaccination period compared to the pre-vaccination period. G1P[8] peaked during the year 2014 (62.6%) and has declined steadily since then. G3P[8] started appearing in 2015 and was the predominant genotype in the following years. No novel strains were detected during the post-vaccination period. Mixed genotype infections occurred in a higher proportion in the post-vaccine period (17.4%) compared to the pre-vaccine period (6.4%). G1 (33%) was the most common G-type found in mixed infections, mainly in combination with G12 (10.8%) and G3 (9%). Similarly, P[8] (93.4%) was the most common P-type in mixed infections, along with P[4] (55.6%) and P[6] (37.8%).Pre- and post-introduction surveillance at five sites in India indicate that vaccination is impacting severe rotavirus gastroenteritis. The overall prevalence of rotavirus in children with hospitalized gastroenteritis decreased after vaccine introduction, reaching 13.3% by the third year post-vaccine introduction, indicating the effectiveness predicted by clinical trials and modeling ,15. The During the study period, from 2012 to 2020, the major genotypes were G1P[8], G3P[8], G2P[4], G9P[4], G9P[8], G12P[6], and G1P[6], which include some reassortants that are not common in other parts of the world. There was marked temporal fluctuation, with G9P[8] detected at a high frequency in 2013/2014, only to disappear by 2019/2020, while G12P[6] was high in 2014/2015. Our findings are consistent with surveillance data from India and neighboring countries that also saw the emergence of G12 strains . We alsoChanges in genotype distribution and increased diversity are seen with other rotavirus vaccines. In Brazil, G2P[4] emerged as the major strain, while no change in genotype distribution was seen in Kenya after Rotarix introduction ,21. An iGlobally, the rate of mixed rotavirus infections is similar to our findings ,27. MixeTo conclude, our study showed a reduction in rotavirus diarrhea across five sites in India after Rotavac vaccine introduction. Changes in circulating strains with an increased rate of mixed infections were also seen in the post-vaccine period. In our study from 2016, additional methods were used for the genotyping of samples that remained untyped with standard laboratory protocols. Some of the differences in genotypes before and after vaccination introduction may have been caused by the change in genotyping methods. Due to the short period of surveillance, it is difficult to determine whether the changes were due to natural strain variations or vaccine pressure. Continued surveillance is warranted to determine the long-term effects of rotavirus vaccination.Active hospital-based surveillance for diarrhea was established in five sentinel sites consisting of major referral hospitals from September 2012 to June 2020. The hospitals included were Christian Medical College , Sri Venkateshwara Medical College , Hi-Tech hospital , Pt. Bhagwat Dayal Sharma Postgraduate Institute of Medical Sciences , and Rajendra Prasad Government Medical College .Sample collection and laboratory methods are detailed in the study protocol . In brie


Advances achieved with molecular biology and genomics technologies have permitted investigators to discover epigenetic mechanisms, such as DNA methylation and histone posttranslational modifications, which are critical for gene expression in almost all tissues and in brain health and disease. These advances have influenced much interest in understanding the dysregulation of epigenetic mechanisms in neurodegenerative disorders. Although these disorders diverge in their fundamental causes and pathophysiology, several involve the dysregulation of histone methylation-mediated gene expression. Interestingly, epigenetic remodeling via histone methylation in specific brain regions has been suggested to play a critical function in the neurobiology of psychiatric disorders, including that related to neurodegenerative diseases. Prominently, epigenetic dysregulation currently brings considerable interest as an essential player in neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic lateral sclerosis (ALS) and drugs of abuse, including alcohol abuse disorder, where it may facilitate connections between genetic and environmental risk factors or directly influence disease-specific pathological factors. We have discussed the current state of histone methylation, therapeutic strategies, and future perspectives for these disorders. While not somatically heritable, the enzymes responsible for histone methylation regulation, such as histone methyltransferases and demethylases in neurons, are dynamic and reversible. They have become promising potential therapeutic targets to treat or prevent several neurodegenerative disorders. These findings, along with clinical data, may provide links between molecular-level changes and behavioral differences and provide novel avenues through which the epigenome may be targeted early on in people at risk for neurodegenerative disorders. Neurodegenerative (ND) disorders are among the leading bases of disability and death worldwide ,2,3. TheIn the past decade and a half, ND has been the topic of an immense number of investigations. Much progress has been made, but for patients with ND disorder, these ailments remain overwhelming and deadly, emphasizing an urgent need to develop more effective treatments. Recent progress suggests that epigenetic modifications are highly relevant targets, and thus, they represent a potential site for pharmaceutical intervention. Aberrant epigenetic modifications related to ND disorders are starting to emerge and emphasize the great need to assess recent advancements in epigenetic mechanisms in ND disease research. We and others have recently discussed other mechanisms involved in severe ND disorders, including Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), and Amyotrophic lateral sclerosis (ALS) ,8,9. TheThe term “epigenetic” denotes modification of gene expression without directly altering the DNA sequence . These cHistones are a highly conserved set of nuclear proteins that assemble in an octamer composed of pairs of H2A, H2B, H3, and H4. Nuclear DNA is highly condensed and wrapped around these nuclear proteins, nucleosomes, in the form of chromatin to accommodate chromosomes in the nuclei . The hisHistone methylation usually occurs at the arginine or lysine N-terminal region, which leads to the activation or suppression of gene expression. Each arginine residue in histones can be subjected to monomethylation and symmetric/asymmetric dimethylation. Likewise, each lysine residue can be mono-, di- and tri-methylated, and also nearby residues can form different methylation combinations ,21,22,232+ dioxygenase Jumonji-C (JmjC) domain (KDM2 family proteins) was identified as a specific H3K36me1/2 demethylase (KDM2A and B), which does not demethylate H3K36me3 [The amine oxidase containing lysine-specific demethylase 1 (LSD1/KDM1A) and lysine-specific demethylase 2 (LSD2/KDM1B)  remove mH3K36me3 . The KDMH3K36me3 . The KDMH3K36me3 ,58,59. IH3K36me3 ,61. KDM5H3K36me3 ,64,65,66H3K36me3 ,67. KDM6H3K36me3 ,69,70,71H3K36me3 ,77,78,79H3K36me3 ,79,80. KH3K36me3 ,82,83. RH3K36me3  and may H3K36me3  and/or eAD is the most established neurodegenerative disorder in the world. Emerging findings indicate that epigenetic dysregulation of gene expression may play a significant role in aging and ND ,87,88,89HD is a late-onset, autosomal progressive neurodegenerative disorder caused by the trinucleotide CAG repeat in the coding for glutamine (Q) in exon 1 of the Huntingtin (Htt) gene , leadingEnhanced H3K9me2 has been found in murine models of HD ,111,112,Htt WT and targeted inactivation of both copies of Htt (dKO) genotypes suggested that the loss of Htt triggered a significant reduction in the total number of H3K27me3-marked promoters [Besides, studies have also shown a functional interaction of the Htt gene with protein arginine methyltransferase 5 (PRMT5), an enzyme mediating the dimethylation of arginine (R) of essential cellular proteins, including histones and spliceosomal proteins . Htt hasromoters . These fromoters , such asromoters ,124.The additional function of PRC2 in HD was supported by H3K27me3 ChIP-sequencing in neuronal chromatin obtained from the HD postmortem PFC and non-neurologic controls. The findings suggest the loss of neuronal PRC2-H3K27me3 sites and the upregulation of some PRC2 target genes associated mainly with Hox gene clusters and developmentally related proteins in the HD-affected human brain ,127,128.BDNF gene locus [Htt (Q150) knock-in mice [Notably, the diminished enrichment of H3K4me3, a mark of active gene transcription, has been associated with impaired transcription of target genes  in both human HD postmortem brains as well as the cortex and striatum of R6/2 mice . Reducedne locus . These one locus ,133. Fur-in mice . Notably-in mice ), and th-in mice . A simil-in mice ,135 was -in mice , furtherPD is the second most predominant neurodegenerative disorder in the world after AD . The mosSNCA promoter region in postmortem brain samples from patients with PD and matched controls [SNCA promoter reduced α-synuclein levels in neuronal cell lines and PD-derived induced pluripotent stem cell lines (iPSCs) [SNCA promoter in the neuronal nuclei (NeuN) of positive neurons of substantia nigra (SN) tissue samples from PD patients. Interestingly, significant reductions in H3K4me3 and H3K27me3 marks were observed in SH–SY5Y cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA) [SNCA gene, directly regulates the accessibility of transcription factors that can access gene regulatory regions. Finally, there are still undiscovered histone methylation marks that may regulate synaptic and pathological abnormalities found in PD. Those may also have a considerable impact on regulating chromatin structure and overall effects on gene transcription in PD and should be of future interest.A mounting number of studies overwhelmingly suggest the critical function of the histone modification and PD-associated α-synuclein coding gene SNCA expression , meaningcontrols . Similar (iPSCs) . Signifi(6-OHDA) . In the (6-OHDA) ,148, sig(6-OHDA) . In cont(6-OHDA) , indicatALS is a fatal ND distinguished by the selective loss of both upper and lower motor neurons . There ir 43 kD (TDP-43)-based pathology [C9orf72 exposed the likelihood that epigenetic changes might also involve ALS pathology. The studies of epigenetic changes in ALS are still in infancy and much remains to be studied. Because other epigenetic modifications in ALS have been recently reviewed [Few epigenetic changes were reported related to ALS before discovering noncoding GGGGCC hexanucleotide repeat (HRE) in the gene C9ORF72 that is strongly associated with ALS disease ,157, whiathology , the obsreviewed ,159, we C9orf72 mRNA in the frontal cortices and cerebella of c9FTD/ALS patients is associated with increased binding of C9orf72 to H3K9me3, H3K27me3, and H4K20me3 marks [C9orf72 binding to trimethylated histones , but also enhanced C9orf72 gene expression [R521C mouse model of ALS, overexpression of PRMT1 rescues neurite growth associated with oxidative stress [R521C mouse model of ALS [The reduced e3 marks , associapression . In yeaspression , whereaspression ,163. In e stress  through l of ALS . Consistl of ALS . TDP-43 l of ALS . It is, l of ALS . There wl of ALS . Similarl of ALS , suggestCollectively, the findings discussed above  suggest Because alcohol is a neurotoxic agent, its use has been associated with neurodegeneration ,173,174 GIPC1, BCL2L1, and UBE1) expressions were observed in the PFC and HP of postmortem alcoholic brain tissues [Investigation on histone methylation in AUD is in infancy, and studies are beginning to explore the function of each different histone methylation regulated gene expression. As discussed earlier, histone methylation could regulate gene repression  or activation . Postmortem brains of alcoholic individuals  exhibite tissues . H3K27me tissues . Consist tissues . These lMag, Mbp, Mobp, and Plp) and H3K36me levels was found in AIE adolescent PFC [Arc gene, enabling increased negative elongation factor (NELF) binding to the Arc promoter was observed in AIE adult rats. These events inhibited Arc expression in the amygdala. Inhibition of Kdm6b or Arc eRNA expression in the CeA significantly increased H3K27me3 occupancy at the Arc SARE site and recapitulated the molecular and chromatin phenotypes of AIE [Smyd3 (di- and trimethylates H3K4), Setdb1 (trimethylates H3K9) and Setd6 (mono-methylates H2AZK8) gene expression. This paradigm also increased Setd7 (mono-methylates H3K4), Setd3 (methylates H3K4 and H3K36), Ash1l (methylates H3K36) [Similar observations were also observed in animal studies decreased H3K27me3 at the Mt1 gene promoter and increased global H3K4me3 levels at the Mt2 gene promoter in the cerebral cortex were observed when mice were exposed to acute alcohol for 6 h . Increascent PFC . A recencent PFC . In the cent PFC . Consists of AIE . Alcohols H3K36) . These fAlcohol exposure during early development has been shown to induce neuronal loss at different stages of brain development, leading to neuronal dysfunction at the adult stage. In addition to histone acetylation and DNA methylation changes, histone methylation changes have been shown in many developmental alcohol studies. GD 7 acute ethanol exposure increased H3K9me2, decreased H3K27me3 and altered G9a, Setdb1, Kdm1a, Kdm4c, Uhrf1 and Ezh2 mRNA levels . ReducedIn summary, with the available findings, it is clear that suppressive chromatin mediated by various histone marks regulated by a dynamic balance between specific HMT and HMD significantly contributes to the neurodegeneration and progression of disease pathology . Given t