The adenoviral E1A protein induces cell proliferation, transformation, andtumor formation in rodents, on the one hand. On the other hand, E1A expressionincreases cell sensitivity to a number of cytotoxic agents. Therefore, E1A is acandidate for use as a component of combination therapy for malignant tumors.The highest augmentation in the cytotoxic effect was achieved by a combined useof E1A expression and histone deacetylases (HDAC) inhibitors. However, HDACinhibitors do not induce apoptosis in cells transformed with E1A and cHa-rasoncogenes. In this study, it was shown that HDAC inhibitors reduce theexpression of adenoviral E1A. However, under unregulated E1A overexpression,these cells undergo apoptosis in the presence of HDAC inhibitors. Treatmentwith a HDAC inhibitor, sodium butyrate (NaBut), was shown to activate theanti-apoptotic factor NF-kB in control cells. However, NaBut was unable tomodulate the NF-kB activity in E1A overexpressed cells. Therefore, it is fairto postulate that cells transformed with E1A and cHa-ras oncogenes avoid theapoptosis induced by HDAC inhibitors thanks to a NaBut-dependent decrease inE1A expression. The E1A gene of human adenovirus type 5 is an early response gene that isexpressed in infected cells and provides the necessary conditions for virusreplication , 3. It wThe transforming activity of E1A is determined by its ability to deregulate thecell cycle by binding to and altering the activity of such cellular factors aspRb family proteins -7 and thAdenoviral E1A promotes apoptotic cell death by modulating the expression ofthe genes regulating apoptosis -19, the ras oncogene, which activates theanti-apoptotic PI3K/Akt cascade and NF-κB via the stimulation of theRas/Raf/MEK/ERK kinase cascade [The level of apoptosis in cells expressing E1A can be reduced by thecomplementary transforming  cascade . Theant cascade . Thus, t cascade .Histone deacetylase inhibitors (HDIs) inhibit tumor cell growth, thus causingcell cycle arrest, senescence, or apoptosis, without having a toxic effect onnormal cells , 26. ThecHa-Ras and E1A oncogenes [cHa-ras to avoid death underthe action of HDIs is due to the HDI-dependent decrease in E1A expression andactivation of the NF-κB anti-apoptotic factor. Therefore, induction ofapoptosis in E1A+Ras-transformed cells by HDIs is possible only underunregulated E1A expression.We have previously shown that HDIs cause cell cycle arrest and senescence ofcells transformed by ncogenes -29but dncogenes , 26. TheCell linescHa-ras and with p1Aplasmid that contained nucleotides 1–1634 of the genome of humanadenovirus type 5 encoding the E1A protein [Our studies were performed using mouse embryonic fibroblasts that had beenstably transformed with a vector encoding  protein . Cells wCell distribution according to DNA content2 HPO4 , 1.5 mM KH2 PO4 , pH7.2), permeabilized with saponin at a final concentration of 0.01% for 20 min,and repeatedly washed with the PBS solution to remove saponin. The cells werethen incubated with RNase A (100 µg/mL) and propidium iodide  and analyzed on a Coulter Epicks XL flowcytometer .The distribution of cells by DNA content was studied by flow cytometry. Thecells were washed with a PBS solution -2,5-diphenyltetrazolium bromide (MTT) (Sigma) toinsoluble purple formazan. The cells were incubated in a MTT solution in PBS ata final concentration of 0.5 mg/mL (1.5 h at 37°C in a CO2incubator). The culture medium was then removed, and the cells were suspendedin dimethyl sulfoxide (DMSO). The optical density in each well was determinedat a wavelength of 570 nm using a Multiscan-EX plate reader (Labsystems). DMSOwas used as a blank control.Cell viability was measured by MTT assay. The cells were plated in 96-wellplates at a density of 2 × 10Protein immunoblottingThe cells were lysed in a buffer containing 1% NP40, 0.5% sodium deoxycholate,0.1% sodium dodecyl sulfate (SDS), protease, and phosphatase inhibitors.Proteins were separated by electrophoresis, transferred to a PVDF membrane(Millipore), and analyzed using the appropriate specific antibodies. Proteinson the membranes were detected using the enhanced chemiluminescence method. Antibodies raised against E1A (M73) proteins , Gapdh (14C10)  and pan-Rasproteins  were used.Gene transcription analysisCellular RNA was isolated using a Trizol reagent . The reversetranscription reaction was performed using 2 µg of RNA. The amplificationreaction (PCR) was performed in the presence of 100 ng of the correspondingprimers to cDNA of the mouse e1a and gapdh genes:5’-TGTGATGGGTGTGAACCACG-3’/5’-CCAGTGAGCTTCCCGTTCAG-3’.Linear PCR amplification of DNA fragments was performed during 25–35cycles. The specific reaction product was analyzed by electrophoresis in 2%agarose gel.Caspase-3 activityn-nitroanilide at a wavelength of 405 nm using a Multiscan-EXspectrophotometer (Labsystems).Caspase-3 activity was assayed in vitro based on the cleavage of the specificcolorimetric substrate Ac-DEVD-pNA . Cells were lysed for 20 min at+4°C in a buffer containing 50 mM Tris-HCl, pH 7.5; 120 mM NaCl; 1 mMEDTA; 1% NP-40, and protease inhibitors. Caspase activity was determined in96-well plates in 40 µL of lysates mixed with 160 µL of a reactionbuffer  containingAc-DEVDpNA substrate. The efficiency of Ac-DEVD-pNA cleavage was determinedspectrophotometrically based on the accumulation ofTemporary transfection and analysis of luciferase activityCells were transfected using the Lipofectamine-2000 reagent (Invitrogen),according to the manufacturer’s protocol. A luciferase reporter vectorcarrying three copies of NF-κB-binding sequences (3 × κB-luc)was used for transfection. Renilla luciferase expression was used as aninternal control. Cells were treated with 4 mM NaBut 24 h post-transfection andthen processed according to the manufacturer’s instructions for measuringluciferase activity after 48 h. Luciferase activity was determined using aTD-20/20 luminometer (Turner Designs). Each experiment was repeated at least 3times.Histone deacetylase inhibitor sodium butyrate inhibits the expression ofadenoviral E1AFig. 1 showthat E1A expression is reduced in the presence of NaBut. In mouse embryo fibroblaststransformed with the E1A and cHa-Ras oncogenes (mERas line),transcription of the e1a gene and the levelof E1A protein decreased as soon as during the first hoursof exposure to NaBut. Is this effect specific to the particular cell line andHDIs employed? In order to answer this question, we analyzed the E1A expressionin the transformed human cell lines and used alternative HDIs. We found thatvalproic acid (VA), trichostatin A (TSA) (data not shown), and vorinostat alsoreduced the amount of E1A in mERas transformants.The results shownin Fig. 1C demonstratethat the decrease in E1A expression under the action of HDIs is not specific onlyto the mERas line. Immunoblotting demonstrated that the amount of E1A protein inthe transformed human renal epithelial HEK-293 cells decreased in the presenceof NaBut. The results of immunoblotting showed that the expression of the Rasprotein did not change in the presence of NaBut.Thus, HDIs were found to suppress the expression of adenoviral E1A,whereas Ras expression was not modulated by HDIs.In order to clarify the reasons for the absence of the expected cytotoxiceffect of HDIs in E1A-expressing cells, we analyzed the effect of a HDI, sodiumbutyrate (NaBut), on the expression of transforming oncogenes. The data presentedin The detected decrease in the E1A protein level in the presence of HDIs canshift the equilibrium between the activities of transforming proteins in mERascells. Meanwhile, the action of oncogenic Ras becomes dominant. We consideredthat the low level of apoptosis in the E1A+Ras transformed cells treated withHDIs was associated with the HDIs-mediated decrease in pro-apoptotic E1Aprotein expression and activation of the anti-apoptotic Ras/Akt/NF-κBcascade.Production of a E1A+Ras-transformed cell line with E1A expressionunregulated by HDIs e1a gene. In order to test the hypothesisthat reduction in E1A expression is required to reduce the HDIs-inducedapoptosis, we produced a MER-E1A cell line based on mERas cells. The MER-E1Acell line additionally expressed the E1A 12S protein under the control of anunregulated cytomegalovirus (CMV) promoter. Regulation and activity of CMV andAd5 viral promoters differ significantly: so, they can be used in target cellsfor different purposes. The high-activity CMV promoter is convenient forefficient transgene expression.The mERas cell line was obtained using a p1A plasmid carrying the nucleotides1–1634 of human adenovirus type 5 encoding the E1A protein . In thesFigure 2 showsthe immunoblotting results that demonstrate howNaBut affects the expression of the E1A protein in the original mERas cell line(upper panel) and in the new MER-E1A cell line with constitutive E1A expressionunder the control of the CMV promoter (lower panel). In the control mERascells, E1A expression decreased to almost zero already during the first hoursof exposure to NaBut and remained at a low level throughout the entire study(up to 72 h). However, in MER-E1A cells, the E1A 12S protein was expressed at ahigh level independently of NaBut.Thus, we obtained a line of transformed rodent cells expressing the adenoviralE1A gene under the control of the CMV promoter in which E1A expression did notdecrease in the presence of HDIs.Sodium butyrate induces apoptosis only in E1A+Ras-transformed cells wherethe amount of E1A does not decrease in the presence of HDIsFigure 3A demonstratesthat the viability of control mERas cells treated with NaBut decreased more thanthat of the untreated ones. However, the amount of formazan, whichcharacterizes cell viability, rose with an increase in the duration of exposureof mERas cells to NaBut. The increased amount of formazan attests to the factthat the cells did not divide but remained alive. Meanwhile, the number ofviable MER-E1A cells decreased below the baseline, indicating cell death.Next, we compared the effect of HDIs on the proliferation ofE1A+Ras-transformed cells where E1A is expressed under the control of anintrinsic promoter, and in cells where the CMV promoter regulates E1Aexpression. We evaluated the effect of NaBut on cell viability depending on E1Aexpression. The control mERas cells and MER-E1A cells with unregulated E1Aexpression were treated with NaBut for 24–72 h; their viability wasdetermined using the MTTassay. Fig. 4A.One can see that NaBut did not increase the sub-diploid peakin cells transfected with the control empty pcDNA3 vector. At the same time, the percentage of cells with a sub-diploid DNAcontent in cells transfected with CMV-E1A increased twofold already 48 h afterthe exposure to NaBut. The findings demonstrate that there is a significant differencein cell response to HDIs depending on how HDIs modulate E1A expression.In order to test the hypothesis about the induction of death of cells with E1Aexpression not regulated by NaBut, we analyzed the distribution of cells by DNAcontent using a flow cytometer. Cell distribution after transient transfection is shownin Fig. 4B).Meanwhile, 35% of MER-E1A cells contained fragmented DNA 72 h after the exposure to NaBut.The corresponding results were obtained in stable clones with E1Aoverexpression under the control of the CMV promoter (MER-E1A). In the controlmERas cells, NaBut did not increase the sub-diploid peak in the distributionhistogram of DNA content, which is characteristic of dying cells, even after 72 h.The differences in the regulation of caspase-3 activity by HDI depending onmodulation of the E1A expression are consistent with our data demonstratingdifferences in the proliferative response of these cells to HDI.We analyzed the activity of the caspase-3 mediating the transduction of theapoptotic signal. For this purpose, cells transfected with pcDNA3 or CMV-E1Awere either left untreated or treated with NaBut for 24 h; the as cells.MeanwhiNaBut does not increase NF-κB activity in cells with unregulated E1AexpressioncHa-ras [Fig. 4D).We found that unregulated high expression of adenoviral E1A from the CMV promoterprevented HDI-dependent activation of the anti-apoptotic factor NF-κB.Therefore, inhibition of NF-κB activity by adenoviral E1A is one ofthe reasons for the induction of apoptosis by HDIs in these cells.It was shown earlier that HDIs activate the anti-apoptotic factor NF-κB incells transformed with E1A and cHa-ras . This acCells with unregulated E1A expression do not accumulate senescence markerSA-β-Gal in the presence of NaButcHa-Ras andE1A oncogenes [Fig. 5)show that there is SA-β-Gal in almost allcontrol mERas transformants after treatment with NaBut for 72 h, thusindicating that cellular senescence was induced. In MER-E1A cells, theSA-β-Gal marker had not accumulated. After exposure to NaBut for 72 h,very few MER-E1A cells remained attached to the slides for furtherSA-β-Gal staining; most cells died and were floating. Hence, it is fair toconclude that NaBut induces cellular senescence in cells transformed withE1A and Ras if E1A expression decreases inthe presence of NaBut. Meanwhile, the transformants with constitutive E1Aexpression die rather than senesce.Cellular senescence and apoptosis are the alternative anti-proliferativeprograms induced by cytotoxic and stress factors. It was previously shown thatHDIs induce senescence of cells transformed with ncogenes -29. The ncogenes . In ordeRas or other oncogenes,adenoviral E1A immortalizes and transforms primary rodent cells [in vitro [In cooperation with activated nt cells , 3. Intnt cells , 17-19.in vitro ,16. Morin vitro . Clinicain vitro , 33,34.in vitro . AccordicHa-ras and E1Aoncogenes used in this study. However, sodium butyrate inducedapoptosis in these cells when adenoviral E1A was expressedunder the control of a HDI-unregulated promoter. In studies reporting thatcombined use of HDIs and E1A enhanced the cytotoxic effect, E1Aexpression was controlled not by a native promoter, but by thecytomegalovirus (CMV) promoter or by the catalytic subunit of telomerase (TERT)[Fig. 1A).The regulation of E1A expression is currently understudied. No data on the roleof acetyltransferases or deacetylases in the regulation of E1A transcription areavailable. Inhibition of histone deacetylases activates gene transcription via arelaxation of the chromatin structure. On the other hand, HDIs can inhibit oractivate transcription by changing the acetylation level of transcriptionfactors [E1Atranscription. Earlier, we showed that NaBut suppresses thetrans-activating ability of the E2F factor [Many studies have demonstrated that combined use of HDIs and adenoviral E1A intumor cells increases the cytotoxic effect , 36, 37.e (TERT), 39-40.e (TERT), 38.Confactors . Hence, factors . The absF factor , 43. TheF factor , whereasF factor ,46. TheFig. 1B).Moreover, the content of the E1A protein decreasesmore intensively than E1A transcription does. This findingindicates that deacetylase inhibitors modulate the stability of the E1Aprotein. Like many cellular proteins, virus-encoded proteins also act assubstrates for acetyltransferases and deacetylases. The E1A protein is able tobind to p300/ CBP and can be acetylated by p300 and PCAF[Second, our data suggest that HDIs reduce the content of the E1A protein bothin mERas cells and in a transformed human embryonic kidney (HEK) 293 cell line.We have shown that avoidance of apoptotic death by control mERascells is associated with the downregulated expression of the pro-apoptotic E1Aprotein and activation of the anti-apoptotic NF-κB cascade. Meanwhile, theoncogenic Ras inducing senescence starts to play a predominant role. In turn,induction of apoptotic death in the presence of NaBut in cells with E1Aoverexpression is associated with a suppressed and unregulated activity of theanti-apoptotic NF-κB complex. Data on the repression of NF-κBactivity by E1A oncoprotein have been reported[Comparison of the responses of transformed cells with regulated and unregulatedE1A expression to HDIs showed that apoptosis was induced only in cells with anincreased unregulated E1A expression. In the control mERas cells with a reducedcontent of E1A, the cell senescence program was initiated(reported, 20, 21.reported, it is fcHa-Ras and E1Aoncogenes in which E1A is expressed under the control of a native viralpromoter. In the present study, we have shown that HDIs suppress the expressionof adenoviral E1A. Apoptotic death of E1A/Ras-transformed cells can be inducedby HDIs if E1A is expressed at a high unregulated level. In other words, theavoidance of apoptotic death by Ras-transformed cellsexpressing E1A is associated with downregulation of E1A expression in thepresence of HDIs. In turn, the forced HDI-independent expression of E1A pavesthe way for apoptosis induction.Expression of adenoviral E1A increases the sensitivity of tumor cells toapoptosis-inducing agents .Therefo


Since 2013, the H7N9 avian influenza A virus (AIV) has caused human infections and to the extent of now surpassing H5N1. This raises an alarm about the potential of H7N9 to become a pandemic problem. Our compilation of the amino acid changes required for AIVs to cross the species-barrier discovers 58 that have very high proportions in both the human- and avian-isolated H7N9 viruses. These changes correspond with sporadic human infections that continue to occur in regions of avian infections. Among the six internal viral genes, amino acid changes do not differ significantly between H9N2 and H7N9, except for V100A in PA, and K526R, D627K, and D701N in PB2. H9N2 AIVs provide internal genes to H7N9. Most of the amino acid changes in H7N9 appear to come directly from H9N2. Seventeen amino acid substitutions appear to have fixed quickly by the 5th wave. Among these, six amino acid sites in HA1 are receptor binding sites, and PB2-A588V was shown to promote the adaptation of AIVs to mammals. The accelerated fixation of mutations may promote the adaptation of H7N9 to human, but need further functional evidence. Although H7N9 AIVs still cannot efficiently transmit between humans, they have the genetic makeup associated with human infections. These viruses must be controlled in poultry to remove the threat of it becoming a human pandemic event. The H7N9 avian influenza virus (AIV) is a triple reassortant virus having hemagglutinin (HA) and neuraminidase (NA) genes from migratory birds and six internal genes from chicken H9N2 AIVs , the Global Initiative on Sharing Avian Influenza Data (GISAID) database (www.gisaid.org) and the Influenza Research Database (FluDB) (www.FluDB.org). Redundant sequences were removed and laboratory strains were excluded. GenBank/GISAID accession numbers were listed in Supplementary Table All previously published genomes of H7N9 and H9N2 influenza A viruses (1994–2017) were obtained from the Influenza Virus Resource at the National Center for Biotechnology Information (NCBI) , then the amino acid proportion f  at the jth position at wave w was given by f  = n/N(w)  and f , there was a proportion switch between jam and jan between waves w and w+1 when both of the following conditions were met: (i) f  + f  > 0.8 and  + f  > 0.75; and (ii) [n – n] and [n – n] had opposite signs or zero in absolute value  in HA had very high proportions in both human- and avian-isolated H7N9 Figure . These cE/D627K/N and D701N in PB2 had much higher proportions in human-isolated than avian-isolated H7N9 in all 5 waves  of H9N2 has amino acid change E/D627K/N in wave 4, and one in four sequences (25%) have change D701N in wave 3. However, the relatively high proportions of these two mutations may reflect a bias of having few available sequences.A trend for fixation occurred in the 5th wave in 17 sites: A118T, S123N, R136K, A131V, L173I, and M232I in HA1 (H5 numbering); E383A, V426I, and S486R in HA2 (H5 numbering); I16T, S247P, and N327S in NA (N9 numbering); N394D in PA (N9 numbering); E24D in M2 (N9 numbering); and K191E, N559T and A588V in PB2 (N9 numbering)  protein structures of the HA [Protein Data Bank (PDB) code:4LCX, but then their proportions increased quickly reaching 100 and 97.43%, respectively, by the 5th wave. Amino acid change N327S was first identified in the 2nd wave at a proportion of 9.90%. This change increased to 97.44%, and almost replaced 327N (2.56%) by the 5th wave.Recent research on gain-of-function has identified genetic changes required for AIVs to cross the species-barrier and adapt to replication and transmission in mammals. Our compilation of these genetic changes (Supplementary Table AIVs must adapt to their new host (humans) to infect successfully. To initiate infection, hemagglutinin (HA), which is the major surface glycoprotein of influenza viruses, binds to host cell surface complex glycans via a terminal sialic acid. The switch of the preference from avian- to human-type sialic acid receptors  is a key element necessary for AIVs to cause human pandemics (Matrosovich et al., A series of amino acid changes suggested to enhance polymerase activity and increase virulence in mammals have very high proportions in both the human- and avian-isolated H7N9 viruses (Supplementary Table Amino acid changes E/D627K/N and D701N in PB2 were shown to be critical adaptations for infecting mammals (Subbarao et al., Six internal genes of H9N2 AIVs have very high proportions in a series of amino acid changes that associate with changes in host tropism or increased pathogenicity (Supplementary Table The 5th wave of human infection saw a rapid growth in the number of cases (Wang et al., H7N9 AIVs have continuously evolved since 2013. Although they have triggered five epidemics of human infections, they still do not have efficient human-human transmission, and only fix to avian. Our analyses identify a series of amino acid changes that associate with cross-species transmission from avian to human in high proportions in both human- and avian-isolated H7N9 AIVs. Avian-isolated H7N9 viruses have the genetic makeup associated with human infections and this suggests that if we cannot control this subtype in poultry, an impending human pandemic is still on the doorstep.YS conceived, designed, and supervised the study. ZP, DX, XL, TL, and XS collected and analyzed the data. YS and RM wrote the drafts of the manuscript. ML commented on and revised drafts of the manuscript. All authors read and approved the final report.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.


LDLR) result in failure of receptor mediated endocytosis of LDL leading to its elevated plasma levels and onset of familial hypercholesterolemia (FH). In the current study we investigated the role of PON1 polymorphisms rs662; c.575A > G (p.Gln192Arg) and rs854560; c.163T > A (p.Leu55Met) in a large family having FH patients harboring a functional mutation in LDLR. Genotypes were revealed by RFLP, followed by confirmation through Sanger sequencing. PON1 activity was measure by spectrophotometry. Our results show significantly reduced serum paraoxonase and arylesterase activities in FH patients compared with the healthy individuals of the family (p < 0.05). PON1 QQ192 genotype showed a significantly higher association with FH (p=0.0002). PON1 Q192 isoform was associated with reduced serum paraoxonase activity by in silico analysis and PON1 R192 exhibited higher serum paraoxonase and arylesterase activity than the other polymorphs. Our results highlight that the combination of LDLR mutations and PON1 MMQQ genotypes may lead to severe cardiac events.Paraoxonase 1 (PON1) is a serum enzyme associated with high density lipoprotein (HDL) regulation through its paraoxonase and arylesterase activity. PON1 inhibits the oxidation of HDL and low density lipoprotein (LDL), and is involved in the pathogenesis of a variety of diseases including atherosclerosis. Conversely, mutations in the low density lipoprotein receptor ( The human paraoxonase (PON) gene family located on the long arm of chromosome 17 consists of three members, each of which coding for three different calcium dependent esterases: PON1, PON2, and PON3 . PON1 anPON1) genotype status, anti-oxidant, and anti-atherogenic capacity of the enzyme has been suggested previously  mice significantly inhibits progression of atherosclerosis (LDLR. A large Pakistani family with LDLR associated FH was investigated in this study to understand the role of PON1 in the protection against atherosclerosis (PON1 coding sequences of single nucleotide polymorphisms (SNPs) rs662  and rs854560 (c.163T > A (p.Leu55Met) in relation to resultant paraoxonase and arylesterase activity in hypercholesterolemia patients with mutated LDLR. The structural and functional aspects of these SNPs have also been studied to explore how different allozymes affect and mediate the paraoxonase and arylesterase activities of the enzyme.Functional mutations in LDLR gene cause the monogenic form of familial hypercholesterolemia (FH) (LDLR mutation (c. 2416_2417InsG) presenting clinical FH was identified, including 10 patients suffering from FH and 24 healthy individuals (A large consanguineous Pakistani family (n=34) with Blood (5 mL) was drawn after 12–14 h fasting and collected in separate tubes for DNA extraction by organic method (PON1 gene as described by PON1 Q192R (rs662) and PON1 L55M (rs854560) were as follows: PON1 Q192R-forward primer TTG AAT GAT ATT GTT GCT GTG GGA CCT GAG and PON1 Q192R-reverse primer CGA CCA CGC TAA ACC CAA ATA CAT CTC CCA GaA; PON1 L55M -forward primer GAG TGATGT ATA GCC CCA GTT TC and PON1 L55M-reverse primer AGT CCATTA GGC AGTATC TCC g. The reverse primers contained mismatched nucleotides as indicated by lower case letters in italics. This allowed a restriction site for HinfI (G/ANTC) to be introduced into the DNA amplification products in the presence of the polymorphisms arginine-PON1-192 or leucine-PON1-55. PON1 gene segments were amplified by PCR using 0.3 mM deoxyribonucleotide triphosphates (dNTPs), 1x PCR buffer , 2.0 mM MgCl2, 0.5 mM of each primer (forward and reverse), 1.5 U of Taq Polymerase, and 50 ng of genomic DNA. The thermal cycling consisted of an initial denaturation at 95 °C for 4 min, followed by 35 cycles of amplification consisting of denaturing at 95 °C for 45 s, primer annealing at 55 °C for 1 min and chain extension at 72 °C for 1 min. A final extension step was performed at 72 °C for 10 min.Genomic DNA was extracted from peripheral blood leukocytes using standard procedures . The results were analyzed to compare the prevalence of PON1 L55M and PON1 Q192R SNPs. To confirm the DNA sequence, amplified products were also subjected to bidirectional sequencing by the Sanger sequencing method to reveal the genotypes of all the individuals.PCR products were purified using a DNA extraction kit  and subjected to restriction with Het al., 2009). The assay mixture contained 1.0 mM paraoxon and 1.0 mM CaCl2 in 50 mM glycine/NaOH buffer (pH 10.5). The amount of 4-nitrophenol liberated was calculated from the molar coefficient 18,290/Mcm.Paraoxonase activity was determined by measuring the increase in absorbance at 412 nm (Thermo Scientific GENESYS 10 UV Scanning UV/Visible Spectrophotometer) due to the formation of 4-nitrophenol (using paraoxon as substrate) (Zehra 2 in 20 mM Tris-HCl buffer (pH 8.0). The rate of hydrolysis was monitored at 270 nm. The results were calculated using extinction coefficient 1310/Mcm.Arylesterase activity was determined using an assay mixture containing 1.0 mM phenylacetate and 0.9 mM CaClThe lipid profile, including total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C) and HDL cholesterol (HDL-C), of all the subjects was obtained using a Roche/Hitachi automated system with commercial kits for CHOL (Cholesterol CHOD-PAP), LDL-C plus 2nd generation (LDL Cholesterol), HDL-C plus 3rd generation (HDL-Cholesterol) and TG (Triglyceride GPO-PAP) .2) and Fisher’s Exact test. P-values less than 0.05 were considered statistically significant. Continuous variables were analyzed using ANOVA whereas categorical variables were compared by Chi-squared and Fisher’s Exact test.Results for continuous variables are reported as mean ± SD. Differences among patients and healthy family members were assessed using Chi-square  and LDL-C (p < 0.0001) while differences between the TG (p = 0.28) and HDL-C (p = 0.084) were not significant. Paraoxonase (p = 0.001) and arylesterase activities (p = 0.002) were significantly lower in patients compared to healthy individuals of the family.There was no significant difference between patients and healthy individuals with respect to mean ± SD values of age, gender and body mass index (BMI). Routine laboratory findings of lipid profile indicated significant differences between the patients and healthy individuals for TC (PON1 SNPs and combinations) is given in PON1 M55 and Q192 allele prevalence was highly correlated with individuals with FH. Similarly, 80% of patients showed M55M55 genotype whereas 90% showed Q192Q192 genotype. PON1 L55 and R192 alleles were deficient in patients compared to healthy individuals of the family. Conversely, PON1 M55 and Q192 alleles were found significantly higher in patients. Similarly, the haplotype MM/QQ was prevalent in 80% of the FH patients whereas 10% of the FH patients exhibited LM/QQ and LM/QR haplotypes.PCR amplification results and restriction fragment length polymorphism (RFLP) results of amplified products were in accordance with the specific band sizes reported previously , the biding affinity for the allozyme R192 was observed to be 6.3 kcal/mol; however, it was 5.7 kcal/mol for Q192 .2 of paraoxon or OH of phenylacetate through hydrogen bonds. This arrangement helps positioning the acyl bond in proximity to the catalytic dyad of histidine residues H115 and H134, which has been reported to deprotonate a water molecule to generate a hydroxyl ion, thereby hydroxylating the lactones (paraoxon) and arylesters (phenylacetate) 2) mediates with acyl oxygen atoms then a small sized lysine sidechain with just one –NH3+, R192 thus helps in positioning the acyl bond of the substrate to the catalytic dyad of H115 and H134, thus the higher arylesterase and paraoxonase activity of R192 allozyme  and LDLC (728 mg/dL) were markedly high and HDL-C (22 mg/dL) was lower compared to other FH patients in the family. The presence of tendon xanthomas is high risk factor of CVD among patients with FH, which along with decreased paraoxonase and arylesterase activities may lead to atherosclerosis (PON1 coding region SNPs). The L55M polymorphism affects the enzyme concentration (plasma PON1 protein levels indicated by serum arylesterase activity), whereas the Q192R polymorphism affects the catalytic efficiency (serum paraoxonase activity), but not the concentration . M is not favored in human populations, but it is kept in populations in heterozygous individuals. The L55M mutation may considerably affect PON1’s stability and thereby account for the lower enzymatic activity because M55 isoform is an unstable form (sensitive to proteolysis)  has been shown previously to be a better predictor of vascular disease than PON1 Q192R or PON1 L55M genotypes due to possible effects of other genetic and non-genetic factors . The role of PON1 and its various polymorphs has not been studied previously in FH subjects. This work sought to investigate paraoxonase and arylesterase activity of various PON1 SNPs in individuals with mutated


Cumulative evidence indicates that activation of innate immune responses in the central nervous system (CNS) induces the expression of type 1 interferons (T1 IFNs), a family of cytokines. The T1 IFNs (IFN-α/β), through activation of the JAK/STAT-signaling in microglia, astrocytes, and neurons, induce the expression of IFN-inducible proteins, which mediate the pro- and anti-inflammatory functions of IFNs. Accordingly, T1 IFN-inducible Absent in Melanoma 2 proteins (murine Aim2 and human AIM2) negatively regulate the expression of TI IFNs and, upon sensing higher levels of cytosolic DNA, assemble the Aim2/AIM2 inflammasome, resulting in activation of caspase-1, pyroptosis, and the secretion of pro-inflammatory cytokines . Of interest, studies have indicated a role for the Aim2/AIM2 proteins in neuroinflammation and neurodegenerative diseases, including Alzheimer’s disease (AD). The ability of Aim2/AIM2 proteins to exert pro- and anti-inflammatory effects in CNS may depend upon age, sex hormones, cell-types, and the expression of species-specific negative regulators of the Aim2/AIM2 inflammasome. Therefore, we discuss the role of Aim2/AIM2 proteins in the development of AD. An improved understanding of the role of Absent in Melanoma 2 proteins in AD could identify new approaches to treat patients. T1 Is (ISGs) , 5. Altes (ISGs) –11. Accos (ISGs) .Depending on the neurodegenerative disease state, T1 IFNs can be protective or deleterious . For exa−/− mice [Ifnb gene (encoding for IFN-β) exhibited a loss of the dopaminergic neurons, development of Lewy bodies and Parkinson’s-like disease [SWE /PS1ΔE9 AD mice that were deficient in the IFN-α receptor gene (Ifnar1) exhibited a reduced pathology and an altered microglial phenotype [Aim−/−.(B6.Sv129); 5XFAD mice [Generation of the IFNAR1-null  or IFN-β−/− mice  improved disease . Of inte disease , 22. Acchenotype . SimilarFAD mice .Type 1 (T1) interferonopathies are characterized by the constitutive production of T1 IFNs and activation of T1 IFN response (increase in the expression of ISGs) in cells –26. The Accumulating evidence indicates that an inflammasome activation in glial and neuronal cells modulates neuroinflammation , 32. TheDevelopment of aging-associated Alzheimer’s disease (AD) is characterized by synaptic loss and neuronal death, which results in cognitive decline, dementia, and loss of motor functions with time –38. DepoCumulative evidence indicates a role for the T1 IFN-signaling in the development and progression of AD –23, 42. AIM2-like receptor (ALR)-family genes (ALR genes) [Aim2 and Ifi202) and human genes . Notably, the murine Aim2 and human AIM2 protein sequences are conserved between the mouse and humans. Most proteins in the family contain the N-terminal Pyrin signaling domain (PYD) and the C-terminal DNA-binding HIN domain [The T1 IFN-inducible PYHIN-protein family includes the structurally related Aim2/AIM2 proteins that are encoded by the R genes) –55. The N domain . The murN domain  and humaN domain  proteinsN domain . InteresN domain . SimilarN domain , 58. BasN domain , 51, 52.N domain .Aim−/− female mice on the mixed (B6.Sv129) [Aim2-deficient mice on mixed genetic background exhibited inflammation and adipogenesis in white adipose tissue with age, leading to obesity and insulin resistance [Aim2-deficiency in the B6.Sv129 and B6 mice resulted in an increase in the expression of the IFN-inducible p202 protein [Aim2−/−.(B6.Sv129) mice are not very informative with respect to the precise role of the Aim2 protein in the development of neuroinflammation and neurodegenerative diseases.Bone marrow-derived macrophages from 6.Sv129)  or pure 6.Sv129)  genetic sistance . Notably protein , 51, 59. protein , studiesAIM2 gene expression in peripheral blood mononuclear cells (PBMCs) from vascular patients  revealed a significant positive association with age [AIM2 expression between patients with advanced atherosclerosis and other vascular diseases. Similarly, human normal lung fibroblasts (WI-38) in culture, upon aging, exhibited a measurable increase in the levels of the AIM2 protein [AIM2 gene expression in PBMCs from healthy young  and elderly  individuals without any treatment or after in vitro stimulation of cells with poly(dA:dT), an AIM2 ligand, noted that the stimulation of cells from elderly individuals resulted in reduced expression of the AIM2 gene and pro-inflammatory cytokines than the young donors [AIM2 gene and the functions of the AIM2 protein in the CNS decrease with the age.Analysis of the with age . Notably protein . However protein , which ag donors . Becauseg donors , it rema+) and total sleenocytes from the B6, New Zealand black (NZB) and B6.Nba2 congenic males, as compared with age-matched females, the basal levels of the Aim2 mRNA and protein were significantly higher [Aim2 mRNA and protein [AIM2 gene in PBMCs from males (n = 62) was higher than females (n = 38) [AIM2 mRNA levels in naïve macrophages were higher in SLE men (n = 6) than women (n = 9) [Aim2 and AIM2 genes in microglia and astrocytes is regulated by the sex hormones in a cell-type dependent manner. Further, expression of the p202 protein [In purified splenic B cells (B220y higher . Further protein . Similar(n = 38) . Additio (n = 9) . Conside protein  and IFI1 protein , the neg protein , studiesAim2/AIM2 genes [Aim2/AIM2 gene expression) in macrophages/microglia. In turn, activation of the Aim2/AIM2 inflammasome by higher levels of the cytosolic DNA in macrophages negatively regulates the ISD pathway to suppress T1 IFN response. Therefore, a mutual regulation between activation of ISD pathway and inflammasome pathway in the CNS may be critical to maintain a homeostasis  in macrophages/microglia , 52, 69.M2 genes , 61 and M2 genes . HoweverM2 genes . NotablyM2 genes . These oAim2-deficient male or female mice expressed higher basal levels of the IFN-β mRNA and activated the T1 IFN response as compared with the age- and sex-matched wild-type B6 mice [Immune cells  from  B6 mice , 51. Fur B6 mice . However B6 mice , resulte B6 mice . Additio B6 mice . Further B6 mice . TogetheConstitutive expression of the POP3 protein, a negative regulator of the AIM2 inflammasome , in muriAim2 gene. Interestingly, in a murine model of chronic neurodegeneration, the expression of Aim2 gene was upregulated in vivo in a T1 IFN-dependent fashion [S. aureus in mice resulted in the production of IL-1β and other inflammatory chemokines and cytokines (including IL-6 and CXCL1) [Aim2 gene expression in the CNS by T1 IFNs or by an infection by bacteria may contribute to neuroinflammation.An extensive expression analysis of T1 IFN-inducible cytosolic DNA sensors in highly purified primary astrocytes and microglia indicated that both cell-types express mRNAs encoding for the PYHIN-family proteins, including the Aim2 protein . Treatme fashion . Furtherd CXCL1) . TogetheAim2 mRNA and treatment of neurons with synthetic double-stranded DNA induced IL-1β secretion in Aim2-dependent manner [Aim2 gene expression in neurons indicated that Aim2 protein acted in cell-autonomous manner to regulate the neuronal morphology. Notably, the behavioral analyses of the Aim2−/−.(B6.Sv129) mice revealed that the mice exhibited lower locomotor activity, increased anxious behaviors, and a reduced auditory fear memory [−/−.(B6.Sv129) mice is due to activation of the T1 IFN response, which increases the expression of the p202 protein.Cultured cortical and hippocampal mouse neurons expressed higher basal levels of t manner . Interesr memory . HoweverUsing a controlled cortical impact (CCI) mouse model for traumatic brain injury (TBI) and manipulating the extent of pyroptosis in blood-brain barrier (BBB) cells, one study noted that TBI resulted in Aim2 inflammasome-mediated pyroptosis in brain microvascular endothelial cells (BMVECs) within injured cerebral cortex region . FurtherAim2 gene was detectable in neurons, astrocytes, oligodendrocytes, and microglia in the sham-operated spinal cord. In rats with SCI, Aim2 protein was also detectable in leukocytes and activated microglia/macrophages (CD68+ cells) in the spinal cord. These observations are consistent with the possibility that Aim2 protein is expressed in the normal spinal cord, and after SCI, expression of the Aim2 gene increases in activated astrocytes and microglia due to the secretion of certain cytokines by infiltrated leukocytes.In a rat model of T9 spinal cord contusive injury (SCI), levels of the Aim2 protein were measurably higher than sham-operated rats after 1 h, 6 h, and 1 day of the injury . Further−/−.(B6.Sv129), 5XFAD, and Aim2−/−.(B6.Sv129); 5XFAD mice, a study noted that Aim2 gene deficiency in the Aim2−/−.(B6.Sv129); 5XFAD mice mitigated the Aβ deposition in the cerebral cortex and hippocampus region as compared with the parental 5XFAD mice [Aim2−/−.(B6.Sv129); 5XFAD mice as compared with age-matched 5XFAD mice. Surprisingly, the Aim2−/−.(B6.Sv129) mice did not exhibit an improved memory and reduced anxiety as compared with 5XFAD mice in behavioral tests [Aim2−/−.(B6.Sv129); 5XFAD mice, thus suggesting a role for other inflammasomes in the observed phenotype. Of interest, IL-6 and IL-18 expression was higher in brains from the Aim2−/−(B6. Sv129); 5XFAD mice than the 5XFAD mice [Using the wild-type B6, Aim2FAD mice . Furtheral tests . CompareFAD mice .Aim2 gene deletion in APP/PS1 mice increased spatial memory in behavioral tests, facilitated the long-term potentiation in hippocampal slices, altered the structure of dendrites, and increased the dendritic spine densities. Notably, a transcriptional analysis revealed that Aim2 gene deletion in the APP/PS1 mice altered the expression levels of certain proteins, including Pten, Homer1, and Ppp2r3a, and increased activation of the AKT protein kinase [In another study , the APPn kinase . These oAim2−/−.(B6.Sv129); 5XFAD AD mouse model, the lack of Aim2 protein function reduced Aβ deposition in the brain [Aim2 gene deficiency in the mice is predicted to increase the expression of T1 IFN-β and activate a T1 IFN response [Trem2 mRNA levels [Aim2 gene deficiency reduced the Aβ deposits in the Aim2−/−.(B6.Sv129); 5XFAD AD mouse model through upregulation of the Trem2 gene expression. Therefore, it is important to determine the role of Aim2 protein and its negative regulators in the regulation of Aβ deposition.In he brain . Consideresponse , 51 and A levels , which eA levels , it is cAim2−/−.(B6.Sv129); 5XFAD mice, Aim2 gene deficiency increased the levels of certain pro-inflammatory cytokines (including IL-6) in the brain and had limited effect on the cognitive behavior of the mice. As noted above, Aim2 gene deficiency in the B6.Sv129 or B6 mice activated a T1 IFN response, resulting in an increase in the expression of p202 protein (encoded by the Ifi202 gene) [Aim2 gene exhibited the transcriptional changes in certain genes [In 02 gene) , 51. The02 gene) . Therefoin genes Aim2 gene deficiency in the B6.Sv129 mice resulted in inflammation in white adipose tissue and associated with an increase in the expression of the Ifi202 gene [Ifi202 gene expression in stromal vascular fraction (SVF) inhibited adipogenesis and inflammation [Aim2-deficient B6.Sv129 mice, upon challenge with LPS, expressed significantly higher levels of IL-6 mRNA [Interestingly, 202 gene . Furtherammation . AdditioL-6 mRNA . TogetheATM (A-T mutated) gene [Atm-deficient mice and certain AD mouse models exhibited a loss of ATM protein kinase functions in neurons [Neurodegeneration in ataxia telangiectasia (A-T) patients is caused by inheriting mutations in the ed) gene , 82, whied) gene . Accordi neurons . Interes neurons . Further neurons . As noteAim2 gene in epithelial cells [Aim2−/−.(B6.Sv129) mice [Aim2-deficient mice, as compared with age- and sex-matched wild-type B6 mice, exhibited activation of the ATM protein kinase [The ATM protein kinase family also includes DNA-dependent protein kinase (DNA-PK) . Interesal cells  and hipp29) mice  activate29) mice . Similarn kinase . Becausen kinase  and actin kinase , studiesAim2-deficient 5XFAD AD mice on the pure B6 genetic background  could provide novel insights into the precise role of the Aim2 protein in modulation of neuroinflammation and the development of AD. An improved understanding of the mechanisms by which Absent in Melanoma 2 proteins and their negative regulators modulate neuroinflammation could identify new approaches to treat AD.Suppression of T1 IFN response by Aim2 and AIM2 proteins in the CNS may be important to keep the levels of the T1 IFN-inducible negative regulator proteins (murine p202 and human IFI16-β and POP3) below a threshold to suppress neuroinflammation-related neurodegenerative diseases (Table


Low pathogenic avian influenza (LPAI) viruses can silently circulate in poultry and wild aquatic birds and potentially mutate into highly pathogenic avian influenza (HPAI) viruses. In the U.S., recent emergence and spread of H7N8 and H7N9 HPAI viruses not only caused devastating losses to domestic poultry but also underscored the capability of LPAI viruses to mutate into HPAI viruses. Therefore, in this study, we evaluated pathogenicity and transmissibility of H7N8 and H7N9 LPAI viruses (the progenitors of HPAI viruses) in chickens and turkeys. We also included H7N2 isolated from an outbreak of LPAI in commercial chickens. H7 viruses replicated more efficiently in the respiratory tract than in the gastrointestinal tract, suggesting that their replication is restricted to the upper respiratory tract. Specifically, H7N2 replicated most efficiently in two-week-old chickens and turkeys. In contrast, H7N8 replicated least efficiently in those birds. Further, replication of H7N2 and H7N9 was restricted in the upper respiratory tract of four-week-old specific-pathogen-free (SPF) and broiler chickens. Despite their restricted replication, the two viruses efficiently transmitted from infected to naïve birds by direct contact, leading to seroconversion of contacted chickens. Our findings suggest the importance of continuous monitoring and surveillance of LPAI viruses in the fields.  Orthomyxoviridae and the genus Influenzavirus A. The virus has a negative-sense, single-stranded and segmented RNA genome and contains eight gene segments encoding at least 10 proteins: Polymerase basic 1 (PB1), PB2, polymerase acid (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix 1 (M1), M2, nonstructural 1 (NS1) and 2 (NS2) [Avian influenza virus (AIV) belongs to the family Most AIVs cause little to no disease in birds. These low pathogenic avian influenza (LPAI) viruses contain an HA cleavage site which can only be cleaved by proteases available in the intestinal and respiratory tracts . In contH7 subtype viruses are widely distributed and cause high mortality in gallinaceous poultry with substantial economic losses for the poultry industry and sporadic human infections ,7. LPAI H7 HPAI viruses have emerged multiple times in commercial poultry in the U.S. . In 2016The detection and spread of H5 and H7 LPAI viruses in gallinaceous poultry are considered as an indicative emergence of HPAI viruses. This highlights the importance of controlling LPAI viruses in the field and need for routine and frequent testing of poultry for AIV. For a better control of LPAI viruses, we evaluated replication and bird-to-bird transmissibility of North American H7N8 and H7N9 LPAI viruses that were isolated from commercial poultry farms during AI outbreaks in 2016 and 2017, respectively. In this study, we included one of the H7N2 isolates from commercial chicken farms in 2004.6), the viruses were additionally passaged in the eggs. The HA titers of the two viruses were increased to 28 after the third passage in eggs. After each passage of the viruses, we conducted nucleotide sequence analyses of the HA and NA genes by RT-PCR and did not detect occurrence of any mutations during viral passages. LPAI viruses used in this study were A/Chicken/Delaware/VIVA/04 (H7N2), A/TY/IN/1573-2/2016 (H7N8), and A/chicken/Tennessee/17-007431-3/2017 (H7N9). H7N2 was isolated from a commercial broiler chicken in Delaware during outbreaks of AIV in 2004 . H7N8 anTo compare their cytopathic effect (CPE), Madin–Darby canine kidney (MDCK) cells in a 6 well plate were individually infected with H7N2, H7N8, and H7N9 at a multiplicity of infection (MOI) of 0.1 PFU/cell. After 1 h of adsorption, the culture medium was changed with Dulbecco’s modified Eagle’s medium (DMEM) with or without the addition of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (1 μg/mL). After 12 h post-infection (hpi), the infected cells were visualized directly by photomicroscopy. Further, efficient cleavage of HA protein in virus-infected MDCK cells was evaluated by Western blot analysis. Cell lysates were separated by SDS-PAGE, and transferred protein into a nitrocellulose membrane was detected by using anti-H7 HA, A/Netherlands/219/2003 (H7N7) (BEI resources). Plaque formation of H7 viruses in MDCK cells was conducted by plaque assay . n = 6 for each virus) were intranasally inoculated with 200 µL of each virus (107 pfu/mL). Tissue samples  were collected from three birds for virus titration in MDCK cells on days 3 post-infection (pi). Collected samples were homogenized, diluted, and inoculated into MDCK cells. Virus titers were expressed as 50% tissue culture infectious dose (TCID50/g) by the end-point method of Reed and Muench [Replication of H7N2, H7N8, and H7N9 viruses was determined in 2-week-old chickens (SPF) and turkeys. One-day-old turkeys (White Holland) were obtained from Murray McMurray farm  and housed in our enhanced BSL-2 (ABSL-2) facility. All turkeys were confirmed to be seronegative to H7 viruses. Birds (d Muench . In addin = 6 for each virus) were intranasally inoculated with 200 µL of each virus (107 pfu/mL). On days 3 pi, tissue samples (lung and trachea) were collected from three chickens for virus titration in MDCK cells. In addition, trachea samples were fixed in phosphate-buffered formalin (10%), processed for section and stained with hematoxylin and eosin. Sections from mock-infected birds were used as controls.Replication and transmissibility of H7N2 and H7N9 viruses were determined in four-week-old SPF (specific-pathogen-free) and broiler chickens. One-day-old broiler chickens were obtained from a commercial hatchery  and housed in ABSL-2 facility. All chickens were seronegative to H7 viruses. Chickens (n = 3) were placed in an isolator. At 24 dpi, three contact exposure birds (naïve birds) of the same species were placed into the same isolator. After the first day of exposure, swab  samples were collected daily up to 5 days, and virus shedding was determined as described above. Birds were monitored daily for clinical signs up to 14 days post-exposure (dpe). Seroconversion was determined by HI assay.In parallel with viral replication, transmissibility of H7 viruses was determined in SPF and broiler chickens. Each experiment, virus-infected birds  using the Turkey’s multiple comparison test. All of the results were analyzed using Prism 5.0  with a significance level of Since H7N8 and H7N9 strains are recently emerged viruses, little information is available for viral infectivity. We first evaluated cytopathic effect (CPE) in virus-infected cells A. In 12 4 TCID50/g). H7N9 replicated in the trachea of one chicken. The titer of H7N9 was lower than those of H7N2 infected chickens. In contrast, replication of H7N8 was not detected in the collected tissue samples. In general, shedding of H7 viruses was only detected in oral swab samples at 3 dpi, indicating that viral replication was limited to the upper respiratory tract.Virus replication was determined in chickens and turkeys, two major poultry associated with the outbreaks of AIV. In SPF chickens, replication of H7 viruses was mostly restricted to the trachea. Virus replication was not detected in lungs (except one chicken), spleens, and brain of infected chickens. H7N2 replicated well in the trachea of all three chickens A. One chIn turkeys, H7N2 replicated in the trachea of two birds B. The vi5). In H7N8 infected groups, seroconversion was found in turkeys (HI titer 26). In contrast, significantly low HI titers was detected in the chickens infected with H7N8, suggesting that H7N8 strain may not be adapted for efficient replication in chickens. This in vivo replication result suggests inefficient replication of H7N8 in poultry. Therefore, we further compared replication and transmissibility of only two H7 viruses (H7N2 and H7N9) in 4-week-old chickens. All the remaining chickens and turkeys were healthy without exhibiting any clinical signs up to 14 dpi. Despite their restricted replication, we found seroconversion of all the chickens and turkeys infected with H7N2 or H7N9  between infected and direct-contact exposed chickens and between SPF and broiler chickens. In contrast, groups of chickens with H7N9 showed different pattern in seroconversion. HI titers of SPF chickens were significantly lower than those of broiler chickens. Interestingly, we found comparable levels of HI titers of broiler chickens infected with H7N9 to those of SPF and broiler chickens infected with H7N2. This suggests that H7N9 might replicate more efficiently in broiler chickens than in SPF chickens. All chickens were monitored for exhibition of clinical signs for 14 dpe. They were healthy except for one broiler chicken infected with H7N2. Mortality of the chicken was found at 10 dpi, although we did not find specific lesions associated with avian influenza infection by necropsy. At 14 dpe, serum samples were collected from all the remaining chickens to determine seroconversion. All chickens were seropositive . In H7N2LPAI is an economically important disease and remains a continuous threat to commercial poultry operations . H5 and DRKSRHRRIRGLF; underline). The change in the cleavage site sequence of HA protein affects pathogenesis of the viruses in poultry [Our study suggests that H7 LPAI viruses are avirulent in domestic poultry. We further found attenuation of H7N8 and H7N9 compared to H7N2. Multiple outbreaks of LPAI H7N2 occurred in chickens and turkeys between 1996 and 2004 in the Eastern U.S. . However poultry ,23. In f poultry ,13. It iDirect contact is one of the important ways for bird-to-bird transmission of LPAI viruses . In this


Persistent human papillomavirus (HPV) infection is the leading cause of cervical cancer. Although the fundamental link between HPV infection and oncogenesis is established, the specific mechanisms of virus-mediated transformation are not fully understood. We previously demonstrated that the HPV encoded E6 protein increases the activity of the proto-oncogenic transcription factor STAT3 in primary human keratinocytes; however, the molecular basis for STAT3 activation in cervical cancer remains unclear. Here, we show that STAT3 phosphorylation in HPV positive cervical cancer cells is mediated primarily via autocrine activation by the pro-inflammatory cytokine Interleukin 6 (IL-6). Antibody-mediated blockade of IL-6 signalling in HPV positive cells inhibits STAT3 phosphorylation, whereas both recombinant IL-6 and conditioned media from HPV positive cells leads to increased STAT3 phosphorylation within HPV negative cervical cancer cells. Interestingly, we demonstrate that activation of the transcription factor NFκB, involving the small GTPase Rac1, is required for IL-6 production and subsequent STAT3 activation. Our data provides new insights into the molecular re-wiring of cancer cells by HPV E6. We reveal that activation of an IL-6 signalling axis drives the autocrine and paracrine phosphorylation of STAT3 within HPV positive cervical cancers cells and that activation of this pathway is essential for cervical cancer cell proliferation and survival. Greater understanding of this pathway provides a potential opportunity for the use of existing clinically approved drugs for the treatment of HPV-mediated cervical cancer. Persistent infection with HPV is the predominant cause of anogenital and oral cancers. Transformation requires the re-wiring of signalling pathways in infected cells by virus encoded oncoproteins. At this point, a comprehensive understanding of the full range of host pathways necessary for HPV-mediated carcinogenesis is still lacking. In this study we describe a signalling circuit resulting in the aberrant production of the IL-6 cytokine. Mediated by the HPV E6 oncoprotein, it requires activation of the NFκB transcription factor. The autocrine and paracrine actions of IL-6 are essential for STAT3 activation in HPV positive cervical cancers and loss of the pathway results in increased cancer cell death and a reduction in proliferation. This study provides molecular insights into the mechanisms by which a virus encoded oncoprotein activates an oncogenic pathway and identifies potential targets for therapeutic intervention. High-riin vivo . EGFR acin vivo –7. In coin vivo . The E7 in vivo , resultiin vivo . Additioin vivo ,12. HPV in vivo . HR-E6 pin vivo ,15. In ain vivo –18.bona fide oncogene and its aberrant activation has been observed in a growing number of malignancies [The transcription factor signal transducer and activator of transcription (STAT) 3 is an essential regulator of cellular proliferation, differentiation and survival . It is agnancies . As suchgnancies .Oncogenic viruses can activate STAT3 to drive cellular proliferation, necessary for viral replication and tumourigenesis . Using aA number of extracellular stimuli including cytokines and growth factors induce STAT3 phosphorylation and signalling . This reHere, we show that HPV positive cervical cancer cells have higher levels of phosphorylated STAT3 protein when compared with those that are HPV negative. This results from increased IL-6 production and release, leading to autocrine and paracrine activation of STAT3 via a signalling pathway requiring the IL-6 co-receptor gp130. Mechanistically, we show that IL-6 production is controlled by E6-mediated stimulation of NFκB signalling, which appears to be dependent on an upstream signalling pathway requiring the Rac1 GTPase and the AKT protein kinase. Finally, we demonstrate a correlation between NFκB activation, IL-6 expression and cervical disease progression, suggesting that targeting the IL-6 pathway to prevent STAT3 activation may have therapeutic benefits in cervical cancer.We analysed the level of STAT3 phosphorylation in a panel of six cervical cancer cell lines to establish whether HPV augmentation of STAT3 phosphorylation was evident. The panel included two HPV negative , two HPV16 positive  and two HPV18 positive  lines. Both HPV16+ and HPV18+ cancer cells retained markedly higher levels of STAT3 phosphorylation at both Y705 and S727 residues compared to the HPV negative cell lines . The oveAs STAT3 can be regulated by extracellular stimuli, we investigated whether HPV promotes the secretion of factors capable of inducing STAT3 phosphorylation. C33A cells (HPV-) incubated with conditioned media (CM) from HeLa (HPV18+) or CaSKi (HPV16+) cells showed an increase in STAT3 phosphorylation on both Y705 and S727 residues over time compared to treatment with CM from C33A cells . STAT3 pIL6, IL10, LIF (Leukaemia inhibitory factor) and OSM (Oncostatin M) mRNA levels were significantly higher than in C33A cells –gp130 co-receptor complex . IL-6 anTo confirm that autocrine IL-6/gp130 signalling was also required for STAT3 activation in HPV+ lines, HeLa cells were pre-incubated with IL-6 and gp130 neutralizing antibodies. Incubation with either neutralising antibody reduced STAT3 dual phosphorylation and nuclear translocation , confirmIL6 mRNA expression  targeting the IKKα/β complex, which phosphorylates and activates NFκB, or transfected with a plasmid encoding a mutant IκBα protein (IκBm), which cannot be degraded and as such retains inactive NFκB in the cytosol . Inhibitpression , IL-6 prpression  and secrpression . Importapression .IL6 mRNA expression, IL-6 protein levels and secretion , targeting the AKT1 and 2 isoforms  or transpression , coupledpression . To confpression  and a paIL6 mRNA expression  or co-transfected with a transdominant mutant Rac1 (Rac1 T17N - Rac1-DN) and IL-6 mRNA levels were measured by RT-qPCR. Inhibition of Rac1 activity with either NSC or Rac1-DN potently inhibited IL-6 mRNA expression in E6 expressing cells . InhibitKi cells . We alsoKi cells . CollectWe examined whether Rac1 was also necessary for STAT3 activation by E6. Chemical inhibition of Rac1 or over-expression of the Rac1-DN mutant decreased the dual phosphorylation of STAT3 in HPV18 E6 expressing C33A cells . SimilarWAF1/KIP1  and Survivin (birc5) to be at the transcriptional level  and IL-6 low . As our mechanistic analysis revealed a significant role for NFκB in IL-6 production, we wished to determine whether expression and activation of this critical transcription factor was also increased in the patient samples available. Western blot analysis revealed an increase in both the levels of total and phosphorylated p65 protein that correlated with disease progression -dependent angiogenesis in a STAT3 dependent manner  and has We identified NFκB as an essential upstream mediator of IL-6 expression. NFκB is a key component of the inflammatory response and a key hallmark of cancer . The indThe role of NFκB in cervical carcinomas remains controversial, with HPV showing the potential to both activate and inhibit the transactivation function of NFκB ,73,74. HPIK3CA gene is extensively mutated, with the most common mutation (E545K) resulting in constitutive PI3K/AKT signalling [PIK3CA, CaSKi cells encode the E545K mutant [PIK3CA [Our study uncovered a signalling circuit linking E6 to NFκB activation and IL-6 production. Chief amongst these, we found that the Rac1 GTPase was crucial in mediating the activation of NFκB. Cells expressing E6 were enriched for the GTP bound form of Rac1, indicating that E6 has the ability to activate the GTPase function of Rac1. The mechanism by which this occurs is currently not known, although it does not require interactions with E6AP, p53 or cellular PDZ domain containing proteins as classical E6 mutants deficient in these abilities are still able to increase IL-6 expression and STAT3 phosphorylation. Use of small molecule inhibitors or dominant negative forms of Rac1 support the idea that Rac1 GTPase function and interaction with downstream targets are crucial for the E6-mediated activation of NFκB. In this regard, we also identified a role for the protein kinase AKT in HPV-mediated NFκB activation. AKT can be activated by Rac1  and has gnalling . This ongnalling . InteresK mutant . It may  [PIK3CA .XL and Survivin, reinforcing the concept that STAT3 inhibition is deleterious to HPV cancer cell survival [Loss of active STAT3 had a significant impact on both cervical cancer cell proliferation and survival. The increased apoptosis observed was coupled with a loss of expression of key pro-survival factors such as Bcl-survival ,25. HoweThe NFκB–IL-6—STAT3 signalling axis is important to cancer biology. We previously demonstrated increased STAT3 phosphorylation in cervical disease  and we nThe data presented here demonstrate that NFκB is essential for the induction of IL-6 and the autocrine/paracrine induction of STAT3 phosphorylation in HPV+ cervical cancer cells. We identify that the Rac1 GTPase and protein kinase AKT lie upstream of NFκB/IL-6 signalling. Although therapies are not currently available, strategies to target the NFκB–IL-6 –STAT3 signalling axis may benefit the treatment of HPV+ cancers.http://www.shine/mvm.ed.ac.uk/archive.shtml), a biobank of over 20,000 samples designed to facilitate HPV associated research. The East of Scotland Research Ethics Service has given generic approval to the Scottish HPV Archive as a Research Tissue Bank (REC Ref 11/AL/0174) for HPV related research on anonymised archive samples. Samples are available for the present project though application to the Archive Steering Committee (HPV Archive Application Ref 0034). RNA and protein were extracted from the samples using Trizol as described by the manufacturer  and analysed as described.Cervical cytology samples were obtained from the Scottish HPV Archive , DoTc2 4510 , SiHa , CaSKi , SW756 (HPV18 positive squamous carcinoma) and HeLa  cells were purchased from ATCC and grown in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% Foetal Bovine Serum (FBS)  and 50 U/ml penicillin/streptomycin (Lonza). NHK cells were purchased from Lonza and maintained as described [The IKKα/β inhibitor IKK inhibitor VII was purchased from Calbiochem and used at a final concentration of 5 μM unless otherwise stated. The AKT1/2 inhibitor AKT VIII was purchased from Calbiochem and used at a final concentration of 5 μM unless otherwise stated. Recombinant human IL-6 was purchased from R&D Systems and used at a final concentration of 20 ng/mL unless otherwise stated. Recombinant TNFα was purchased from PeproTech EC Ltd and used at a final concentration of 10 ng/mL. All compounds were used at concentrations required to minimise potential off-target activity. Neutralising IL-6 antibody (ab6628) was purchased from Abcam and used at a final dilution of 1:400. Neutralising gp130 antibody (MAB228) was purchased from R&D Systems and used at a final concentration of 1 μg/mL.SalI and XmaI restriction enzymes [5’CUAACACUGGGUUAUACAA‘3 and 5’CTAACTAACACTGGGTTAT‘3. For HPV16, a single siRNA targeting the HPV16 E6 protein was purchased from Santa Cruz Biotechnology . For each experiment, 40 nM of pooled siRNA was used and cell lysates were harvested after 72 hours.Plasmids expressing HPV18 E6 sequences were amplified from the HPV18 genome and cloned into peGFP-C1 with  enzymes . The pla enzymes ; the Con enzymes  and a co enzymes . The IκBTransient transfections were performed with a DNA to Lipofectamine 2000 (ThermoFischer) ratio of 1:2.5. 48 h post transfection, cells were lysed in Leeds lysis buffer for western blot .48 hr post-transfection, cells were trypsinised and reseeded in a six well plate at 500 cells per well and left to incubate for 14–21 days. Colonies were then stained  and colonies were counted manually. Each experiment was repeated a minimum of 3 times.Cells were transfected as required. 60 mm dishes were coated with a layer of 1% agarose  in 2X DMEM  supplemented with 20% FBS. 48 hr post-transfection, cells were trypsinised and added to 0.7% agarose in 2X DMEM  supplemented with 20% FBS at 1000 cells/mL. Once set, DMEM supplemented with 10% FBS and 50 U/mL penicillin was added. The plates were then incubated for 14–21 days. Each experiment was repeated at least three times unless stated otherwise. Visible colonies were counted manually.6 cells/mL and were treated as required per experiment. Cells were then trypsinised and collected by centrifugation at 700 x g for 5 mins. Cells were then washed in cold PBS and re-centrifuged. 1x106 cells were then incubated in 100 μL Annexin V reagent  and 880 μL ddH2O) for 15 mins at room temperature in the dark. 400 μL of 1 x binding buffer was then added before analysis by flow cytometry. Samples were processed on an LSRFortessaTM cell analyzer (BD) and the PI histograms analysed on modfit software.Annexin V apoptosis assay  was performed as indicated on the product datasheet. Briefly, cells were seeded in 6 well plates at a density of 1 x 10xL . Western blots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL (Thermo/Pierce). Densitometry analysis was performed using ImageJ analysis software .Total protein was resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for phospho-STAT3 (S727) , phospho-STAT3 (Y705) ), STAT3 , phospho-NFκB p65 (S536) , NFκB p65 , phospho-AKT (T308) , phospho-AKT (S473) , AKT , IL-6 , HA , GFP , FLAG , GAPDH , PARP-1  and Bcl-pLNCX AKT vector  and MSCV-HA-18E6 were transfected into HEK293TT cells with murine retrovirus envelope and GAG/polymerase plasmids  using PEI transfection reagent as previously described . After 4Total RNA was extracted using the E.Z.N.A. Total RNA Kit I (Omega Bio- Tek) according to the manufacture’s protocol. One μg of total RNA was DNase treated following the RQ1 RNase-Free DNase protocol (Promega) and then reverse transcribed with a mixture of random primers and oligo(dT) primers using the qScriptTM cDNA SuperMix (Quanta Biosciences) according to instructions. RT- qPCR was performed using the QuantiFast SYBR Green PCR kit (Qiagen). The PCR reaction was conducted on a Corbett Rotor-Gene 6000 (Qiagen) as follows: initial activation step for 10 min at 95°C and a three-step cycle of denaturation (10 sec at 95°C), annealing (15 sec at 60°C) and extension (20 sec at 72°C) which was repeated 40 times and concluded by melting curve analysis. The data obtained was analysed according to the ΔΔCt method using the Rotor-Gene 6000 software . SpecifiIL-6 promoter or the ConA promoter, which contains tandem repeats of a κB-response element [Cells were seeded into 12 well dishes and transfected the following day using PEI with reporter plasmids expressing firefly luciferase under the control of the  element ,90. Wher element .Cells were seeded onto coverslips and, 24 hr later, were transfected as required. 24 hr after transfection, cells were fixed with 4% paraformaldehyde for 10 min and then permeabilised with 0.1% (v/v) Triton for 15 minutes. Cells were then incubated in primary antibodies in PBS with 4% BSA overnight at 4°C. Primary antibodies were used at a concentration of 1:400. Cells were washed thoroughly in PBS and then incubated with Alex-fluor conjugated secondary antibodies 594 and Alexa 488 (1:1000) (Invitrogen) in PBS with 4% BSA for 2 hours. DAPI was used to visualise nuclei. Coverslips were mounted onto slides with Prolong Gold (Invitrogen). Quantification of nuclear localisation was quantified as described .The human IL-6 DuoSet® ELISA was purchased from R&D Systems and was used according to the manufacturer’s instructions.The activation of Rac1 was determined by pulldown assay as previously described  and follFor microarray analysis, a dataset of 27 cervical cancer cases and 23 normal cervix samples was utilised. Microarray data was obtained from GEO database accession number GSE9750.Where indicated, data was analysed using a two-tailed, unpaired Student’s t-test.S1 FigA) Normal human keratinocytes (NHK) were transfected with GFP or GFP tagged HPV18 E6 and analysed for IL-6 mRNA expression by RT-qPCR. Samples were normalized against U6 mRNA levels. B) Representative western blot of NHK cells transfected with GFP or GFP tagged HPV18 E6 and analysed for the expression of IL-6 protein. Expression of GFP was confirmed with an antibody against GFP whilst the GFP E6 fusion was detected using a HPV18 E6 antibody. GAPDH served as a loading control. C) NHK cells were transfected with GFP or GFP tagged HPV18 E6. The culture medium was analysed for IL-6 protein by ELISA. Data are representative of at least three biological independent repeats. Error bars represent the mean +/- standard deviation of a minimum of three biological repeats. *P<0.05, (Student’s t-test).(TIFF)Click here for additional data file.S2 FigA) CaSKi cells were transfected with HPV16 E6 specific siRNA and analysed for IL-6 mRNA expression by RT-qPCR. Samples were normalized against U6 mRNA levels. B) Representative western blot of CaSKi cells transfected with a pool of two specific siRNAs against HPV16 E6 and analysed for the expression of IL-6. Knockdown of HPV16 E6 was confirmed using antibodies against HPV16 E6 and p53. GAPDH served as a loading control. C) CaSKi cells were transfected with a pool of two specific siRNAs against HPV16 E6. The culture medium was analysed for IL-6 protein by ELISA. Data are representative of at least three biological independent repeats. Error bars represent the mean +/- standard deviation of a minimum of three biological repeats. *P<0.05, ***P<0.001 (Student’s t-test).(TIFF)Click here for additional data file.S3 FigA) C33A cells were transfected with GFP, GFP tagged HPV18 E6 wildtype, HPV18 E6 ΔPDZ, HPV18 E6 F4V and HPV18 L52A. Lysates were probed with antibodies against IL-6 and GAPDH served as a loading control. Expression of the GFP E6 fusions was confirmed by anti-GFP western blot and p53 western blot validated the inability of the F4V and L52A mutants to degrade p53.(TIFF)Click here for additional data file.S4 FigA) Representative western blot of C33A cells treated with 20 ng/mL recombinant human TNF⍺ for the indicated time points. Cell lysates were analysed for phosphorylated and total p65, phosphorylated and total STAT3 and IL-6 expression. GAPDH served as a loading control. Data are representative of at least three biological independent repeats. B) C33A cells treated with 20 ng/mL recombinant human TNF⍺ for 60 mins were fixed and were analysed by immunofluorescence staining for total STAT3 (green) and total p65 (red) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm.(TIFF)Click here for additional data file.S5 FigA) Representative western blot of CaSKi cells treated with increasing doses of IKKi. Cell lysates were analysed for the expression of phosphorylated and total p65, phosphorylated and total STAT3 and IL-6 expression. GAPDH served as a loading control. B) Representative western blot of CaSKi cells transfected with mutant IκB (IκBm). Cell lysates were analysed as in A).(TIFF)Click here for additional data file.S6 FigA) Scatter dot plot of percentage nuclear STAT3 from B) Scatter dot plot of percentage nuclear STAT3 from (TIFF)Click here for additional data file.S7 FigA) Representative western blot of CaSKi cells treated with increasing doses of the AKTi. Cell lysates were analysed for the levels of phosphorylated and total AKT and STAT3 and IL-6 protein. GAPDH served as a loading control. B) Representative western blot of CaSKi cells transfected with dominant negative AKT (AKT-DN). Cell lysates were analysed as in A).(TIFF)Click here for additional data file.S8 FigA) Scatter dot plot of percentage nuclear STAT3 from B) Scatter dot plot of percentage nuclear STAT3 from (TIFF)Click here for additional data file.S9 FigA) Representative western blot of CaSKi cells treated with increasing doses of NSC. Cell lysates were analysed for the levels of phosphorylated and total STAT3 and IL-6 protein expression. GAPDH served as a loading control. B) Representative western blot of CaSKi cells transfected with Rac1 N17 (Rac1-DN). Cell lysates were analysed as in A).(TIFF)Click here for additional data file.S10 FigA) Scatter dot plot of percentage nuclear STAT3 from B) Scatter dot plot of percentage nuclear STAT3 from (TIFF)Click here for additional data file.S11 FigA) Colony formation assay (anchorage dependent growth) of HeLa and CaSKi cells after treatment with IKKi or transfection with IkBm. B) Colony formation assay (anchorage dependent growth) of HeLa and CaSKi cells after treatment with AKTi or transfection with AKT-DN. C) Colony formation assay (anchorage dependent growth) of HeLa and CaSKi cells after treatment with NSC or transfection with Rac1-DN. D) Soft agar assay (anchorage independent growth) of HeLa cells after treatment with the indicated inhibitors or transfection with the indicated constructs. E) Soft agar assay of CaSKi cells after treatment with the indicated inhibitors or transfection with the indicated constructs. Error bars represent the mean +/- standard deviation from the appropriate control (DMSO or pcDNA) of a minimum of three biological repeats. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).(TIFF)Click here for additional data file.S12 FigA) HeLa and CaSKi cells were transfected with a pool of four specific siRNAs against STAT3 and analysed for IL-6 mRNA expression by RT-qPCR. Samples were normalized against U6 mRNA levels. B) Representative western blot of HeLa and CaSKi cells transfected with a pool of four specific siRNAs against STAT3. Cell lysates were analysed for total STAT3 and IL-6. GAPDH served as a loading control. C) HeLa and CaSKi cells were transfected with a pool of four specific siRNAs against STAT3. The culture medium was then analysed for IL-6 expression by ELISA. Data are representative of at least three biological independent repeats. Error bars represent the mean +/- standard deviation. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).(TIFF)Click here for additional data file.


The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies. The polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation.IMPORTANCE The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies. Virus transmission from its natural host species to a different species reflects mechanisms of molecular restriction, evolution, and adaptation. Birds harbor most of the influenza A viruses, which are typically replication-restricted in human hosts because of receptor incompatibility and limited viral polymerase activity in human cells. Although many host factors have been reported to be involved in viral replication –8, the k–Influenza A viral ribonucleoprotein (vRNP), the viral minimum replicon, comprises the viral genome, the heterotrimeric RNA polymerase PB1, PB2, PA, and the nucleoprotein (NP), and carries out viral RNA transcription and replication in infected cells. Avian influenza A polymerases have very limited activity in mammalian cells, indicating an unknown host-specific restriction mechanism that directly affects viral RNA replication. The adaptation of avian viruses to mammals, such as occurred with the H5N1 and H7N9 avian viruses, occurs along with substitutions on the viral polymerase, mainly on the PB2 subunit (E627K and other signatures), which enhance viral replication 928. Many 29–The acidic (leucine-rich) nuclear phosphoprotein 32-kDa (ANP32) family comprises several members, including ANP32A, ANP32B, and ANP32E, which have various functions in the regulation of gene expression, intracellular transportation, and cell death . Althoug(This article was submitted to an online preprint archive .)SC09) (AH13)   on theseSC09) . HoweverSC09) . We did SC09) , 41, 42.SC09) . The tarSC09) , althougSC09) . We then) (AH13) , and H1N) (AH13)  and F. W) (AH13) . These rSC09, H7N9AH13, and WSN viruses , which is distinct from a previous report that used a gene knockdown method and resulted in an approximately 3- to 5-fold reduction in viral polymerase activity . We foun viruses  to C. Ext manner . We conft manner , suggestt manner . Reconstt manner , indicatInfluenza A virus replication starts from the transcription and replication of the negative single-stranded viral RNA (vRNA) by the vRNP complex. vRNA is transcribed into positive cRNA (cRNA) and mRNA; the cRNA is then used as a template to amplify into new vRNA , 48. We SC09 and tested the virus production in the supernatant of the transfected cells using an antigen capture enzyme-linked immunosorbent assay (ELISA) (We next used an eight-plasmid reverse genetics system in H1N1 (ELISA) . The res (ELISA) . The NP  (ELISA) . Taken tSC09, human isolate H7N9AH13, H3N2 canine influenza virus A/canine/Guangdong/1/2011(H3N2GD11), H3N8 equine influenza virus A/equine/Xinjiang/1/2007(H3N8XJ07), A/equine/Jilin/1/1989(H3N8JL89), or H9N2 chicken virus A/chicken/Zhejiang/B2013/2012(H9N2ZJ12) in DKO cells (ZJ12 or the H3N8JL89 (an avian-like virus). This result is consistent with previous reports that avian ANP32A can promote avian viral polymerase activity in human cells  dramatically changed viral fitness to mammalian ANP32 proteins  in an extra 33-amino-acid insert region which connects to host SUMOylation to specifically promote avian viral polymerase activity . HoweverTogether, our data give new insights into the functions of ANP32A and ANP32B. The 129-130 substitution could be used as a novel target to modify the genomes of animals to develop influenza A-resistant transgenic chickens or other animals, which will benefit the husbandry industry, as well as animal and human health. Further investigation into the molecular mechanisms that determine how ANP32 proteins work with the viral polymerase complex, the structure of the ANP32 and vRNP complex, and the fitness of different virus subtypes, for example, would contribute to our understanding of viral pathogenesis and host defense.2. Certain reagents were kindly provided by the indicated individuals: polymerase plasmids of H1N1 human influenza A virus A/Sichuan/01/2009 (H1N1SC09) and H7N9 A/Anhui/01/2013 (H7N9AH13) were provided by Hualan Chen; H1N1 human influenza virus A/WSN/1933 (WSN) was provided by Yoshihiro Kawoaka; H3N2 canine influenza virus A/canine/Guangdong/1/2011 (H3N2GD12) was provided by Shoujun Li from China Southern Agriculture University; and H9N2 avian influenza virus A/chicken/Zhejiang/B2013/2012 (H9N2ZJ12) was provided by Zejun Li from Shanghai Veterinary Research Institute of CAAS. H3N8 equine influenza viruses A/equine/Jilin/1/1989 (H3N8JL89) and A/equine/Xinjiang/1/2007 (H3N8XJ07) were preserved in our lab. The reverse genetics system based on the pBD vector for the H1N1SC09 virus was established in Hualan Chen’s lab. The pCAGGS plasmids containing full-length ANP32A isoforms of several species were generated by gene synthesis  according to the sequences deposited in GenBank, including chicken ANP32A , human ANP32A , zebra finch ANP32A , duck ANP32A , turkey ANP32A , pig ANP32A , mouse ANP32A , equine ANP32A , chicken ANP32B , and human ANP32B . Site-directed mutants of these sequences were generated using overlapping PCR and identified by DNA sequencing. Mutants of pcAGGS-huANP32B-Δ216/190/165 and pcDNA3.1-PA-V5 were constructed according to the online In-Fusion HD cloning kit user manual (http://www.clontech.com/CN/Products/Cloning_and_Competent_Cells/Cloning_Kits/xxclt_searchResults.jsp). Briefly, the fragments of the pcAGGS/pcDNA3.1 vector and each target gene were amplified with a 15-bp homologous arm and then fused using In-Fusion HD enzyme . To create the pcAGGS-huANP32B-Δ216/190/165 plasmids, pcAGGS-huANP32B was used as a template to amplify the pcAGGS vector. This sequence was then fused with different truncated huANP32B fragments (huANP32B-Δ216/190/165). To obtain pcDNA3.1-PA-V5 plasmid, pBD-H1N1SC09-PA was used as the template to amplify the PA-V5 sequence and then fused with pcDNA3.1 vector.Human embryonic kidney  and Madin-Darby canine kidney  cells were maintained in Dulbecco modified Eagle medium  with 10% fetal bovine serum , and 1% penicillin and streptomycin (Gibco) and kept at 37°C in 5% COAF081496.1), CLTC (NM_004859.3), CYC1 (CR541674.1), NIBP (BC006206.2), ZC3H15 (NM_018471.2), C14orf173 (DQ395340.1), CTNNB1 (NM_001904.3), ANP32A (NM_006305.3), ANP32B (NM_006401.2), SUPT5H (U56402.1), HTATSF1 (NM_014500), and DDX17  (http://crispr.mit.edu/) was used for gRNAs design and off-target prediction  , the gRNediction . DNA fraediction . BrieflyHigh-efficiency guide sequences for ANP32A and ANP32B that bind upstream and downstream with close proximity to the target (129/130 ND) were chosen. The gRNA expression plasmids were constructed as described above. An 80-nucleotide oligonucleotide with the desired mutations at the target site was used as the donor DNA. 293T cells were transfected with 1 μg of pMJ920 plasmids, 1 μg of gRNA expression plasmids, and 50 pmol of donor DNA. After 48 h, GFP-positive cells were isolated by FACS, and site-directed mutagenesis clones were identified after screening by sequencing and Western blotting with anti-PHAP1 antibody (ab51013) and anti-PHAPI2/APRIL antibody EPR14588 (ab200836). Cell lines with double gene mutations were generated by second-round transfection and selection.Renilla luciferase expression plasmids , using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Cells were incubated at 37°C. The cells were lysed with 100 μl of passive lysis buffer (Promega) at 24 h after transfection, and the firefly and Renilla luciferase activities were measured using a dual-luciferase kit (Promega) with a Centro XS LB 960 luminometer (Berthold Technologies). The function of ANP32 was examined using a polymerase assay by cotransfection of DKO cells with different ANP32 proteins for 24 h. All of the experiments were performed independently at least three times. Results represent means ± the standard errors of the mean (SEM) of the replicates within one representative experiment. The expression levels of polymerase proteins on different cell lines were detected by Western blotting, using specific mouse monoclonal antibodies for the NP and PB1 proteins and anti-V5 tag antibody (ab27671) for the PA-V5 protein.A minigenome reporter, which contains the firefly luciferase gene flanked by the noncoding regions of the influenza hemagglutinin gene segment with a human polymerase I promoter and a mouse terminator sequence  was tranTotal RNA from 293T cells was extracted using an RNeasy minikit (Qiagen) according to the manufacturer’s instructions. For the synthesis of first-strand cDNA derived from firefly luciferase RNAs driven by influenza polymerase, equal concentrations of RNA were subjected to cDNA synthesis using a reverse transcription (RT) kit . Primers used in the RT reaction were as follows: 5′-CATTTCGCAGCCTACCGTGGTGTT-3′ for the firefly luciferase vRNA, 5′-AGTAGAAACAAGGGTG-3′ for the firefly luciferase cRNA, and oligo-dT20 for the firefly luciferase mRNA . The cDN2SO4, and the optical density at 450 nm was measured using a VersaMax microplate reader . Dilution buffer was taken as a blank control, and the purified IAV-NP protein was double diluted eight times in dilution buffer to obtain the standard curve. The virus production was calculated using the standard curve.The ELISA has been previously described . BrieflyHuman influenza virus WSN was rescued from a 12-plasmid rescue system . Brieflyg and 4°C for 10 min. After centrifugation, the crude lysates were incubated with anti-FLAG M2 magnetic beads  or anti-NP magnetic beads (MCE protein A/G magnetic beads and NP antibody from our lab) at 4°C for 2 h. After incubation, the resins were collected using a magnetic separator and washed three times with PBS. The resin-bound materials were eluted using a 3× Flag peptide or by boiling in the SDS-PAGE loading buffer, subjected to SDS-PAGE, and then transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk powder in Tris-buffered saline (TBS) for 2 h. Incubation with the first anti-mouse antibody  was performed for 2 h at room temperature, followed by three washes with TBST. The secondary antibody  was then applied, and the samples were incubated at room temperature for 1 h. Subsequently, the membranes were washed three times for 10 min with TBST. Signals were detected using an Odyssey imaging system .For immunoprecipitation and Western blotting, transfected cells were lysed using an ice-cold lysis buffer  and centrifuged at 13,000 × P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).Statistical analysis was performed in GraphPad Prism . Statistical differences between groups were assessed by one-way analysis of variance (ANOVA), followed by a Dunnett’s post test. All the experiments were performed independently at least three times. Error bars represent standard deviations (SD) or standard errors of the mean (SEM) in each group, as indicated in the figure legends (NS, not significant;