In humans, both SAMD9 and SAMD9L are poxvirus restriction factors, although the latter requires interferon induction in many cell types. While knockout of SAMD9 with Crispr-Cas9 was sufficient for abolishing the restriction for vK1L-C7L- in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. Both paralogs are antagonized by VACV K1, C7 and C7 homologs from diverse mammalian poxviruses, but mouse SAMD9L is resistant to the C7 homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in SAMD9/SAMD9L genes serves as a barrier for cross-species poxvirus transmission.Host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. Human SAMD9, a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (VACV) K1 and C7. A paralog of SAMD9, SAMD9L, is also encoded by some mammals, while only one of two paralogs is retained by others. Here, we show that SAMD9L functions similarly to SAMD9 as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. In mice, which naturally lack SAMD9, overcoming SAMD9L restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. While a VACV deleted of both K1 and C7 (vK1L Zoonotic viral infections represent a major threat to public health. For many viruses, host species-specific difference in viral entry receptors presents a major hurdle for cross-species transmission. Poxviruses, however, can enter nearly any animal cell. Why many poxviruses show strict host species specificity and what it would take for them to jump to new hosts are less clear. Here, we present data suggesting that SAMD9 and its paralog, SAMD9L, constitute a critical host barrier against poxvirus infection and pathogenesis. We also discovered some host species-specific difference in SAMD9/SAMD9L and some poxvirus-specific difference in antagonizing SAMD9/SAMD9L, suggesting that these differences serve as a barrier for cross-species poxvirus infection. The knowledge is fundamental for understanding the determinants of poxvirus host-range. Emerging and reemerging infectious diseases have continued to pose a major threat to public health. In particular, zoonotic viral infections have caused such lethal human diseases as SARS, avian influenza, human monkeypox, and Ebola . For manPoxvirus host range at the cellular level is governed by a group of poxvirus genes referred to as the host range genes , 6. The SAMD9 was initially identified as a tumor suppressor whose loss-of-function mutations in humans cause normophosphatemic familial tumoral calcinosis (NFTC) , 16. HumAlthough SAMD9 has been established as a critical poxvirus restriction factor in human cell lines, whether this restriction is important at the organismal level and in other mammalian species is unknown. Moreover, whether SAMD9L plays a role in host defense is unknown. We studied the function of SAMD9L from a host that lacks SAMD9 (mice) as well as a host that maintains both paralogs (humans). We found that SAMD9L functions nearly identically to SAMD9 as a host restriction factor and that overcoming SAMD9/SAMD9L (SAMD9/L) restriction is essential for poxvirus replication and pathogenesis. We also discovered some host species-specific difference in SAMD9/L and some poxvirus species-specific difference in antagonizing SAMD9/L, suggesting that these differences contribute to the barrier for cross-species poxvirus infection.-C7L-) as the model poxvirus to study poxvirus restriction factors in different host species. Mouse is one of the mammalian species that have lost SAMD9 gene, and a previous study suggested that mouse SAMD9L was not a functional paralog of human SAMD9 in terms of the tumor suppressor function [-C7L- [-C7L-, we used the CRISPR-Cas9 technology to knock out mSAMD9L from 3T3 cells. To control for potential off-target effect, we performed two independent knockouts with different guide sequences. To confirm the gene knockout, cell clones were isolated, and the mSAMD9L genotype of representative clones from the two knockouts (named ΔmSAMD9L#1 and ΔmSAMD9L#2) was determined by sequencing. All mSAMD9L alleles of the cell clones were found to contain indels, resulting predominantly in frameshift  in permissive human cells [The importance of human SAMD9 as a poxvirus restriction factor became evident only when viral inhibitors of SAMD9 were removed from poxviruses, so we used a VACV mutant deleted of both K1L and C7L  of vK1L-C7L- did not cause any disease symptom or sustained body weight loss, similar to the mock infection . Again, neither group of mice developed any disease symptoms or had sustained body weight loss . Since knockdown of human SAMD9 (hSAMD9) was sufficient for abolishing the host restriction for vK1L49 cells , 26, hSA-C7L-, as reported previously [-C7L-, reducing the viral yield by more than 100-fold compared to that in untreated cells (-C7L- after IFN-β treatment (-C7L- was similar to that of the wild type (WT) VACV WR strain , no hSAMD9 was detected by Western blot even after the cells were treated with IFN-β . ΔhSAMD9eviously . Howevered cells . To tested cells . In contreatment , and theR strain , indicatK1L-C7L- .-C7L-. These include normal human foreskin fibroblasts (HFFs) and cancer cells derived from skin (A431), breast (HS578T and MDA-MB-231), cervix (HT-3), prostate (PC-3) and ovary (SKOV3). We transduced these cells with a lentivirus expressing a gRNA targeting either hSAMD9 or hSAMD9L and pooled the stably transduced cells. The SAMD9 and SAMD9L protein level in the pooled cells was reduced but not eliminated as in the clonally selected HeLa knockout cells  grew in IFN-treated ΔhSAMD9 cells . 3T3 cells with CRISPR knockout of SAMD9L as well as SAMD9L-/- MEFs were permissive for the replication of vK1L-C7L-. Furthermore, vK1L-C7L- caused lethal intranasal infection only in SAMD9L-/- mice but was completely avirulent in SAMD9L+/+ and SAMD9L+/- mice, demonstrating the potency of SAMD9L as a restriction factor at the organismal level.Mouse is one of the mammalian species that have lost SAMD9 but maintained SAMD9L. Mouse SAMD9L is 53% and 71% identical to human SAMD9 and SAMD9L at amino acid level, respectively. Through gene knockout with both the conventional technique as well as the CRISPR-Cas9 technique, we found that mouse SAMD9L was essential for restricting the replication of a model poxvirus, a vaccinia virus mutant deleted of both K1L and C7L  mutations of SAMD9 or SAMD9L cause multisystem developmental disorder. GoF SAMD9 mutations cause MIRAGE  disorder , while Gelection .Corresponding to the conservation of SAMD9/L in mammals, the distribution of SAMD9/SAMD9L inhibitors in mammalian poxviruses are also very broad. Nearly all mammalian poxviruses encode a C7 homolog that provides a host-range function similar to VACV C7 , 24, 31,The animal studies reported in this paper were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio (Protocol no. 14040x), and adhere to the guidelines and recommendations from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Office of Laboratory Animal Welfare. The Institution has an Animal Welfare Assurance on file with the NIH Office of Laboratory Animal Welfare (assurance number A3345-01). The care and use of animals is in accordance with the NRC Publication, as revised in 2011, “Guide for the Care and Use of Laboratory Animals,” and other applicable Federal regulations.−/− and SAMD9L+/+ mice according to standard protocols. Human foreskin fibroblasts (HFFs), described in [VERO (ATCC CCL-81), HeLa 229 (ATCC CCL-2.1), NIH/3T3 (ATCC CRL-1658), A431 (ATCC CRL-1555), HS578T (ATCC HTB-126), HT-3(ATCC HTB-32), MDA-MB-231 (ATCC HTB-26), SKOV3 (ATCC HTB-77), and PC-3 (ATCC CRL-1435) were originally from ATCC. HEK 293FT was from Thermo Fisher Scientific (cat. no. R70007). Mouse embryonic fibroblasts (MEFs) were generated from embryos of SAMD9Lribed in , were ki-C7L-) and a panel of vK1L-C7L--derived recombinant viruses expressing VACV-K1 (vVACV-K1L) or a C7 homolog from different poxviruses  were described before [WT VACV WR strain, K1L and C7L deletion VACV  according to the published protocol . In brie library . They weThe lentiCRISPRv2-derived plasmid was either transfected directly to the target cells or used in making lentiviruses for transduction of the target cells. In the latter case, the lentiviral plasmid and the packaging plasmids pMD2.G and psPAX2  were transfected into HEK 293FT cells with lipofectamin 3000 (Thermo Fisher Scientific). 60 hr post transfection, culture supernatants were collected, clarified by centrifugation, passed through a 0.45 μm filter, and used for transduction. For lentiviral transduction, the lentiviruses and the target cells in medium containing 10 μg/ml polybrene were centrifuged at 1,500 rpm for 2 hr. 24 hr after either transfection or transduction, the cells were subjected to puromycin selection for 2 (transfection method) or 7 (transduction method) days. The puromycin concentrations are 3 μg/ml for HeLa, 10 μg/ml for 3T3, and 2 μg/ml for all other cells.For each gene knockout in 3T3 and HeLa cells, two separate knockouts with different guides were performed. Clones of the knockout cells were isolated and validated by Western blotting for hSAMD9 or hSAMD9L or genotyping for mSAMD9L. For genotyping, the genomic DNA of the clones was extracted using the QuickExtract DNA extraction kit (Epicentre). ~500 bp of DNA flanking the target site was PCR-amplified with the primer pair (5’-GGCCACTCAATCTCATTGACCCAAT-3’ and 5’-TGCCCAGGATATTCTTAGAGCTAGC-3’) and cloned to pGEM vector (Promega). The sequence of 10–20 pGEM clones was determined by Sanger sequencing.For knockout of hSAMD9 or hSAMD9L in additional human cell lines and HFFs, only guide #1 described above was used for the knockout. The stably transduced target cells were pooled without clonal selection, and the knockout was validated by Western blot.SAMD9L+/- mice [Mice with different SAMD9L genotypes used in the infection experiment were generated from breeding pairs of +/- mice . At arou+/- mice . The vir+/- mice . Individ+/- mice . All moumSAMD9L ORF was PCR-amplified with the primer pair (5’-AGTGGACAAGTAACTCAACCAAAATTG-3’ and 5’-GATCACTTTTATGCCATATGCC-3’) from cDNA synthesized from 3T3 cellular mRNAs. A 3XFlag tag sequence and a HA tag sequence were then appended to the 5’ and 3’ end of the mSAMD9L ORF by recombinant PCR, and the final PCR product was inserted between KpnI and SacII sites of the pcDNA3.1/V5-His-topo vector (Thermo Fisher Scientific). The mSAMD9L ORF was completely sequenced and found to have one amino acid difference  compared to the mSAMD9L reference sequence in GenBank (NP_034286.2). The difference was not corrected, as SAMD9L from most rodents also has Val at this position.Cells in 12-well plates were incubated with 1 PFU per cell of different viruses for 2 h at room temperature. Following adsorption, the cells were washed twice with phosphate-buffered saline (PBS). One set of the cells was harvested immediately as the 0 hr post infection sample, while the other set were moved to 37°C incubator to initiate viral entry and harvested at 24 hr post infection. The viral titers in the cell lysates were determined by plaque assays on VERO cells. For testing the effect of IFN, human or murine cells were treated with 200 U/ml of human or murine IFN-β  for 24 hr, before the cells were infected with VACV as described above. For assessing viral late protein expression, Western blot with a mAb against the VACV late protein WR148 (clone HE7)  was perfFor immunoprecipitation of hSAMD9 or hSAMD9L, HeLa cells or IFN-treated ΔhSAMD9 HeLa cells were infected with different VACV viruses. For immunoprecipitation of mSAMD9L, 293FT cells were transfected with the mSAMD9L expression plasmid and then infected with different VACVs at 48 hr post transfection. After 8 hr of infection, the cells were lysed on ice with a lysis buffer (0.1% (w/v) NP-40, 50 mM Tris, pH 7.4, 150 mM NaCl) supplemented with protease inhibitor cocktail tablets . The cleared cell lysates were mixed with 50 μl of 50% (vol/vol) V5-agarose beads (Sigma-Aldrich) for 30 min at 4°C. After washing with lysis buffer, the beads were resuspended in SDS sample buffer, the eluted proteins were resolved by SDS-PAGE and detected with Western blot as described previously . The detS1 Fig(A). Genotyping ΔmSAMD9L 3T3 cells. The mSAMD9L knockout cell lines (ΔmSAMD9L) were constructed by transient transfection of 3T3 cells with a plasmid encoding Cas9 and a gRNA targeting mSAMD9L (guide#1 or guide#2). The targeted SAMD9L sequence is underlined with the PAM sequence in bold italics and the encoded amino acid sequence shown above the DNA sequence. Shown below the target are the genomic sequences from representative cell clones (ΔmSAMD9L#1 and ΔmSAMD9L#2). Gray and crossed-out sequence indicates deletion. ^ indicates insertion. The number after the + and − denotes the number of indels, and the number before the “x” denotes the number of times the sequence was detected from a total of 10–20 cloned PCR products. (B). The restriction of K1L and C7L deletion vaccinia virus (vK1L-C7L-) in 3T3 cells was abolished by knocking out mSAMD9L with CRISPR-Cas9. A validated mSAMD9L knockout cell clone (ΔmSAMD9L #2) and the parental 3T3 cells were infected with vK1L-C7L- at an MOI of 1 PFU/cell. Viral growth was determined by measuring viral titers at 0 and 24 hour-post-infection (hpi).(PDF)Click here for additional data file.S2 Fig(A). vK1L-C7L- infection induced anti-VACV antibody response in mice. Mice described in (B). Mice described in (PDF)Click here for additional data file.S3 FigRelated to (PDF)Click here for additional data file.S4 Fig(A). Knockdown of hSAMD9 but not hSAMD9L in human foreskin fibroblasts (HFFs) rescued viral late protein expression by vK1L-C7L-. HFFs were transduced with a lentivirus expressing a gRNA targeting either hSAMD9 or hSAMD9L, and stably transduced cells were pooled. The knockdown of hSAMD9 or hSAMD9L was confirmed by Western blot (left). A set of the cells were infected with vK1L-C7L- and the level of a representative VACV late protein WR148 was determined by Western blot (right). Par., parental; Δ9, hSAMD9-knockdown; Δ9L, hSAMD9L-knockdown. (B). IFN restored host restriction for vK1L-C7L- in hSAMD9-knockdown HFFs. The parental and the knockdown cells were left untreated or treated with IFN-β and infected with vK1L-C7L-. Viral growth was determined by measuring viral titers at 0 and 24 hpi.(PDF)Click here for additional data file.S5 Fig(A). Either hSAMD9 or hSAMD9L was knocked down from various human cells with CRISPR-Cas9 as described in (B). Knockdown of hSAMD9 but not hSAMD9L abolished the restriction for vK1L-C7L- in human cells from diverse tissues. The parental and the knockdown cells were infected with vK1L-C7L- at an MOI of 1 PFU/cell. (C). IFN restored host restriction for vK1L-C7L- in hSAMD9-knockdown (ΔhSAMD9) cells. Various ΔhSAMD9 cells were left untreated or treated with IFN-β and infected with vK1L-C7L-. Viral growth was determined by measuring viral titers at 0 and 24 hpi.(PDF)Click here for additional data file.S6 Fig-C7L--derived virus that expressed MYXV-M63 (negative control) or SPPV-063 (WT or mutated). The C7 homolog was precipitated with an anti-V5 antibody, and the co-precipitated hSAMD9 or hSAMD9L was detected by Western blot.IFN-treated ΔSAMD9 HeLa cells (A) or untreated parental HeLa cells (B) were infected with vK1L(PDF)Click here for additional data file.


The season was characterized by dominant circulation of A(H1N1)pdm09 viruses, accounting for 66% of detected influenza viruses, followed by B/Victoria-lineage viruses (24%) and A(H3N2) viruses (10%). All sequenced influenza A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses belonged to the 6B.1, 3C.2a and 1A genetic groups, respectively. Amino acid analysis of 57 A(H1N1)pdm09 isolates revealed the presence of 16 changes in hemagglutinin (HA) compared to the vaccine virus, five of which occurred in four antigenic sites, together with 16 changes in neuraminidase (NA) and a number of substitutions in proteins MP, NP, NS and PB2. Despite the many amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically closely related to A/California/7/2009 vaccine virus. Bulgarian A(H3N2) strains (subclade 3C.2a) showed changes at 11 HA positions four of which were located in antigenic sites A and B, together with 6 positions in NA, compared to the subclade 3C.3a vaccine virus. They contained unique HA1 substitutions N171K, S312R and HA2 substitutions I77V and G155E compared to Bulgarian 3C.2a viruses of the previous season. All 20 B/Victoria-lineage viruses sequenced harboured two substitutions in the antigenic 120-loop region of HA, and 5 changes in NA, compared to the B/Brisbane/60/2008 vaccine virus. The results of this study reaffirm the continuous genetic variability of circulating seasonal influenza viruses and the need for continued systematic antigenic and molecular surveillance.Influenza virological surveillance is an essential tool for early detection of novel genetic variants of epidemiologic and clinical significance. The  •6B.1, 3C.2a and 1A were the most prevalent genetic subclades of A(H1N1)pdm09, A(H3N2) and B/Victoria viruses, respectively.•N-glycosylation motifs in HA and NA between epidemic and vaccine viruses were found.Differences in antigenic sites, RBS and •Amino acid substitutions in internal proteins M, NP, NS1 and PB2 were identified.•All influenza A viruses were resistant to M2 blockers but both A/B viruses were susceptible to oseltamivir and zanamivir. Each year 600 million influenza cases occur worldwide, with 3 million having severe disease and 250,000–500,000 resulting in death . PeriodiInfluenza vaccines were developed to reduce the substantial influenza-associated morbidity and mortality but their effectiveness declines over time due to emerging genetic and associated antigenic differences between vaccine and circulating viruses. Influenza viruses are one of the most variable and rapidly evolving human viruses because of their high mutation rate, rapid replication, segmented genome (which facilitates the reassortment of genes between different influenza viruses) and zoonotic events for type A viruses. Evolution of influenza viruses proceeds by continuous replacement of genetic groups with new ones leading to increases in the antigenic distances from the current vaccine viruses.H3 (A-E) and in type B viruses  located on the globular head of the HA1 subunit are targets of neutralizing antibodies , A/tibodies . Amino aimmunity .aim of the present study was to analyse influenza virus circulation in Bulgaria during the 2015/2016 season and determine the genetic and antigenic characteristics of the detected viruses related to amino acid changes at antigenic, N-glycosylation and functionally significant sites of HA and NA.The World Health Organization (WHO) encourages National Influenza Centers (NICs) to conduct ongoing influenza virologic surveillance, to monitor spread of viruses and their continuous evolution to inform twice yearly recommendations on vaccine composition and assessing other risks associated with circulating influenza viruses. 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 reduced sensitivity to antivirals. Such combined genetic, antigenic and phenotypic analyses provide improvements in the process of vaccine virus selection and inform patient treatment regimens. The 22.1From October 2015 to May 2016, patients, who were ambulatory treated or hospitalized either for influenza like illness (ILI) or acute respiratory illness (ARI), were enrolled in different regions of the country. Combined nasal and pharyngeal specimens from the enrolled patients were collected with the help of commercial polyester collection swabs . Swabs were stored at 4 °C for up to 72 h before shipment to the NIC. Specimens were processed immediately or stored at − 80 °C before testing.2.2t value < 38 was regarded as positive.Virus RNAs were extracted automatically from the respiratory specimens using a commercial ExiPrep Dx Viral DNA/RNA kit  in accordance with the manufacturer's instructions. Detection and typing/subtyping of influenza viruses were carried out by a Real Time RT-PCR method with the use of a kit — SuperScript III Platinum ® One-Step qRT-PCR System . All samples were first tested for the presence of influenza A and B viruses. Those that were positive for influenza A were subsequently screened for A(H1N1)pdm09 and A(H3N2). The genetic lineage of detected influenza B viruses was also determined by Real Time RT-PCR. Primers, probes and positive controls were provided by WHO-CC, Atlanta. Amplification was performed with a Chromo 4 thermal cycler (Bio-Rad) in accordance with the protocol of WHO-CC, Atlanta . A C2.3t values < 28 were inoculated onto Madin Darby canine kidney (MDCK) and MDCK-SIAT1  assay, in accordance with the WHO Manual, using vaccine viruses/antigens and their corresponding antisera provided by the WHO-CCs in London and Atlanta  with sequences of all but three viruses being derived from virus isolates that were characterized antigenically. For phylogenetic analyses all sequences, including those of reference viruses whose genetic group identities were known and viruses representing different countries of Europe during the 2015/2016 season, were retrieved from GISAID. Phylogenies for HA and NA genes were constructed using the maximum likelihood method within Molecular Evolutionary Genetics Analysis software . Best nucleotide substitution models were used: the Hasegawa-Kishino-Yano model with a gamma distribution (HKY + G) for HA; and the Tamura 3-parameter model with gamma distribution (T92 + G) for NA. Reliability of the tree topology was assessed by bootstrap analysis with 1000 replications. HA amino acid numbering was applied after removing the signal peptide. Amino acid identity was calculated using flusurver (http://flusurver.bii.a-star.edu.sg).Full-genome or HA and NA gene sequences of influenza viruses detected in Bulgaria during the 2015/2016 season were determined at WHO-CC, London. Full-genome sequencing was carried out at WHO-CC, Atlanta. Sequences have been deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database (2.5http://www.cbs.dtu.dk/services/ NetNGlyc) to identify sequence motifs N–X–S/T (sequon), where X can be any amino acid except proline.Putative N-glycosylation motifs in the HA and NA were predicted using the NetNGlyc 1.0 web Server (2.6Screening of A(H1N1)pdm09 viruses for the presence of point mutations conferring H275Y oseltamivir resistance was carried out using a Real Time RT-PCR assay that allowed discrimination of a single nucleotide difference between oseltamivir sensitive and resistant viruses. Two TaqMan probes differing in position 823 of the NA gene were used simultaneously: the first probe contained a cytosine at position 823 and was labeled with VIC (H275), while the second probe contained thymine in the same position and was labeled with FAM (275Y). Primer/probe sequences and protocol were kindly provided by Public Health England , London. Reference influenza viruses A/Denmark/524/2009  and A/Denmark/528/2009  provided by WHO-CC, London were used as positive controls. A phenotypic analysis (MUNANA test) of influenza virus susceptibility to neuraminidase inhibitors (oseltamivir and zanamivir) was performed at WHO-CC, London.2.7p values of < 0.05 were considered statistically significant.Age and gender of patients, the clinical features of their illness and the incidence of each virus were compared using the Chi square or Fisher's exact tests for categorical variables. 3Bulgaria is a country with a total population of approximately 7.2 million people and an ARI surveillance system is used to monitor influenza. It comprises a national sentinel network of general practitioners and pediatricians working in 208 health care facilities situated in all 28 major cities — regional centers covering 5.3% of the population in the country. Primary care physicians report the weekly number of clinical cases of ARI by age group, collect respiratory specimens and send them to the National Reference Laboratory. The Laboratory is recognised as a WHO NIC. It is the only laboratory in the country that conducts research on influenza viruses and performs testing of clinical samples from severely ill patients hospitalized in different regions of the country.www.grippe.gateway.bg).The first influenza detection, an A(H1N1)pdm09 virus, occurred in week 51/2015 and the 2015/2016 influenza season was characterized as being of average duration and moderate intensity but with a lower incidence rate compared to the previous two seasons. The epidemic lasted seven weeks (from week 2 to week 8) and peaked in week 6/2016, slightly later than the 2014/2015 season, with an incidence rate of 158.74 cases per 10,000 people. As in previous years, the ILI and ARI morbidity rate was the highest in young children < 4 years of age, followed by the 5–14 years age group (3.1The study population consisted of 1127 patients demonstrating symptoms of ILI or ARI: 218 (19.3%) of these were persons attending outpatient healthcare centers; 909 (80.7%) were inpatients, of which 36 were in intensive care units (ICU). The patients' ages ranged from 25 days to 92 years old (y.o.) (average age 21.7 y.o.) and 51.6% were male. Influenza viruses were detected in 318 (28%) patient samples. Of these, 241 (75.8%) were positive for influenza type A virus and 77 (24.2%) for type B. Among the influenza A viruses, 210 (87%) were A(H1N1)pdm09 and 31 (13%) A(H3N2) viruses . All det3.2p < 0.05) among hospitalized patients. For patients infected by A(H1N1)pdm09 virus, these proportions were 12.4% (27/218) and 20% (183/909), and 4.6% (10/218) and 7.4% (67/909) for influenza type B virus infected patients, respectively. All age groups were infected by influenza viruses but the highest influenza virus-positivity (31.3%) was found in the 5–14 y.o. age group. Lower percentages of detected viruses were observed in adults aged 30–64 y.o. (29%) and in children 0–4 y.o (27.8%). The predominant A(H1N1)pdm09 viruses were most frequently detected in the 30–64 y.o. (22.7%) and 0–4 y.o. (20.7%) age groups. The proportions of detected A(H1N1)pdm09 viruses in patients 5–14 y.o. and 15–29 y.o. were similar, 13.7% and 15.3% respectively. The highest rate of influenza B detection (13.7%) was in the 5–14 y.o. age group, which showed an equal proportion of A(H1N1)pdm09 virus detection  and 53.9% were male. Among outpatients, 17.9% (39/218) were identified as positive for influenza virus infection, rising to 30.7% (279/909) (etection .Influenza viruses were detected in 31% (60/194) of the studied patients diagnosed with pneumonia and in 24% (32/128) of patients with CNS involvement . In patients with pneumonia, the detection rates for influenza A(H1N1)pdm09, A(H3N2) and type B viruses were 76.7% (46/60), 5.0% (3/60) and 18.3% (11/60), respectively; and in patients with neurologic complications — 56.3% (18/32), 6.3% (2/32) and 37.4% (12/32), respectively. Among the 36 patients treated in ICUs, A(H1N1)pdm09 and type B viruses were detected in 27.8% (10/36) and 8.3% (3/36), respectively. Of eight deaths, two each were infected A(H1N1)pdm09 or type B viruses.3.3t values < 28 were inoculated onto MDCK and MDCK-SIAT1 cell cultures and 99 of them were successfully cultured after the first or second passage. In total, 81 representative influenza isolates were sent to WHO-CC, London where they were characterized in detail. Fifty-seven Bulgarian A(H1N1)pdm09 isolates, all but two obtained from hospitalized patients, were tested by HI using post-infection ferret antisera raised against the reference viruses presented in The first five detected influenza viruses  were sent to WHO-CC, Atlanta for further characterization. In the course of the 2015/2016 season, a total of 171 Real Time RT-PCR positive clinical specimens with CThe three Bulgarian A(H3N2) viruses were unable to bind red blood cells and therefore no HI analyses were performed.Nineteen Bulgarian type B viruses were assessed by HI using post-infection ferret antisera raised against reference viruses indicated in 3.4HA and NA gene sequences were recovered from GISAID and phylogenetic trees constructed to determine the genetic relationships of Bulgarian isolates with reference viruses and viruses circulating in other countries in the same period. The 57 HA genes of Bulgarian A(H1N1)pdm09 viruses fell within a new genetic subclade, 6B.1, and clustered with the reference virus A/Slovenia/2903/2015 , as did 3.5Complete HA and NA amino acid sequences of Bulgarian influenza isolates were compared to those of vaccine viruses to identify substitutions that might impact vaccine effectiveness .3.5.1N-glycosylation motifs in HA . S162N substitution, specific to subclade 6B.1 viruses, generated a new potential N-glycosylation motif within the Sa antigenic site.HA and NA sequences of the 57 Bulgarian A(H1N1)pdm09 isolates were compared to those of the vaccine virus and representatives of particular genetic groups/subgroups. HA amino acid sequence identity of studied isolates ranged from 97% to 97.4% compared to vaccine virus, A/California/7/2009. All 57 A(H1N1)pdm09 HA sequences contained 11 amino acid changes in HA1 polypeptide and three in HA2: HA1 substitutions P83S, S203T and I321V that were fixed and present in viruses from all genetic groups; substitutions D97N and S185T in HA1 with E47K and S124N in HA2 defining group 6 (A/St. Petersburg/27/2011); HA1 substitutions K163Q, A256T and K283E with E172K in HA2 defining group 6B (A/South Africa/3626/2013) and HA1 substitutions S84N, S162N and I216T defining subclade 6B.1 (exemplified by the reference virus A/Slovenia/2903/2015). In addition, sixteen (28%) viruses carried HA1 A141T substitution and nine (16%) viruses had HA2 I91V substitution. Thirteen viruses carried additional singlе amino acid changes in HA. None of these substitutions were associated with known adaptation to propagation in MDCK culture . Among tI222, E227, H274, E277, N294, E425) residues comprising the NA catalytic site were conserved among all 57 viruses .NA sequences of the same 57 A(H1N1)pdm09 viruses differed from that of A/California/7/2009 by 15 substitutions: N248D and Y351K in viruses of all genetic groups; N44S, N200S, V241I and N369K in genetic group 6 viruses; I34V, L40I, I321V, N386K and K432E in genetic group 6B viruses; V13I, K264I, N270K and I314M in genetic group 6B.1 viruses. T48A substitution was detected in 7 (12%) Bulgarian viruses and 16 viruses harboured additional NA singlе amino acid changes. The eight functional  and 11 framework pdm09 viruses were compared to those of A/California/7/2009, reference viruses  and epidemic strains detected in Armenia, Ukraine and the Russian Federation, where severe and fatal influenza cases were reported at the beginning of the season . In comm3.5.2N-glycosylation motif), N144S (resulting in the loss of N-glycosylation motif), F159Y, K160T (resulting in the gain of a N-glycosylation motif), N171K, Q311H, S312R, I406V (corresponding to HA2-subunit position 77), G484E (G155E in HA2), D489N (D160N in HA2). Substitutions A128T and N144S are located in antigenic site A while S159Y and K160T are in antigenic site B. The substitutions N144S, F159Y, K160T, N225D and Q311H in HA1, compared to a previous vaccine virus, A/Texas/50/2012, define subclade 3C.2a; Bulgarian viruses fell into a cluster having HA proteins with HA1 N171K and S312R, and HA2 I77V and G155E substitutions. Thirteen potential N-glycosylation motifs in HA  were identified. Compared to A/Switzerland/9715293/2013, loss and gain of potential N-glycosylation motifs were observed at HA1 positions144 and 128, respectively, while the HA2 G155E substitution was within a N-glycosylation motif (NGT → NET).The similarity of Bulgarian viruses to the Northern hemisphere 2015/2016 vaccine virus A/Switzerland/9715293/2013 (subclade 3C.3a) at the HA amino acid level ranged from 97.5% to 97.6%. All three Bulgarian strains (subclade 3C.2a) showed variations at eight HA1 positions and three HA2 positions compared to the vaccine virus: HA1 — L3I, A128T (resulting in the gain of a N-glycosylation motif), S247T, T267K, P339N, I380V and P468H. Nine potential N-glycosylation motifs in NA  were identified, with two (146 and 367) being located around the enzymatic active site  in the 120-loop (positions 116–137) antigenic site; no amino acid substitutions occurred in the remaining antigenic sites — 150-loop (positions 141–150), 160-loop (positions 162–167) and 190-helix (positions 194–202) . Seven vN-glycosylation motifs were identified, at positions 56, 64, 144 and 284, all of which were conserved except in B/Bulgaria/790/2016 which carried N144D substitution.All 20 Bulgarian B/Victoria-lineage viruses carried five NA amino acid substitutions and 11 carried single, unique, substitutions. The catalytic and framework residues in NA were conserved among all 20 viruses studied . Four pu3.6In the National Laboratory “Influenza and ARD”, all 210 detected A(H1N1)pdm09 viruses were analyzed by real-time RT-PCR with respect to the H275Y oseltamivir resistance substitution — all viruses carried 275H indicative of retained susceptibility. All NA sequences generated were screened for known markers of reduced susceptibility to NA inhibitors  — none w4The antigenic and molecular characteristics of influenza viruses detected in Bulgaria during the 2015/2016 season are described here. In this surveillance period, the total number and percentage (28% vs 26%) of influenza positive cases were slightly higher compared to the previous season when A(H3N2) viruses predominated . The stuThere have been year-on-year fluctuations in the distribution and frequency of influenza types/subtypes in Bulgaria since the emergence of A(H1N1)pdm09 viruses in 2009/2010: during the 2010/2011, 2013/2014 and 2015/2016 epidemics, there was clear-cut dominance of A(H1N1)pdm09 viruses; in 2011/2012 and 2014/2015, A(H3N2) viruses prevailed strongly; in 2012/2013 unusually high activity of type B influenza was registered. No A(H1N1)pdm09 viruses were detected in 2011/2012 but they represented 18% of detected viruses in 2011/2012. Vaccine coverage in Bulgaria is very low (approximately 3%) and so is unlikely to significantly affect the circulation of various types/subtypes/lineages of influenza viruses.p < 0.05) and a higher percentage of influenza-associated cases of pneumonia (31% vs 14%) and ICU admissions (36% vs 23%) (p < 0.05) was found compared to the previous season pdm09 viruses was greatest in people of active middle age, while type B viruses were most prevalent among children aged 5–14 y.o. in agreement with other studies . As for s season . While is season .N-glycosylation motif located within the HA Sa antigenic site. Evolutionary studies of A(H1N1) viruses indicate that the number of N-glycosylation motifs in the HA has increased over time while the number in NA is relatively stable. In line with this A(H1N1) viruses from the 1918 pandemic had five potential HA1 N-glycosylation motifs and seven in NA, while subclade 6B.1 A(H1N1)pdm09 viruses have seven in HA1 and eight in NA. Despite the above mentioned changes in functionally important sites of HA and NA, no indication of antigenic drift of A(H1N1)pdm09 viruses was observed for Bulgarian isolates, and those worldwide, during the course of the 2015/2016 season, as assessed by use of post-infection ferret sera raised against the A/California/7/2009 vaccine virus in HI assays. However, recent studies have shown that genetic group 6B A(H1N1)pdm09 viruses, including those in subclades 6B.1 and 6B.2, were antigenically distinguishable from the vaccine virus by some human post-vaccination sera pdm09 virus has undergone significant genetic change, and eight genetic groups and several subgroups have been defined . Phylogeion sera .Analyses of the A(H1N1)pdm09 HA and NA were unable to explain the occurrence of many serious disease and several death cases, mostly in children, during 2015/2016 in Bulgaria. None of the studied viruses carried HA1 D222G/N/S or Q293H substitutions, reported to be found more frequently in patients with severe disease or fatal outcome earlier in the pandemic period . D222G sN-glycosylation motif at positions 158–160 within the same antigenic site could potentially alter antigenicity. For A(H3N2) viruses a steady increase in the number of HA1 N-glycosylation motifs has been typical since their appearance in humans in 1968  viruses played a lesser role in the 2015/2016 influenza season . The small number of Bulgarian viruses sequenced belonged to subclade 3C.2a as was the case for the vast majority of A(H3N2) viruses in European countries . While s in 1968 : progeni viruses . Due to In Bulgaria, all type B viruses detected in 2015/2016 were of the Victoria-lineage (clade 1A), a reversal compared to the three previous seasons when all belonged to the Yamagata-lineage. The Bulgarian B/Victoria-lineage viruses showed two amino acid substitutions in HA1, located in antigenic the 120-loop (positions 116–137), and 5 substitutions in NA compared to the vaccine strain B/Brisbane/60/2008 . It has been reported that the 120-loop appeared to be one of the most frequently altered regions, and amino acid substitutions in this region may strongly affect virus antigenicity . Our finConstant monitoring of the susceptibility of influenza viruses to antivirals is necessary to allow rapid detection of viruses with reduced sensitivity. Testing of influenza viruses circulating in Bulgaria, carried out by means of genotypic and phenotypic methods, showed that all studied type A viruses were resistant to M2 blockers but both type A and type B viruses were susceptible to oseltamivir and zanamivir. In our previous study, we detected a single A(H1N1)pdm09 virus carrying NA H275Y substitution in a child treated with oseltamivir . GloballIn summary, A(H1N1)pdm09 viruses of subclade 6B.1 predominated in Bulgaria during the 2015/2016 season. Despite many genetic changes, they remained antigenically closely related to the vaccine virus. In contrast, all detected type B viruses were members of the Victoria-lineage (clade 1A) differing from the B/Yamagata-lineage vaccine component, B/Phuket/3073/2013. This suggests that the 2015/2016 trivalent vaccines provided suboptimal protection against influenza B viruses. The results of this study confirm the genetic variability of circulating seasonal influenza viruses and demonstrate the need for continual antigenic and molecular surveillance for the purpose of early detection of novel genetic variants of epidemiological and clinical significance. Such information is important for the development of optimal strategies for prevention and control of influenza.The authors declare no conflict of interest.


Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation.Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH.We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment.Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. Gliomas make up about 80% of all malignant brain tumors. The exacBralten, L. B. et al. found that IDH1 R132H mutation in U87 cell line significantly decreased cell proliferation, accompanying changes in cell morphology and cell migration patterns.  In additWe further extend our study to explore future treatment options for IDH1 mutated tumor. Fonnet et al found that in glioblastoma tumor samples occurrence of IDH1 R132H mutation reduced this capacity to produce NADPH by 38% and furthermore mutated IDH1 consumes rather than produces NADPH.  ThereforU87MG cells were purchased from ATCC. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM)  containing 10% fetal bovine serum (FBS) supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL). These cells were cultured in accordance with the suppliers’ instructions.The IDH1-Flag and IDH1 R132H-Flag plasmids constructed into pCMV-Tag2B vector were purchased from Addgene . The transfection was done using LipofectamineTM 2000 from Invitrogen and stably transfected cells were selected using G418 and tested using anti-Flag antibody by western blotting.Control SiRNA (sc-37007), siRNA targeting IDH1 (sc-60829), siRNA transfection reagent (sc-29528) and siRNA transfection medium (sc-36868) were purchased from Santa Cruz (USA). Briefly, U87 cells were plated and transfected with either SiRNA control or siRNA targeting IDH1 using siRNA transfection reagent (sc-29528) following the protocol from the manufacture. After 2 days, cells were harvested and protein levels of IDH1 were detected using western blots.The U87MG cells and its stable cells over expression of IDH1 or IDH1 R132H mutant were plated at same cell number and cultured in 96-well flat-bottomed microtiter plates supplemented with DMEM containing 10% fetal bovine serum and kept in a humidified incubator containing 95% air and 5% CO2 at 37°C. After 24 hours, viable cell number was determined by MTT assay at 570nm.Cells were rinsed once with PBS and lysed by using sodium dodecylsulfate (SDS) sample buffer. Equal amounts of protein from each group were separated on 10% SDS-polyacrylamide gels. Proteins were transferred to Immobilon P membranes (Millipore). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline for one hour at room temperature and incubated with the appropriate antibody in TBS-T containing 5% non-fat dry milk overnight at 4°C. After washing in TBS-T the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Proteins were detected using the chemiluminescent substrate. Anti-Flag antibody , p-mTOR site of phosphorylation , mTOR , p-AKT site of phosphorylation , AKT , GAPDH , caspase-3 , cleaved caspase 3 .The cells were plated at 1.5×105 cells per well in a 12-well plate and grown overnight in incubator at 37°C with 5% CO2. After one day, a straight line scratch was made on a confluent monolayer of cells using a sterile 1 ml disposable serological pipette. Then the cells were washed with 1 ml DMEM to remove debris. Photos were taken at 0, 24, and 48 h after adding drug. The distance of migration and migration index were calculated by Image-Pro Plus 6.0 software  and the following formula: inhibition of cell migration (%) =  × 100%.4 cells/well in 96-well plates incubated for one hour at 37°C with 5% CO2 in serum-free HBSS with added Ca2+ and Mg2+.GSH and GSSG were quantified using a GSH-Glo™ Glutathione Assay Kit  according to the manufacturers’ instructions, following incubation of 1×106 per well) were incubated for one hour at 37°C with 5% CO2 in HBSS. Wash cells with cold PBS. Pellet cells in a tube by spinning at low speed for 5 minutes, and discard supernatant. NADPH was quantified using a NADP+/NADPH assay kit , according to the manufacturers’ instructions.Cells  and consumes NADPH  at the sIn conclusion, our study demonstrates the role of IDH1 R132H mutation in gliomagenesis by activating AKT-mTOR signaling pathway and its pivotal role in promoting cell migration and invasion. The AKT-mTOR signaling pathway activation by IDH1 R132H will provide new evidence on future application of other combined targeted therapy for glioma with IDH1 R132H mutations. Furthermore, we confirmed the role of IDH1 R132H mutations in affecting cellular redox status using glioma cell lines, and provided evidence to further explore the application of chemotherapy such as 5-FU in the treatment of gliomas with IDH1 R132H mutation.


Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry.N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys Posttranslational modifications (PTMs)1–cis and in trans binding occur when one multivalent reader protein (or an effector complex) binds histone modifications on the same or on different histones, respectively  . IDRs ar2+ ions are coordinated by Cys4-His-Cys3 ) form the third antiparallel β-strand when bound to the PHD domain, whereas subsequent H3 residues are folded to an α-helix , albeit with reduced affinity  protein is a multidomain histone-binding protein that contains a PHD zinc finger and a BRD domain connected via a 72-amino acid linker. In addition, BAZ2B contains several DNA-binding domains, such as an MBD (methyl-CpG–binding domain), AT hook, and DDT (DNA binding homeobox and different transcription factors). It shares 29% sequence identity with BAZ2A, also called TIP5 (transcription termination factor I-interacting protein 5), which is part of the nucleolar remodeling complex . BAZ2A bHis-Cys3 . Residue α-helix , 24. In  α-helix  and PHF2 α-helix , the BAZaffinity . The BAZaffinity . The intB), which raises the possibility of competitive interaction of the linker with the PHD/BRD. Hence, the main aim of this work was to investigate the importance of the size and composition of the BAZ2B linker for binding of the N-terminal histone H3 tail to BAZ2B. Using isothermal titration calorimetry (ITC) and NMR spectroscopy, we show that mutation or deletion of the conserved parts of the BAZ2B linker alters H3-binding affinities due to of changes in its charge. Surprisingly, removal of the positively charged residues or introduction of the negative charge via phosphomimicking mutations increases BAZ2B-binding affinity toward the H3 substrate. This points to an active role of the linker in regulating substrate binding to the active site. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the BAZ2B linker may adjust the linker net charge in vivo and thereby regulate the multivalent binding of the BAZ2B PHD and BRD to histone substrates.The charged linker between the PHD and BRD of BAZ2B is difficult to map structurally using X-ray crystallography , which iFig. 415N HSQC spectra of uniformly 15N-labeled BAZ2B PHD domain or PHD–BRD were recorded in the presence of unmodified H3(1–21) . When comparing the chemical shifts of amino acids in the spectra of the native PHD (without the peptide) and PHD bound to H3, we confirmed involvement of the residues in the 310 helix and along the β1-strand in the binding of H3 . The amplitude of changes of 1H and 15N chemical shifts were color-coded from blue (lowest) to red (highest) and mapped onto the BAZ2B PHD structure . Residues that exhibited the strongest chemical shift changes upon H3 binding to the BAZ2B PHD were Glu1943, Glu1944, Leu1945, Leu1947, Asp1950, and Gly1951 (A and B). They were already implicated in H3 binding based on the BAZ2A PHD-H3 structures and 15N HSQC spectra as well as BAZ2B PHD-H3(1–5aa) 15N HSQC spectra . spectra , 24. GluC and D, and Bsupplemental Fig. 1). The mutation sites were selected based on the proximity to H3 residues and their conservation in similar PHD fingers . The BAZ2B PHD bound H3(1–21aa) with dK = 5.78 μm (C and D).To investigate the contribution of amino acids in the PHD that exhibited chemical shifts upon H3 binding, we introduced a series of point mutations in the PHD and PHD–BRD constructs and measured their effects on H3-binding affinity by ITC (C). The D1941A mutation led to a 10-fold decrease in binding affinity, which may be due to the loss of electrostatic interactions with H3K4 and loss of H-bonding with the H3 backbone (E). Notably, residues Asp1950 and Asp1953 showed a 100- and 10-fold reduction in binding affinity, respectively (C and D). Asp1950 and Asp1953 form an acidic cavity for hydrogen bonding with the guanidine nitrogen (Nη) atoms of Arg2 (F). Asp1950 is conserved in different PHD fingers, which points to its important role in histone H3 binding (G). The results of this extensive mutational analysis of BAZ2B PHD–H3 binding suggest that negatively charged residues such as Asp1941, Asp1950, and Asp1953 are important for binding the positively charged residues (Arg2 and Lys4) in the N-terminal part of the H3 peptide .Notwithstanding the observed chemical shifts, the E1944A mutation had no effect on H3-binding affinity, whereas the E1943A mutation led to a 2-fold reduction C. The D1backbone E. Notablectively , C and D of Arg2 F. Asp195 binding G. The reA). We observed specificity of the PHD finger toward H3(1–10aa) (dK = 23.13 μm) compared with dK > 4 mm for H3(11–21aa) (A). The same is true for the PHD–BRD construct (115 μm for H3(1–10aa) compared with >2 mm for H3(11–21aa)) (A). However, both PHD and PHD–BRD bound H3(1–21aa) with 2- to 4-fold higher affinity than H3(1–10aa), which indicates that H3 makes additional contacts with the PHD beyond the first 10 residues (A). We did not observe the same effect when comparing BRD binding to H3K14ac(1–21aa) versus H3K14ac(11–21aa) (A), as BRD binding relies on the K14ac-X-X-Arg17 motif  A. The sa1–21aa)) A. Howeveresidues A. We did11–21aa) A, as BRDaa) Kd = .13 μm co17 motif .A). The affinity of BAZ2B PHD-BRD toward H3(1–21aa)K14ac was 3.6-fold reduced compared with BAZ2B BRD and 2.4-fold reduced compared with H3(11–21aa)K14ac. These results indicate that the PHD linker domain negatively modulates the binding of the BRD to H3K14ac, suggesting that the two domains act in a combined manner to bind H3K14ac.We observed a significant difference in binding affinity when comparing affinity constants for BAZ2B BRD and PHD–BRD toward H3(1–21aa)K14ac or BAZ2B PHD–BRD toward H3(1–21aa)K14ac and H3(11–21aa)K14ac A. The afB). Addition of methyl groups at Lys4 progressively reduced the binding affinity, in agreement with the published data using BAZ2B PHD (A and B). Given that binding of H3K4 to the PHD reduces the overall binding affinity of PHD–BRD to H3K14ac (A), whereas lack of binding of H3K4me3 to the PHD increases it (B), we conclude that the PHD and BRD bind H3K4K14ac in a multivalent manner in cis.To investigate this further, we compared the binding affinity of BAZ2B PHD–BRD to H3K4(me0–3)K14(ac) peptides B. AdditiAZ2B PHD . InteresAZ2B PHD , A and B H3K14ac A, whereaeases it B, we con6 in the H3(1–21aa) and H3(1–21aa)K14ac peptides with a cysteine and chemically labeled it with the nitroxide spin label MTSL, which results in greatly enhanced NMR relaxation for nearby nuclei . The intensity ratios between the oxidized (paramagnetic MTSL-tagged) and reduced (diamagnetic) BAZ2B-H3(1–21aa)K14(ac) complexes are displayed in C and D, and supplemental Fig. 2. The measurements showed loss of peak intensities near the binding site in the PHD (as expected) as well as losses in the linker close to the BRD and within the BRD itself. The losses were more pronounced for H3, which is not bound by the BRD, suggesting that, in the absence of tandem binding, the BRD still forms transient encounters with the PHD (C and D). Upon tandem binding of H3K4K14ac, the shortest distance of encounter is limited by the H3 stretch between H3T6 and H3K14 (about 35 Å), and the PHD–BRD complex assumes a more extended conformation (C and D). This is in line with the previously published SAXS data comparing PHD–BRD and PHD–BRD in complex with H3K14ac, which showed an increase in radius of gyration and Dmax (maximum diameter) indicating a more extended conformation of the histone peptide complex , which suggests a modulatory effect of either the linker between the two domains or the BRD itself on PHD–H3 binding. To gain more insight into the underlying molecular mechanism, we compared the H3-binding affinity of BAZ2B PHD, PHD linker, and PHD–BRD (A and B). ITC showed that the PHD linker construct has a very similar dissociation constant compared with the PHD–BRD construct , implicating the linker region itself as the main negative modulator of the binding affinity (B).The PHD domain showed a 9-fold higher affinity for H3(1–21aa) compared with the PHD–BRD construct A, which  PHD–BRD , A and Baffinity B.2-strand) when comparing (1H and 15N) chemical shift differences between BAZ2B PHD and PHD–BRD as well as PHD and PHD linker spectra . In particular, Asp1950, known to be crucial for H3 binding, showed pronounced signal shifts . To test whether the linker can interact with the PHD in trans, we added a linker peptide (Ala1982-Lys2007aa) to the 15N-labeled PHD and observed chemical shift perturbations in the PHD, indicating that the linker can weakly but specifically bind the PHD domain (E and F). Although the binding mode of the linker is not identical to H3, Asp1950 is strongly involved in both cases . Collectively, these data indicate transient binding of the linker to the PHD, giving rise to competition between the linker and the H3 peptide for binding to the PHD.Analogously, NMR experiments provided further evidence that the linker induces changes in the spectra of the PHD . Chemical shift perturbations in BRD residues followed a single-site binding model with saturation reached at 1:1 ratio and estimated dK > 8 μm . The dK values determined in this experiment agree reasonably well with the ITC measurement, given the limitations due to the concentration range required for NMR. Shifts in the PHD showed more complex binding and reached saturation at 1.5 ratio with an estimated dK > 190 μm . This confirms the autoinhibitory effect of the linker on H3 binding to the PHD.Considering that, in the case of multivalent interactions, ITC measurements provide only an average of binding events, we used NMR to examine the binding affinities of PHD and BRD residues for H3K14ac. We performed titration of H3K14ac into A). We did not observe an effect of the linker on BRD-binding affinity toward H3K14ac , which confirms that the PHD linker domain, and not the linker per se, exerts negative cooperativity on BRD binding to H3K14ac.Finally, we investigated whether the linker has a similar effect on the binding affinity of the BRD by generating constructs with different N-terminally truncated linkers A. We didA). ITC showed a lower H3-binding affinity of Lm1 (80 μm) and a higher affinity of Lm2 (18 μm) (A). This indicates that the PHD-proximal part of the linker competes with the PHD for H3 binding and that the BRD-proximal part of the linker somewhat neutralizes the inhibitory effects of the PHD-proximal part. Conversely, Lm1 and Lm2 bind H3K14ac with the same affinity as the wild-type BAZ2B PHD–BRD (A), which confirms that the linker does not interfere with the binding of K14ac to the BRD. Reduction of the linker length to 21 aa (BAZ2B Ls) reduced binding affinity for H3K14ac (A), which indicates the requirement of a minimal length and flexibility of the linker to prevent a steric clash between the PHD and BRD and allow their optimal mutual positioning for histone binding.The 72 aa–long linker comprises two prominent stretches enriched in lysine and arginine residues, which are located in the PHD- and BRD-proximal part of the linker. We deleted the BRD-proximal part  as well as the PHD-proximal part of the linker  A. ITC sh (18 μm) A. This i PHD–BRD A, which  H3K14ac A, which Asupplemental Fig. 6). Moreover, charged amino acid motifs between histone recognition domains were also identified in related proteins such as UHRF1, TRIM24 (tripartite motif 24), and TRIM33 (tripartite motif 33) (B). Mutation of the charged residues in the linker region led to a drastic change in H3-binding affinity (C). Alanine mutation of the PHD-proximal positively charged patch (10M+) and the BRD-proximal positively charged patch (7M+) led to a 9-fold and 3-fold increase in binding affinity toward H3, respectively (C). Combined mutation of 10M+ and 7M+ (17M+) did not increase the H3-binding affinity beyond that of 10M+ (C and D).The BAZ2B linker is rich in basic amino acids, especially lysines, which are conserved across species (otif 33) . Strikinotif 33) B. Mutatiaffinity C. Alaninectively C. Combin of 10M+ , C and D+ or 10M+) increased the binding affinity, mutation of negatively charged residues led to a 2-fold reduction in binding affinity (C). The combined mutation of 7M+ and negatively charged residues (7M+ 4M−) restored the wild-type binding affinity (C). The same changes in H3-binding affinity were observed when all of the different mutations were introduced in the PHD linker construct . Given that 17M+ has a similar binding affinity as 10M+ and that mutations of negatively charged residues neutralize the 7M+ effect on binding affinity, we conclude that the positively charged residues in the PHD-proximal part of the linker are the primary contribution to the inhibitory effect of the linker on H3-binding affinity.Although the mutation of positively charged residues (7Maffinity C. The coaffinity C. The saB and C). Additional negative charges led to an increase in affinity and completely neutralized the inhibitory effect of the positively charged residues in the BRD-proximal part of the linker (C).We additionally mutated several phosphorylatable residues  present in the BRD-proximal part of the linker into phosphomimicking Asp residues , B and Ce linker C.+ . The BRD spectra differed from PHD–BRD WT but aligned well with PHD–BRD 10M+, indicating a transient interaction of the PHD-proximal part of the linker with the BRD. This indicates the importance of the linker in modulating not only the binding affinity of the PHD but also the relative positioning of the two domains.Furthermore, we explored the effect of the PHD-proximal part of the linker on the BRD by comparing chemical shift differences between BAZ2B BRD, PHD–BRD, and PHD–BRD 10Min vivo by posttranslational modifications, whereby lysine acetylation or poly(ADP-ribosyl)ation reduce the positive charge and phosphorylation increases the negative charge. Given the effect of the electrostatic properties of the linker on histone binding, we tested whether BAZ2B PHD–BRD is also able to bind H3K14ac in the cellular environment. To this end, we performed a histone peptide binding assay using biotinylated peptides prebound to streptavidin beads and incubated with nuclear extracts from FLAG-BAZ2B PHD–BRD–transfected HEK293T cells (A and B). The 10M+ mutant exhibited 2- to 3-fold stronger binding to H3K14ac compared with the WT, corroborating in vitro ITC data (B and 4C). Reduction of cellular PAR levels with the PARP1/2 inhibitor olaparib reduced the binding of BAZ2B PHD–BRD to both H3 and H3K14ac, confirming that poly(ADP-ribosyl)ation modulates BAZ2B histone-binding affinity in vivo (C and D).Changes in the linker net positive charge may occur 3T cells . Bound p3T cells , A and BITC data B and 4C. in vivo , C and Din vitro PARylation assay indeed showed that BAZ2B PHD–BRD is poly(ADP-ribosyl)ated, as judged by the smear of higher molecular weight (∼55 kDa) relative to the His6-BAZ2B PHD–BRD size (∼30 kDa) detected by an anti-PAR antibody (A). Constructs that lack positively charged residues, such as 10M+ , 17M+ , and Lm2 , were not PARylated (A). Surprisingly, alanine mutations of negatively charged aspartates and glutamates downstream from the PARylated motif (4M−) dramatically increased BAZ2B PARylation (A). This suggests that negatively charged residues form salt bridges with lysines and make them less accessible to modification. Furthermore, we confirmed the interaction between BAZ2B PHD–BRD and PARP1 in an in vitro GST pulldown assay (B) and an in vivo FLAG co-immunoprecipitation assay (C). In accordance with the in vitro PARylation assay, 10M+ and 17M+ mutants showed weak or no interaction with PARP1, respectively (C). The 4M− mutant was PARylated in vivo and showed strong interaction with PARP1 (C).Local sequence concentration of positively charged residues is often prone to poly(ADP-ribosyl)ation (PARylation) . An in vantibody A. ConstrARylated A. SurpriRylation A. This swn assay B and an on assay C. In accectively C. The 4Mth PARP1 C.3–7-K-X-K/R-K/R (B). Therefore, we tested whether BAZ2B PHD-BRD binds different phosphoinositides using commercially available PIP strips (D). BAZ2B PHD–BRD was found to bind phosphatidylinositol 5-phosphate with strongest affinity, followed by PI3P, PI4P, PIP, PIP, and phosphatidic acid (D). Phosphoinositide binding was strongly reduced in the PHD–BRD Lm1 truncation lacking the BRD-proximal part of the linker, whereas the construct lacking the PHD-proximal part of the linker (Lm2) as well as 10M+ and 17M+ mutants did not show any binding (D). Interestingly, mutation of acidic residues in the linker (4M−) showed preferential binding to phosphatidylserine (D).Nuclear PIP-binding proteins share a common motif K/R-(X)-K/R-K/R  also fou-K/R-K/R B. TherefP strips D. BAZ2B dic acid D. Phosph binding D. Intereylserine D.1950 and Asp1953) are crucial for binding histone H3 at position Arg2, that PHD–BRD binds H3K14ac in a polyvalent manner, that the PHD linker domain negatively modulates binding of H3K14ac by the BRD, and that the interdomain linker competitively modulates H3 binding to the PHD through transient interactions with the same binding site  increased the H3-binding affinity of PHD–BRD to the level of the PHD alone, indicating that these linker residues modulate H3 binding to the PHD domain. This suggests that the PHD-proximal part of the linker directly modulates the binding of the PHD domain to H3 and, as a consequence, determines the overall affinity of PHD–BRD toward the acetylated histone substrate due to the polyvalent binding mode of the PHD–BRD.How does the linker compete with H3 for binding to the PHD? The PHD-proximal part of the linker resembles histone H3 and is rich in basic residues (mainly lysines) that are highly clustered, as observed previously for IDRs . Alaninecis or in trans? A previous study concluded that BAZ2B PHD–BRD binds H3K14ac in trans K14ac with lower affinity compared with H3(11–21aa)K14ac; and PHD–BRD binds H3K4me3K14ac with higher affinity comparable with the affinity of BRD for K14ac. Moreover, our paramagnetic relaxation enhancement data explain the observed increase in radius of gyration in SAXS measurements by showing that the separation between the PHD and BRD increases due to bivalent H3K14ac binding. Although our data point to an in cis binding mode as more favorable, we cannot exclude an in trans binding mode.Does BAZ2B PHD–BRD bind H3K14ac in in trans . The autin trans . Our datHow does the BAZ2B linker compare with H3? The resemblance of the PHD-proximal part of the BAZ2B linker to the histone substrate represents an example of intramolecular mimicry. Although molecular mimicry conventionally refers to a phenomenon in immune biology where pathogens mimic host proteins to disrupt host functions, it can also cover a broader concept where a protein (or protein domain) binds a target that is structurally similar to its cognate substrate . A recen2014 and Thr2019 in the BAZ2B linker region were already found to be phosphorylated during embryogenesis (in vivo acetylation or poly(ADP-ribosyl)ation, which would equally neutralize the linker positive net charge. Linker PTMs can change structural properties of the linker, fine-tune linker-mediated interactions by altering its physicochemical properties, and facilitate protein–protein interactions, as they can be recognized more easily by the cognate readers due to higher flexibility and accessibility of the linker region  or because of electrostatic changes induced by PTMs , 43. PTMIn addition to covalent adjustments of the physicochemical properties of the linker, non-covalent binding of the phosphatidylinositol phosphate ligand to the positively charged residues may abrogate the inhibitory effect of the linker on PHD–H3 interactions. PIP5 was already shown to alleviate inhibition of the polybasic linker in UHRF1 on tandem Tudor domain–H3K9me3 binding  and to pCan the linker mediate BAZ2B assembly into macromolecular complexes? IDRs are frequently involved in protein–protein interactions that govern transcription, cell cycle regulation, or cell signaling . IDRs macis binding of the PHD–BRD tandem domains to H3K14ac. We pin down the negative effect of the linker to its positively charged cluster, which competes with H3 for the binding to the PHD. Neutralization of the linker positive charge by posttranslational modifications such as acetylation, PARylation, or phosphorylation may represent a cellular regulatory mechanism to increase the binding affinity of BAZ2B to its histone substrate. Autoinhibition by intramolecular mimicry and its release by posttranslational modifications may therefore represent a more common mechanism to regulate the dynamics of protein interactions than envisaged previously.Collectively, we reveal an example of intramolecular mimicry mediated by an intrinsically disordered linker region of the multivalent histone binder BAZ2B, which negatively regulates histone H3 binding to BAZ2B PHD by acting as a histone mimic. We show that the modulation of PHD-H3 binding by the histone-mimicking linker negatively affects in 6 tagging, BAZ2B constructs were cloned into the pETM11 vector. For N-terminal GST tagging, BAZ2B PHD–BRD was cloned into pGEX-4T1. Site-directed mutagenesis was performed with Phusion polymerase (Escherichia coli Rosetta2 (DE3) strain (Novagen). Expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside (Sigma) in the presence of 0.1 mm ZnCl2 , followed by sonication in binding buffer , and 20 mm imidazole) in the presence of 0.1% Nonidet P-40 and protease inhibitors. Proteins were purified using HisTrap nickel resin (Thermo Fisher Scientific), eluted with elution buffer , and 500 mm imidazole), and dialyzed overnight against 50 mm NaCl and 25 mm Tris-HCl (pH 7.4). For the GST purification, cells were lysed by sonication in 150 mm NaCl, 50 mm Tris-Cl (pH 7.4), protease inhibitors, and 0.1% Nonidet P-40. Following incubation with glutathione-agarose beads (Thermo Fisher Scientific), proteins were eluted with 20 mm glutathione, 50 mm Tris (pH 7.4), and 150 mm NaCl.For N-terminal Hislymerase  accordinlymerase . Protein15N-labeled ammonium chloride (Eurisotop) without or with 13C-labeled glucose (Eurisotop). Protein expression and purification were performed as described above. Purified proteins were dialyzed overnight against 100 mm NaCl in 10 mm sodium phosphate buffer (pH 6.8).Transformed Rosetta2 cells were grown in M9 minimal medium containing Histone peptides were synthesized and purified as described previously  and provm for the sample and 1.5–4 mm for the titrant. All experiments were carried out at T = 298.15 K and a stirring speed of 800 rpm. Control injections were subtracted from sample measurements to correct for heat development during dilution. MicroCal Origin 7.0 software was used for analysis; the data points were fitted according to a one-site binding model. The summary of raw data can be found in supplemental Table 1.All calorimetric measurements were carried out at 25 °C using an iTC200 microcalorimeter  with a 200-μl cell capacity and a 40-μl syringe volume. The initial injection of 0.4 μl was followed by 19 2-μl injections with a spacing time of 150 s. In some cases, a different experimental setup with 10 injections of 1 μl followed by 14 injections of 2 μl was required to gain a more sigmoidal titration curve. Concentrations ranged from 150–260 μ6 of the N-terminal H3(1–21) peptide was replaced by a Cys and subsequently tagged with the nitroxide spin label S-methyl methanethiosulfonate . The peptide was subsequently separated from DTT by a PD-10 desalting column . Absorbance was measured at 412 nm, and a 2.5-fold excess of MTSL was added, followed by incubation at 37 °C for 3 h. The measurements were recorded on a Bruker AV600HD+ at 25 °C. The intensity ratio of the oxidized and reduced (50 mm ascorbic acid) BAZ2B(PHD-BRD)–H3(1–21) complex was measured. Ratios of cross-peaks between oxidized and reduced states were calculated (Iox/Ired) and depicted as a temperature coding using home-written routines based on NMRPipe (Thr NMRPipe .2O was used as a lock solvent and added to the BAZ2B H3(K14ac) complexes dissolved in 10 mm sodium phosphate buffer (pH 6.8) and 100 mm NaCl. Protein concentrations were typically between 0.3–1.6 mm with a 1:1 protein:peptide stoichiometry. All NMR datasets were processed using NMRPipe. For resonance assignment, triple resonance CBCA(CO)NH, HNCACB, HNCA, and HN(CO)CA experiments were performed , rabbit anti-PAR , rabbit anti-PARP1 , mouse anti-His , and mouse anti-GST . Secondary HRP-conjugated antibodies for Western blotting (Jackson ImmunoResearch Laboratories) were used at 1:10,000 dilution.m NAD (Trevigen), activated DNA (Trevigen), 50 mm Tris (pH 7.5), and 50 mm NaCl. The reactions were analyzed by Western blotting with anti-PAR antibody.2 units of PARP1  were incubated with 1.5 μg of BAZ2B proteins in the presence of 200 μm Tris (pH 7.5), 100 mm NaCl, 0.05% Triton, and protease inhibitors) for 2 h with rotation at 4 °C. 20 μl of glutathione-agarose (Thermo Fisher Scientific) was added and incubated for an additional 2 h. The beads were washed four times with 300 μl of pulldown buffer and eluted by boiling in SDS sample buffer.10 μg of GST or GST-BAZ2B PHD–BRD was incubated with 5 units of PARP1  in 250 μl of pulldown buffer (20 ml-glutamine (Sigma), and 1% penicillin-streptomycin (Sigma) under 5% CO2 at 37 °C. 48 h after transfection, the cells were harvested and lysed in 50 mm Tris-Cl (pH 8), 150 mm NaCl, 1% Triton, 1 mm DTT, 50 units/ml benzonase (Novagen), and protease inhibitors . Anti-FLAG M2 magnetic beads (Sigma) were equilibrated by washing the beads twice with TBS. Lysates were incubated with the beads for 2 h at 4 °C with rotation. The beads were washed three times with lysis buffer and eluted with 3× FLAG peptide (Sigma).BAZ2B PHD–BRD was cloned into pDONR221 and transferred into pDEST N3xFLAG by Gateway cloning. 8 μg of FLAG-BAZ2B PHD–BRD was transfected into a 10-cm dish containing HEK293T cells using PEI (Polysciences). HEK293T cells were cultured in DMEM  supplemented with 10% fetal bovine serum (Sigma), 1% m NaCl, 20 mm Hepes (pH 7.9), 10% glycerol, 1% Triton, and 1 mm DTT. Elution was performed at 25 °C in 25 μl of sample buffer by shaking at 1400 rpm for 30 min.100 μg of biotinylated histone peptides  was coupled to 50 μl of Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). HEK293T cells were transfected with FLAG-BAZ2B PHD–BRD, and nuclear extracts were prepared as described in Ref. 6-tagged BAZ2B PHD–BRD wild-type and mutant constructs for 1 h at room temperature. The membranes were washed three times and incubated for 1 h at room temperature with anti-GST as a positive control or anti-His for BAZ2B. Following three washing steps, the membranes were incubated with anti-mouse-HRP for 1 h at room temperature and washed three times before chemiluminescent detection.PIP strips (Echelon) were blocked in 3% BSA for 1 h. The membrane was incubated with 0.5 μg/ml of the PIP-specific binding protein  or Hisin vivo histone binding, GST pulldown, co-immunoprecipitation, PARylation, and PIP strip assays; purified proteins; analyzed the data; and wrote the manuscript. All authors approved the final version of the manuscript.S. K. designed experiments, cloned and purified His6-tagged BAZ2B constructs, performed ITC measurements and analysis, assigned the residues of the BAZ2B BRD, and wrote the manuscript. G. K. designed experiments, performed NMR measurements and analysis, and assigned the BAZ2B PHD. T. K. purified the proteins used in the histone binding screen. E. S. performed and S. K. supervised the histone binding screen. R. K. designed the NMR experiments. D. S. designed the study; performed


Our results support the potential benefit of supplementing IIV with M2eP, to expand the vaccine cross protective efficacy.The current inactivated influenza vaccines provide satisfactory protection against homologous viruses but limited cross-protection against antigenically divergent strains. Consequently, there is a need to develop more broadly protective vaccines. The highly conserved extracellular domain of the matrix protein 2 (M2e) has shown promising results as one of the components of a universal influenza vaccine in different animal models. As an approach to overcome the limited, strain specific, protective efficacy of inactivated influenza vaccine (IIV), a combination of recombinant M2e expressed on the surface of norovirus P particle (M2eP) and IIV was tested in chickens. Co-immunization of birds with both vaccines did not affect the production of M2e-specific IgG antibodies compared to the group vaccinated with M2eP alone. However, the co-immunized birds developed significantly higher pre-challenge hemagglutination inhibition antibody titers against the homologous IIV antigen and heterologous challenge virus. These combined vaccine groups also had cross reactive antibody responses against different viruses  compared to the IIV alone vaccinated group. Upon intranasal challenge with homologous and heterologous viruses, the combined vaccine groups showed greater reduction in viral shedding in tracheal swabs compared to those groups receiving IIV alone. Moreover, M2eP antisera from vaccinated birds were able to bind to the native M2 expressed on the surface of whole virus particles and infected cells, and inhibit virus replication  Avian influenza (AI) virus can cause devastating economic losses in the poultry industry including mortalities, reduced egg production and decreased weight gain. Moreover, the ability of AI to cross the species barrier and cause severe disease and mortality in humans has raised serious public health concerns . VaccinaRecent studies showed that immunity against conserved influenza viral proteins such as M2 (matrix protein 2), HA2 (hemagglutinin subunit 2 protein), M1 (matrix protein 1) and NP (nucleocapsid protein) can provide heterosubtypic protection indicating that these proteins are good targets for the development of universal vaccine , 6. The in vitro [Despite the extensive reports of M2e-based vaccine efficacy in different animal models , 25–27, in vitro . In addiin vitro . Therefoin vitro . Moreovein vitro .Based on the aforementioned studies showing weak neutralizing ability, M2e-based vaccines are not effective enough to be used as stand-alone universal vaccines or as a replacement of the current IIV. Recent studies that tested combined vaccination strategies with IIV and M2e vaccines have demonstrated additive cross-protective effects. In mice, combination of M2e, as either VLP or recombinant protein, with IIV or split vaccine provided long-term cross protection against lethal challenge with heterologous and heterosubtypic viruses in terms of reducing weight loss and lung viral shedding titers , 30, 31.in vitro.The Norovirus P particle has been proposed as a good vaccine platform for antigen presentation , 35. Ins2 chamber. The chamber was filled with CO2 at a rate of 10–30% displacement of chamber volume/minute. The animals were observed for an absence of breathing and lack of heartbeat. The CO2 flow was maintained for at least one minute after respiratory arrest. If any respiration or heart beat was detected, the animal was placed back into the chamber and additional CO2 was given as described above. After confirmation of death, an additional secondary physical euthanasia  was performed before tissue collection and carcass disposal.All animals were maintained, vaccinated, challenged and euthanized as per the protocol #2009AG0002-R2 approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC). This protocol follows the U.S Animal Welfare Act, Guide for Care and Use of Laboratory Animals and Public Health Service Policy on Humane Care and Use of Laboratory Animals. White leghorn chickens were obtained from the SPF flock maintained in our facility . The care, management and euthanasia of chickens were performed as previously reported . BrieflyThe A/Chicken/NJ/150383-7/02 (H7N2), A/Turkey/OR/71 (H7N3), A/Chicken/PA/13609/93 (H5N2), and A/Chicken/CA/431/00 (H6N2) viruses were obtained from the repository of the Food Animal Health Research Program  and passaged once in 10-day-old SPF embryonated chicken eggs (ECEs) to prepare working stocks for the study. Madin-Darby canine kidney (MDCK) cells were purchased from American Type Culture Collection  and maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum (FBS) and 10 μg/ml gentamicin.The M2eP chimeric protein containing the consensus M2e sequence (MSLLTEVETPTRNGWECKCSDSSD) of AI viruses was constructed, purified and characterized as previously described , 36. BriInfectious allantoic fluid containing either A/Chicken/NJ/150383-7/02 (H7N2) or A/Turkey/OR/71 (H7N3) viruses was harvested from ECEs and inactivated with 0.1% beta-propiolactone (BPL)  as previously described [For whole virus ELISA, the allantoic fluids containing H5N2, H6N2, H7N2 and H7N3 viruses were concentrated and purified by equilibrium density centrifugation through a 30% to 60% linear sucrose gradient as described previously .Three different trials were performed. All birds across different experiments were vaccinated via subcutaneous route (SQ) with a mixture of vaccine and Montanide ISA 70 adjuvant  .In Trial 1 , two-week-old SPF chickens were divided into 6 vaccination groups (12 birds/group). One group of birds was immunized with an emulsified solution of phosphate buffered saline (PBS) mixed with adjuvant and served as mock control (Mock) while another group was vaccinated twice with M2eP (5 μg / bird) with a two-week interval (M2eP-2x) . In IIV 2 in trial 2–1 and 2–2, respectively) against homologous vaccine virus to IIV alone groups were used for challenge.In heterologous virus challenge trials, two experiments  were performed with some modification in the vaccination schedule . In Tria6 median egg infective dose (EID50) (in a 0.2 ml volume) of A/Chicken/NJ/150383-7/02 (H7N2) virus two weeks after the last vaccination. Tracheal swabs were collected from the infected birds at 3 and 5 days-post challenge (DPC) and eluted in 1 ml of PBS supplemented with gentamicin (10 μg/ml) for virus detection. At 7 DPC, all birds were euthanized and bled to determine the post-challenge hemagglutination inhibition (HI) titers [Each bird was intranasally challenged with a 10) titers .50 equivalent virus titers of swab samples were interpolated from threshold cycle (Ct) values as described previously [Viral RNA was extracted from 100μl of the eluted swab supernatant using QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Viral genome amounts were determined by quantitative real-time RT-PCR (qRT-PCR) using influenza A virus matrix gene specific primers and probes as previously described , 42. EIDeviously , 37.M2e-specific antibody responses were determined by ELISA using synthetic M2e peptide as a coating antigen  as previously described , 36.450) was measured . Sera from the mock vaccinated group were used as controls. Antigen-specific antibody titers were defined as the reciprocal of the highest dilution with an A450 that is above the mean of the mock sera plus two standard deviations.Immune responses to the different virus strains were determined by ELISA using whole virus as the coating antigen as previously described  with modTwo weeks after the last vaccination, pre-challenge HI antibody titers were determined using BPL-inactivated antigens . At 7 DPC, post-challenge HI antibody titers against H7N2 challenge virus were determined. The HI titers were determined using two-fold serially diluted serum samples after heat inactivation at 56°C for 30 min, 8 HAU of virus antigen and 1% turkey erythrocyte suspension as previously described .6 median tissue culture infectious dose 50% (TCID50) per well of A/Chicken/NJ/150383-7/02 (H7N2), A/Chicken/PA/13609/93 (H5N2), or A/Chicken/CA/431/00 (H6N2) viruses (50 μl/well) in serum-free DMEM, and the other half plate was incubated with serum free DMEM. Following a 60 min incubation period at 37°C, 100 μl of DMEM complete medium with 10% FBS was added to each well and plates were incubated for 20 hours at 37°C. The next day, the plates were washed three times with PBS and fixed with 10% formalin for 10 min at RT. Wells were washed three times with PBS and blocked with 200 μl of PBS + 5% FBS for 1 hour at RT. Selected M2eP-2x sera from Trials 1 and 2–1 (1: 40 dilution), showing the highest M2e-specific antibody titers, were serially diluted, added to the wells and incubated for 2 hours at RT. The wells were washed and incubated with HRP-conjugated goat anti-chicken IgG for 2 hours at RT, followed by incubation with the TMB substrate reagent set for 10 min at RT. The reaction was stopped and the absorbance was measured as described above. The data was expressed as the mean A450 (Δ A450: infected–uninfected cells) of triplicate wells per sample.Sera were tested for reactivity with IAV infected MDCK cells to determine whether the M2e-P specific antibodies can recognize native M2 expressed on virus infected cells. Briefly, MDCK cells were grown in 96-well culture plates in DMEM containing 10% FBS at 37°C until confluence. The growth media was removed and cells were washed 3 times with PBS. Half of the plate was incubated with 1 × 102) were pooled and used as anti-M2e serum. Anti-H7N2 serum stock was prepared by pooling samples from the IIV-2w group  with 12 log2 HI titer. The pool of anti-H5N2 (9 log2 HI titer) was prepared from a previous experiment. This pooling strategy was previously employed to generate sufficient quantities of mixed (consensus) serum pools [Five serum samples from the M2eP-2x group  with high M2e-specific antibody titers (11–13 logum pools –46.6 cells/plate) containing sterile glass cover slips and maintained in DMEM complete medium with 10% FBS until confluence. The growth medium was removed, the wells were washed three times with PBS, and the cells were infected with 1x105 TCID50 doses of A/Chicken/NJ/150383-7/02 (H7N2) virus for 20 hours. The wells were washed three times with PBS then fixed in 4% paraformaldehyde (W/V) for 20 min at RT. From this point onward the PBS wash was left to incubate in the wells for 5 minutes for a total of 3 times, and all procedures were performed at RT. The wells were blocked with PBS + 5% normal goat serum  for 60 min. After PBS wash, the cover slips were removed from the wells, incubated with a 1:300 dilution of chicken sera  for 2 hours and washed to remove unbound antibodies. The cells were stained with a 1:500 dilution of Alexa Fluor 488 conjugated goat anti-chicken IgG antibody  for 2 hours in the dark. The coverslips were washed and counterstained with 1 μg/ml DAPI  for 5 min in the dark. The cover slips were washed and mounted on glass slides. Fluorescence analysis and photography were performed on a Leica microscope  with 63 x magnifications.For IFA, MDCK cells were seeded into 24-well cell culture plates  and A/Chicken/NJ/150383-7/02 (H7N2) viruses were diluted into DMEM containing 0.75 μg/ml TPCK-treated trypsin and mixed (1:1) with serum dilutions that were being tested in micro-centrifuge tubes. The virus-serum mixture was further incubated for 60 min at 37°C for serum antibodies to bind the virus. In the meantime, the plates were washed three times with PBS. At the end of incubation, 200 μl of the mixture containing virus-antibody complexes was added to each well and incubated in the CO2 incubator for 60 min at 37°C with rocking every 15 min. The plates were washed three times with DMEM and overlaid with Minimum Essential Medium plus 0.6% agarose containing 0.75 μg/ml of TPCK-treated trypsin. The plates were incubated at 37°C for 3 days, fixed with 10% formalin, air-dried and stained with 0.1% crystal violet to visualize and count the plaques. Each serum dilution, cell control, and virus control were tested in triplicates.For plaque reduction viral neutralization assay, MDCK cells were seeded in 6-well plates at a density of 2×10t-test was used for statistical comparison in experiments that had only 2 groups while one-way analysis of variance (ANOVA) followed by the LSD post-hoc test was used to compare more than 2 groups. Groups were considered to be significantly different at p value <0.05.Data were analyzed using IBM SPSS Statistics (Version 22.0) software. As indicated in the figure legends, unpaired st vaccination) (st vaccination) were similar to that induced by the priming dose of M2eP in the non-combination group   in all three experiments . In Triaination) . These rination) . Moreoveination) . The antination) .As expected, pre-challenge HI antibodies were not detected in the mock and M2eP groups in all experiments. HI antibody titers were generally higher in birds vaccinated with IIV-H7N2  comparedIn Trial 1, the homologous virus challenge experiment, prime-boost vaccination with M2eP (M2eP-2x) led to reduction in challenge virus shedding at both time points, 3 and 5 DPC  compared to the Mock vaccination . The effBased on the ability of M2eP to improve the performance of IIV against homologous challenge virus , Trial 2–1 was conducted to determine whether priming with M2eP could improve the protective efficacy of IIV-H7N3 against heterologous challenge H7N2 virus. Birds were primed with a single dose of M2eP and boosted with a combination of IIV-H7N3 and M2eP. Other groups were included as controls. Trial 2–2 was conducted to test whether priming with 2 doses of M2eP followed by a combination of IIV and M2eP could enhance the heterologous protection observed in Trial 2–1. Note that the challenge virus replicated better (>500-fold higher titers) in this Trial 2–2 compared to the previous two trials . As shown in p = 0.01) in Trial 2–2 compared to the Trial 1, it was within the expected range (log2 = 6 to 9) based on our previous experience [Post-challenge HI titers were used to assess the level of H7N2 challenge virus replication using sera collected at 7 DPC . Both moperience .2) were observed regardless of whether the coating antigen was pure M2e  groups had higher IgG titers compared to the groups primed and boosted with M2eP alone (M2eP-2x groups). In Trial 1, both ofSerum samples were selected from Trials 1 and 2–1 to determine the ability of M2eP-induced antibodies to recognize the native M2 expressed on virus-infected cells by whole cell ELISA. As shown in The binding of anti-M2e antibody (M2eP-2x immunized chicken sera) to M2e on the surface of infected cells was visualized by fluorescence microscopy as shown in Plaque reduction assay was conducted to determine if the M2e-specific antibodies detected by ELISA Figs , 5 and 6The efficacy of several M2e-based vaccines has been tested in different animal models and demonstrated substantially different results depending on the animal model used, vaccine construct and other experimental conditions. In mice, vaccination with M2e alone confers protection against different subtypes of influenza viruses, including HPAI H5N1 virus. However, the protective efficacy of M2e vaccines in chickens is generally much lower than that observed in mice despite the induction of high M2e specific antibodies , 47, 48.As observed in our previous study with 3 vaccinations of M2eP , a primeWhile combined vaccination did not affect induction of M2e-specific antibodies as evidenced by similar M2e titers observed among vaccinated groups, it significantly boosted induction of HI antibodies that were cross reactive with both homologous and heterologous viruses . These rvs mice). Furthermore, the recognition of anti-M2e antibody (M2eP-2x immune sera) was confirmed by IFA  showed a gradual loss of inhibitory activity. The minimal non-specific inhibitory effect of Mock sera may be due to the presence of non-specific inhibitors in the chicken sera [Previous reports on the effect of anti-M2e antibodies on influenza virus replication are still questionable. Zebedee and Lamb 1988) reported that 14C2 M2e-specific monoclonal antibody has the ability to either reduce the plaque size or the growth rate and the number of plaques of certain influenza strains. In contrast, the humanized single chain Fv 14C2 antibody reduced not only the plaque size, but also the number of plaques for the same strain . The calken sera . Consistken sera . This di88 reportIn Trial 1, M2eP vaccination was able to significantly reduce the H7N2 virus shedding in tracheal swabs compared to the Mock vaccination group . Althouget al. (2014) where chickens were vaccinated with an H9N2 IIV supplemented with recombinant protein containing three tandem copies of M2e and then challenged with a heterologous H9N2 virus [In Trials 2–1 and 2–2 where diluted dose of IIV was used, priming the chickens with M2e, once or twice, followed by the combined vaccination was able to significantly reduce the challenge virus shedding titers compared to IIV alone group indicating that M2eP played an important supplementary role in improving the IIV protection. A similar observation was reported by Park N2 virus . It is wS1 TableAlso included is the consensus sequence used to construct M2eP. Substituted residues are shown in red.(DOCX)Click here for additional data file.