Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase–positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase–negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.Among Shiga toxin (Stx)–producing  Escherichia coli (STEC) with the serotype O157:H7 is characterized by the possession of stx gene(s), the locus of enterocyte effacement (LEE) –encoded type 3 secretion system (T3SS), and a large virulence plasmid (pO157) that encodes enterohemolysin and other virulence factors, such as catalase-peroxidase KatP, a type II secretion system, and the protease EspP  and Stx2 includes 7 subtypes (stx2a to stx2g) –producing ,E. coli (EPEC) O55:H7  through sequential acquisitions of virulence factors and phenotypic traits along with a serotype change  positive, and performed fine phylogenetic and genomic comparisons of these GP STEC O157:H7 strains with typical STEC O157:H7 and SF STEC O157:H– strains. We also analyzed the Stx production levels of GP STEC O157:H7 strains.E. coli O55:H7  for comparisons with previously reported reference sequences . We assembled the reads with Canu version 1.5  in each strain and the SNP sites located therein. We then determined the core genome sequence that was conserved in all strains examined. Only SNPs located in the core genome were subjected to further analysis. After reconstructing the genome sequences of each strain using the SNPs and removing recombinogenic SNP sites using Gubbins (We performed single-nucleotide polymorphism (SNP) detection and phylogenetic analyses as described previously . All intact effector genes and plasmid virulence genes identified in strains Sakai and PV15-279 were clustered using CD-HIT .We determined the complete sequences of the Stx1a and Stx2c phages from strain 980938 (from an asymptomatic carrier) and the Stx2c phage from strain 981447 (from a patient with bloody diarrhea) as described previously  and employed them as detection antibodies. We used reagents supplied in the RIDASCREEN Verotoxin kit for detection. Finally, we measured the absorbance at 450 nm (A450) using Tecan Infinite 200 PRO .We inoculated bacterial cells into 40 mL of CAYE broth  and grew them to mid-log phase at 37°C with shaking. We then added mitomycin C (MMC) to the culture at a final concentration of 500 ng/mL. After MMC addition, we collected 100 μL of the culture every hour and immediately subjected it to sonication using a Bioruptor . We obtained the soluble fractions of each cell lysate via centrifugation at 14,000 × stx2a in addition to stx1a and stx2c  in PV15-279. The virulence plasmid pO157 also showed notable differences, as described in the next section.The repertoires and total copy numbers of IS elements also exhibited notable differences  Table 3.stx1a/stx2c-positive clone circulating in Japan. The phylogenetic relationship of this cluster with other STEC O157 lineages was concordant with the stepwise evolution model  into 2–5 subgroups based on their sequence diversity, we analyzed the effector repertoires of the STEC O157 isolates used in the phylogenetic analysis  in Sakai  Table 4.analysis . This anThe pO157 plasmid of PV15-279 was nearly identical to the plasmid from the GP STEC O157:H7 strain G5101  Figure 3katP and espP. These genes were detected only in typical STEC O157:H7 strains, suggesting that these genes may have been specifically acquired by the typical STEC O157:H7 lineage. Although the roles of katP and espP in STEC virulence in humans have not yet been elucidated, at least the SF STEC O157:H– strain causes severe infections even without these genes.As previously reported , indicating that these phages were recently duplicated in this strain. With 1 exception (the phage from strain 155), the Stx2a phages from the typical STEC O157:H7 strains analyzed in this study were integrated in either wrbA or argW, and all shared a similar genomic structure, although considerable variations were observed in the early region, as reported previously  were nearly identical, showing only minor variations, such as IS integration. The Stx2c phages from typical STEC O157:H7 strains, which were all integrated in sbcB, also exhibited very similar genomic structures and sequences. Although notable sequence similarity was observed between the Stx2c phages from GP STEC O157:H7 and typical STEC O157:H7, the early regions were very different.The Stx2c phages from GP STEC O157:H7 strains  was observed only in EDL933 and PV15-279  that produces Stx2a in addition to Stx1a and Stx2c. The whole-genome sequence-based phylogenetic analysis, which included additional GP STEC O157:H7 strains and representative strains belonging to other in Japan . Most ofin Japan . Moreovein Japan . These fWe also obtained several noteworthy findings concerning the evolution of GP O157:H7 in this study. For example, fine genomic comparisons of Stx phages revealed that Stx phages differ notably between GP STEC O157:H7 and typical STEC O157:H7 . Althougstx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan; the virulence potential of this isolate is similar to that of typical STEC O157:H7. Researchers should pay more attention to this less commonly reported STEC O157 lineage, particularly the spread of this Stx2a-producing GP STEC O157:H7 clone and the emergence of additional stx2a-positive clones. Larger-scale genomic analyses including more GP STEC O157:H7 strains from various geographic regions and more SF STEC O157:H– strains are required to obtain a better understanding of the evolution and genomic diversity of GP O157:H7.In conclusion, we isolated a Stx2a-producing GP STEC O157:H7 strain that emerged from the E. coli used in this study of Shiga toxin–producing E. coli with the serotype O157:H7.Additional information about the various strains of


ICl,vol currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2+-mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat–containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated  Maintenance of a constant volume upon changes in extracellular or intracellular osmolarity is critical for the function and survival of cells . After s4–ICl,vol or ICl,swell) were first reported nearly 30 years ago , which are located in the C terminal portion of the first extracellular loop (EL1), also mildly increased the I−/Cl− permeability ratio of LRRC8A/E heteromers (PI/PCl) were also observed when Thr-44, located at the end of the first transmembrane span, was mutated to cysteine . Detection of GFP-tagged LRRC8C at the cell surface suggested the formation of LRRC8A/C heteromers because LRRC8C needs LRRC8A for plasma membrane expression  or LRRC8C  yielded swelling-activated ICl,vol currents. Currents elicited by LRRC8A-GFP/LRRC8C or LRRC8A/LRRC8C-GFP were indistinguishable and displayed typical slow inactivation of LRRC8A/C currents as reported previously (Cotransfection of isrupted ) with LRpression . Howevereviously , 18. BecC and D). In addition to the obligatory initiator methionine, Glu-6 and Gln-14 are conserved in all subunits, whereas other parts of the N terminus display significant homology (D). We cotransfected various N-terminal deletion or elongation mutants of LRRC8A (E) with LRRC8C, either WT or carrying the same deletion or insertion , into LRRC8−/− HCT116 cells. When coexpressed with WT LRRC8C, LRRC8A deletions Δ2–4 and Δ3–6 abolished ICl,vol, whereas Δ3–4 and Δ5–6 and the insertion of 6 additional amino acids after Gln-14 were tolerated (F). However, when present in both isoforms, the latter deletions and insertions also abolished currents (G). In all cases, LRRC8C-GFP was detected at, or close to, the plasma membrane . Because LRRC8A is required for the surface expression of LRRC8C , only seven mutations gave functional channels when introduced into both isoforms (A). Of note, none of the cysteine mutants of the most N-terminal residues  gave currents.We then systematically mutated N-terminal residues in LRRC8A and LRRC8C and assessed swelling-activated currents after coexpressing both subunits in 16 cells . To expl16 cells . We singisoforms A. Of noti.e. LRRC8A/C heteromers carrying the same mutation in both subunits) and on those functional “single” LRRC8A mutants that gave currents only when coexpressed with WT LRRC8C. Control experiments on WT LRRC8A/C–transfected cells gave slow, variable effects on ICl,vol amplitudes that sometimes included an initial current enhancement followed by a slow decrease to roughly 75% of the previous amplitude (B and D). By contrast, currents from several cysteine mutants were strongly and rapidly reduced by MTSEA application . This effect persisted following the washout of the reagent (C), suggesting that it was caused by a covalent reaction of the engineered cysteine with MTSEA. The blocking effect of MTSEA was particularly striking with Y9C/Q9C channels, which were almost completely and irreversibly inhibited by MTSEA (C and D). Interestingly, exposure of the R8C double mutant to MTSEA, which may restore a positive charge at position 8, strongly increased ICl,vol to values that even exceeded WT currents severalfold .We next explored the effect of the partially positively charged, cell membrane–permeable cysteine-modification reagent MTSEA  on curremplitude , B and D reagent C, suggesby MTSEA , C and D2+ inhibition of cysteine-substituted channels can demonstrate the close vicinity of the substituted residues because Cd2+ binding requires multiple cysteines . By contrast, currents from A11C/S11C, Q14C/Q14C, and P15C/P15C mutant LRRC8A/C heteromers were much less affected (E). Cd2+ binding may occur between the newly introduced cysteines, which are located at equivalent positions, either between homologous (i.e. between LRRC8A and -C) or the same type of subunits (e.g. between two LRRC8A subunits). We therefore tested whether cysteines needed to be inserted into both LRRC8 isoforms of the heteromeric channel. Whereas WT/Q9C channels were unaffected by intracellular Cd2+, Y9C/WT channels were efficiently blocked by Cd2+ (F), suggesting that Cd2+ can be coordinated by cysteines of identical isoforms. Assuming that the N termini of LRRC8 proteins fold back into the pore as in connexin26 (2+ together with transmembrane domain 2 (TM2) residues Cys-139 and Cys-141 in LRRC8A and LRRC8C, respectively, that localize to, or close to, VRAC's permeation pathway . The latter channels were efficiently blocked by Cd2+ (F), demonstrating that the inhibition of ICl,vol is not due to a cross-link of N termini to the interior of VRAC. However, we cannot strictly exclude that Cd2+ binding occurred between the newly introduced and other cysteines present in cytoplasmic parts of the channel that appear to have variable conformations  displayed altered I−/Cl− permeability ratios, which were calculated from the shift of reversal potentials (ΔErev) using the Goldman–Hodgkin–Katz equation  dramatically increased PI/PCl from 1.29 ± 0.01 to 2.29 ± 0.10 (A and D). Concomitantly the E6C/E6C mutation strongly decreased iodide conductance (A), suggesting an increased affinity of iodide to the mutant channel's pore. This double mutation also strongly increased SCN− permeability while having only modest effects on Br− and none on F− permeability and leaving the selectivity sequence intact (E). Mutating Glu-6 in both LRRC8A and -C to alanine, serine, or glutamine also increased PI/PCl, albeit less so (D). Average magnitudes of their ICl,vol were small, and E6K/E6K heteromers failed to yield measurable currents .Mutations in pore-lining residues may change the selectivity of the permeation pathway. We therefore examined whether the functional LRRC8A/C heteromers carrying mutations either in LRRC8A or in both isoforms , whereas adding the membrane-impermeable . Strikingly, MTSES not only restored the ion selectivity but also the current amplitude back to WT levels (F), stressing the importance of a negative charge at this position. Currents from LRRC8AE6C/CE6C channels were efficiently blocked by 4-[oxy]butanoic acid (DCPIB) both in the absence and presence of MTSES, showing that other properties such as inhibitor sensitivity were preserved in these channels . Interestingly, MTSES had virtually identical effects on LRRC8AE6C/CE6C when applied from the outside . This suggests that this membrane-impermeable reagent can penetrate deep into or through VRAC's pore. This was further substantiated by effects on R8C/R8C and Y9C/Q9C mutants . Indeed, MTSES has been suggested to permeate LRRC8A/E channels . This result may not be explained exclusively by the mutation of the common LRRC8A subunit because Glu-6 must be mutated in both subunits to change the anion permeability of LRRC8A/C heteromers. PI/PCl of LRRC8AE6C/C and LRRC8A/CE6C heteromers was barely and not significantly different from that of WT heteromers  by shifting its voltage dependence about 80 mV to more negative potentials (B). When inserted in only one of the subunits, these mutations did not affect inactivation (B), resembling their lack of effect on ion selectivity . With the caveat that MTSES changed the inactivation of WT LRRC8A/C heteromers, MTSES almost abolished the difference in inactivation between WT/WT and E6C/E6C heteromers, pointing again to a critical role of a negative charge at this position .The permeability-changing E6C double mutation also drastically changed the inactivation of LRRC8A/C heteromers A by shiftentials B. When itivation B, resembectivity . E6A/E6AC), no effect was seen for LRRC8A/D (D).The impact of E6C double mutants on inactivation depended on the subunit combination. Whereas a more modest right shift of the voltage dependence of inactivation was observed with LRRC8A/E double mutants C, no effLRRC8A/D D.ICl,vol. Eliminating the positively charged Arg-8 by mutating it to cysteine or alanine in double mutants increased outward rectification (B and D). Likewise, introducing positive charges at Thr-5, either by exposing T5C/WT LRRC8A/C channels to MTSEA or by introducing the T5R mutation in both subunits, increased rectification by selectively decreasing inward currents . Of note, introduction of negative charges, by either treating T5C/WT heteromers with MTSES or by introducing T5E mutations into both subunits, also similarly increased rectification (E and D). It seems unlikely that increased rectification is due to direct electrostatic interactions with the permeating Cl− ion because it was observed with insertions of either positive or negative charges. We hypothesized that inserting charges of equal signs into all six LRRC8 subunits may rather cause conformational changes of the pore by electrostatic repulsion of their N-terminal segments. Indeed, when we inserted positive charges into one isoform but negative charges into the other isoform as in T5E/T5R and T5R/T5E heteromers, rectification was reduced or restored to WT levels, respectively (D).Several charge-modifying mutations in the N terminus or modification of N-terminally inserted cysteines increased the outward rectification of fication , B and Dfication , E and Dectively D.We identified the short hydrophilic N termini of LRRC8 proteins as major determinants of the biophysical properties of VRAC. Mutations of several of the ∼20 amino acids preceding the first transmembrane domain and chemical modification of N-terminal cysteine mutants not only changed current magnitudes but also inactivation gating and, most importantly, pore properties such as iodide/chloride permeability ratios and rectification. Our data are compatible with a model in which the N termini of LRRC8 subunits line the cytoplasmic portion of the pore, possibly by folding back into the ion translocation pathway.ICl,vol reflects averaged properties of differently composed VRACs.Structure–function analysis of volume-regulated anion channels is in its infancy as the molecular makeup of VRAC as LRRC8 heteromers has only been discovered recently , 15. BasPI/PCl permeability ratio from 1.29 to 1.59 when expressed in LRRC8A knockdown cells , demonstrating that the mutated residues are both accessible from the aqueous phase and important for channel function. Although it is often inferred from channel inhibition by MTS reagents that the cysteine-substituted residue lines the pore   . The Cd2PI/PCl from ∼1.3 to ∼2.3 by far exceeds those of the T44C , pannexin, innexins, connexin, and CALHM channels share the same transmembrane topology and have rather large pores that pass not only ions but also organic compounds. Only LRRC8, pannexin, and innexin proteins share sequence similarity , but allunctions . Gap junAnalysis by both the substituted-cysteine accessibility method , 38, 50 jV” gating of connexins is strongly affected by N-terminal charges, which may sense the electrical field across the membrane , LRRC8C (NM_032270), LRRC8D (NM_001134479) and LRRC8E (NM_025061) were used as described before , LRRC8C . Point mLRRC8−/− cells  supplemented with 10% FBS (PAN Biotech) and 1% penicillin/streptomycin at 37 °C and 5% CO2. For recording, trypsin-treated cells were seeded onto gelatin-coated coverslips and transfected using Lipofectamine 2000 (Life Technologies). LRRC8C, -D, and -E were fused at the C terminus to GFP or tdTomato and transfected together with LRRC8A at a 1:1 ratio, respectively. Because LRRC8A is needed for the transport of LRRC8B–E to the plasma membrane , and then incubated sequentially for 1 h each with primary and secondary antibodies in blocking buffer. Images were acquired with an LSM 880 confocal microscope with a 63×, 1.4 numerical aperture oil-immersion lens (Zeiss). Rabbit polyclonal antibody against LRRC8A has been described before  and had a resistance of 2–4 megaohms. For Cd2+ block experiments, CaCl2 and EGTA were omitted from the pipette solution, which then contained 40 mm CsCl, 110 mm cesium methanesulfonate, 1 mm MgCl2, 4 mm Na2ATP, 10 mm HEPES .VRAC currents were recorded in the standard whole-cell configuration at room temperature using an EPC-10 patch-clamp amplifier and PatchMaster software (HEKA Elektronik) or MultiClamp 700B patch-clamp amplifier/Digidata 1550B digitizer and pClamp 10 software (Molecular Devices). Patch pipettes were filled with solution containing 40 mm NaCl, 6 mm KCl, 1 mm MgCl2, 1.5 mm CaCl2, 10 mm glucose, and 10 mm HEPES . To elicit ICl,vol, cells were exposed to a 25% hypotonic solution containing105 mm NaCl, 6 mm CsCl, 1 mm MgCl2, 1.5 mm CaCl2, 10 mm glucose, 10 mm HEPES . Cesium was used instead of potassium in the hypotonic external solution and the pipette solution to block potential potassium current, which could be activated during cell swelling. For measuring ion selectivity, NaCl in the hypotonic solution was substituted with equimolar amounts of NaI, NaSCN, NaBr, or NaF. To record ICl,vol, the standard protocol consisted of a 0.6-s step to −80 mV followed by a 2.6-s ramp from −100 to +100 mV from a holding potential of −30 mV applied at 15-s intervals . Currents at −80 mV were used to analyze current density. To examine the inactivation and rectification of VRAC, voltage protocols consisted of a 2-s step protocol from −120 to +120 mV in 20-mV increment from a holding potential of −80 mV applied every 5 s . Recordings were low pass–filtered at 2 kHz and sampled at 20 kHz.The isotonic extracellular solution contained 150 mm MTSEA or 1 mm MTSES was freshly prepared. All other reagents were bought from Sigma-Aldrich.MTSEA and MTSES were purchased from Biotium and stored at −20 °C as powder. Stock solutions of MTSEA and MTSES were freshly made every day and kept at −20 °C. Solution containing 200 μXP−/PCl−) were calculated from the shifts of reversal potential using a modified Goldman–Hodgkin–Katz equation,Erev is the shift in reversal potential, [Cl]hypo and [Cl]subst are the extracellular Cl− concentrations in the normal and anion-substituted hypotonic saline , and [X]subst is the concentration of the substituting anion. R is the gas constant, T is the absolute temperature, and F is the Faraday constant. Effects of acute MTSEA or MTSES application were assessed as the ratio of current amplitude at the end of application over steady-state current in hypotonic medium immediately before application.Relative anion permeabilities  by the maximal current amplitude at the beginning of the voltage step (Imax) as the measure for inactivation. The time of half-inactivation t½ was defined as the time point where the inactivation reached half of the inactivation after 2 s. Boltzmann curve fitting and calculation of V½ were performed using GraphPad Prism with the following fitting constraints: bottom value less than 0.2 and top value greater than 0.9. Statistical analysis was conducted with GraphPad Prism and SciPy library for Python programming language (Python Software Foundation). For pairwise comparisons of current densities and anion permeability ratios the Mann–Whitney test and Welch t test, respectively, were used to evaluate statistical significance. The obtained p values were corrected for multiple comparisons using the Benjamini–Hochberg procedure to control the false-discovery rate when appropriate. Kruskal–Wallis test was used for comparing multiple groups with Dunn's post hoc test for pairwise comparisons.Liquid junction potentials were measured for all solutions and corrected for in ion selectivity experiments. escribed . BecauseP. Z., M. M. P., and T. J. J. validation; P. Z., M. M. P., and T. J. J. investigation; P. Z. methodology; P. Z. and T. J. J. writing-original draft; M. M. P. and T. J. J. writing-review and editing; T. J. J. conceptualization; T. J. J. supervision; T. J. J. funding acquisition.


OsSEC3A causes a lesion-mimic phenotype and enhanced defense responseRice exocyst subunit OsSEC3A interacts with OsSNAP32, a SNAP25-type SNARE protein, and disruption of  OsSEC3A gene is expressed in various organs, and OsSEC3A has the potential ability to participate in the exocyst complex by interacting with several other exocyst subunits. Disruption of OsSEC3A by CRISPR/Cas9  caused dwarf stature and a lesion-mimic phenotype. The Ossec3a mutant exhibited enhanced defense responses, as shown by up-regulated transcript levels of pathogenesis- and salicylic acid synthesis-related genes, increased levels of salicylic acid, and enhanced resistance to the fungal pathogen Magnaporthe oryzae. Subcellular localization analysis demonstrated that OsSEC3A has a punctate distribution with the plasma membrane. In addition, OsSEC3A interacted with rice SNAP25-type t-SNARE protein OsSNAP32, which is involved in rice blast resistance, via the C-terminus and bound to phosphatidylinositol lipids, particularly phosphatidylinositol-3-phosphate, through its N-terminus. These findings uncover the novel function of rice exocyst subunit SEC3 in defense responses.The exocyst, an evolutionarily conserved octameric protein complex involved in exocytosis, has been reported to be involved in diverse aspects of morphogenesis in Arabidopsis. However, the molecular functions of such exocytotic molecules in rice are poorly understood. Here, we examined the molecular function of OsSEC3A, an important subunit of the exocyst complex in rice. The  N-ethylmaleimide-sensitive factor attachment protein 25)-type SNARE protein in barley, inhibits pathogen penetration into host cells and is required for resistance to powdery mildew  has 47 Exo70 genes; the functions of these copies are largely unknown  using CRISPR/Cas9  technology. Our results showed that lesion spots that resembled the phenotype of lesion-mimic mutants (LMMs) appeared in Ossec3a, along with induced cell death and defense responses. As clues to the mechanism underlying the observed defense responses, we show that OsSEC3A interacted with rice SNAP25-type SNARE protein OsSNAP32 and phosphatidylinositol-3-phosphate [PI(3)P], which are involved in plant disease resistance, through the C-terminus and N-terminus, respectively. These results provide new insight into our understanding of SEC3 in the context of its participation in plant defense, which is a role that has not been identified in previous studies.The role of exocyst subunit SEC3 in plants has been widely investigated. This subunit is required for root hair elongation, embryogenesis, and pollen germination (Oryza sativa L. japonica var. Kitaake) and N. benthamiana plants were used in this study. All rice plants used in this study were grown in paddy fields of the Institute of Crop Science in Beijing, paddy fields of the Institute of Crop Science in Sanya (China), or a greenhouse at 25–28 °C with 16 h of light and 8 h of darkness every day.Rice (http://smart.embl-heidelberg.de/). The protein sequences used for the phylogenetic analysis and sequence alignment were obtained from the NCBI (http://www.ncbi.nlm.nih.gov/pmc/) and assessed using BLASTp software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignment was conducted with the DNAMAN software package .Sequence analysis was performed using SMART (simple modular architecture research tool) (http://www.qiagen.com/). A 1 µg aliquot of mRNA was reverse-transcribed to cDNA using a QuantiTect reverse transcription kit . All qRT–PCR assays were carried out using the SYBR Premix Ex TaqKit  with a 7900HT Real-time PCR System . Expression levels were calculated in three technical replicates consisting of two biological replicates each, with the rice ubiquitin gene serving as an internal control. The primers used in this study are listed in JXB online.RNA extraction and quantitative reverse transcription–PCRs (qRT–PCRs) were performed as previously described  assay, 2.5 kb of the escribed .SacI–SalI sites) to generate the pCAMBIA1305-D35S-OsSEC3A-GFP-NOS vector. This construct was introduced into wild-type and Ossec3a mutant plants to generate transgenic plants. For brefeldin A (BFA) treatment, leaf epidermal cells of N. benthamiana were observed following incubation in 100 µg ml–1 BFA for periods of 20 min and 40 min. For transient expression analysis in N. benthamiana, the binary vector pCAMBIA1305-OsSEC3A-GFP (described above) was introduced into Agrobacterium strain EHA105, which was used to infiltrate N. benthamiana leaves as described previously; N. benthamiana protoplasts were isolated using the same method used for Arabidopsis (To obtain green fluorescent protein (GFP) transgenic plants, the OsSEC3A cDNA fragment was amplified using 1305OsSEC3A-GFP-F/R and cloned into the pCAMBIA1305-GFP vector (produced by insertion of the GFP backbone of pAN580 into the pCAMBIA1305 vector at the http://www.bio.utk.edu/cellbiol/markers/vectors.htm) to produce OsSEC3A–GFP fusion proteins. The recombinant plasmid was co-transformed into rice protoplasts with plasma membrane markers. Transient expression using rice protoplasts from rice leaf sheaths was achieved as described previously (Wild-type OsSEC3A cDNA fragments were amplified with primer pair OsSEC3A-GFP-F/R  and insehttp://cbi.hzau.edu.cn/crispr), was used to select sgRNAs targeting the third and 10th exon of the OsSEC3A gene. Plasmid construction was conducted as previously described. The Cas9 destination vector was driven by the maize ubiquitin promoter for expression in rice, and sgRNA expression was driven by the pol III type promoter of U3 sgRNA. Gateway recombination was used to mobilize sgRNA, after which the binary T-DNA vectors for co-expression of Cas9 and sgRNA were transformed into rice calli.The single guide RNA (sgRNA) design method for the CRISPR/Cas9 system in plants was described previously (Samples of seedling and leaf sheath tissue weighing ~0.3 g (FW) were prepared. Extraction and quantification of salicylic acid (SA) was performed as described previously, with minor modifications  was evaluated for virulence in Kitaake and the Ossec3a mutants. Four-week-old rice seedlings were spray-inoculated following a previously described procedure (2O2) accumulation was detected by 3,3-diaminobenzidine (DAB) staining. H2O2 content was measured with a Hydrogen Peroxide Assay Kit  according to the manufacturer’s protocol.OsSEC3A and various domain deletion variants of OsSEC3A were cloned into Y2H bait vector pGBKT7 (Clontech). The coding regions of other exocyst subunit genes were cloned into prey construct pGADT7 (Clontech)  was plated on SD/-Ade/-His/-Leu/-Trp plates to test the interaction between bait and prey.For the yeast two-hybrid (Y2H) assay, the coding regions of in vitro pull-down assays, the coding regions of OsSEC3A, OsSEC3AC (amino acids 310–888), OsSNAP32, OsExo70A1, and OsExo70B1 were inserted into pMAL-c2X and pGEX4T-1 vectors to generate maltose-binding protein (MBP)–OsSEC3A, MBP–OsSEC3C (amino acids 310–888), glutathione S-trasferase (GST)–OsSNAP32, GST–OsExo70A1, and GST–OsExo70B1 plasmids, respectively .For the luciferase (LUC) reporter assay, the OsSEC3A_N (amino acids 1–450) were amplified using primers  was used to purify MBP-tagged proteins. The PIP strips/arrays  were blocked with 1% non-fat dry milk in phosphate-buffered saline (PBS) for 1 h and incubated in 10 μg ml–1 MBP-tagged recombinant protein in blocking buffer for 1 h at room temperature with gentle agitation. The blots were washed three times with PBS plus 0.1% Tween-20 (pH 7.4) and soaked in this buffer supplemented with 1:1000 murine horseradish peroxidase-conjugated anti-MBP antibodies (New England Biolabs) at room temperature for 1 h. After thorough washing, the signals were detected following the ECL Plus immunoblot method. As a negative control, we also incubated the membrane with the MBP tag only, and proceeded with the same experimental protocol.The coding sequence (CDS) of OsSEC3A and OsSEC3Amers see  and inseSEC3 has two orthologous genes, OsSEC3A (LOC_Os03g42750) and OsSEC3B (LOC_Os11g17600), which share 64% sequence identity at the protein level . Twenty independent transgenic lines for the sgRNA construct were obtained, after which DNA sequencing analysis of these lines was conducted using gene-specific primers. The sequencing analysis showed either loss of peak/gain of peak or overlapping peaks around the target site in the sequencing chromatograms of five lines: #2, #5, #7, #11, and #18  exhibited similarly altered growth in comparison with that of wild-type plants . The proeedlings . The lene plants . Lesionsng stage . The disithering . Howeverithering . When the plants , D. In ae plants .Ossec3a mutant plants was fully recovered when the full-length OsSEC3A cDNA was introduced into Ossec3a mutant line #5 under the control of the 35S promoter (OsSEC3A. The results of the experiments with OsSEC3A RNAi (T3 homozygous) showed a lesion phenotype similar to that of the Ossec3a mutant plants , whereas OsSEC3B and OsSEC5 transcript levels were not affected  higher than that of wild-type leaves , OsGSTU6, and OsGSTF10  genes, including PR1a, NPR1, and PBZ1. At the seedling stage, when lesions had not developed, transcript levels of PR genes were very low and no difference was detected between Ossec3a and wild-type leaf blades. However, 3 d after the appearance of lesions of the tillering stage, PR1a and PBZ1 were up-regulated significantly in the leaf blades of Ossec3a mutants in comparison with corresponding wild-type leaf blades , PAD4 , and PAL   . Therefo. oryzae . No chanculation . These rOssec3a plants show actual resistance to pathogens, we spray-inoculated Ossec3a and wild-type plants with an M. oryzae isolate at the seedling stage. One week after inoculation, Ossec3a mutants and wild-type plants showed typical lesions of similar length , a Vps52 domain (amino acids 243–482), and the C-terminal region (amino acids 549–872) (W protein (whole protein), MBP–OsSEC3AN , and MBP–OsSEC3AC . The results of this assay showed obvious binding of MBP–OsSEC3AW and MBP–OsSEC3AN to phosphatidylinositol, whereas MBP–OsSEC3AC showed no binding signal  . Amino a549–872) . This img signal . These rOsSEC3A participates in plant immunity and defense responses, we assessed whether OsSEC3A interacts with a group of known immunity- and defense-related protein factors, including SPL11, SPL28, OsVAMP721, OsSNAP32, OsPUB51, and ORPM1 , displayed a specific interaction with OsSNAP32 (1–160 (including the PH domain) and OsSEC3A190–510 including Vps52), failed to interact with OsSNAP32 (in vitro pull-down assays and split LUC complementation assays to verify this conclusion. The results of the in vitro pull-down assays confirmed that OsSNAP32–GST pulled down MBP–OsSEC3A and MBP–OsSEC3AC (amino acids 310–888) (N. benthamiana leaves that co-expressed Pro35S:OsSEC3A_C-NLuc and Pro35S:CLuc-OsSNAP32 was ~15-fold greater than that of leaves expressing two negative vector controls (Pro35S:OsSEC3A_C-NLuc and Pro35S:CLuc or Pro35S:NLuc and Pro35S:CLuc-OsSNAP32) .To explore how and why nd ORPM1 , whereasnd ORPM1 . OsSNAP3ce blast , we boldOsSNAP32 , whereasOsSNAP32 . These r310–888) . In the sSNAP32) . These rOssec3a mutant rice plants showed slower growth of the main roots in comparison with that of wild-type plants, no specific inhibition of root hair growth was observed , which was completely redirected to the endoplasmic reticulum  in the absence of pathogen infection. This phenotype of Ossec3a mutants is very similar to that of exo70B1 mutants. It is interesting that mutations of SEC3A in other plants species, such as maize and Arabidopsis, do not produce a similar phenotype P; the N-terminus of OsSEC3A was essential for this binding . StrikinJXB online.Supplementary data are available at Fig. S1. Several exocyst subunits fused with the activation domain displayed no auto-activation in our experiments.OsSEC3A from chromatograms to the reference genome sequence.Fig. S2. Alignment of mutations in OsSEC3A in wild-type and Ossec3a plants .Fig. S3. qRT–PCR analysis of Fig. S4. Performance of whole plants (wild-type and transgenic lines) at different developmental stages.Ossec3a mutant plants.Fig. S5. Agronomic traits  of wild-type and OsLOX2.2 and OsJAR1;2) and signaling pathway genes (OsJAZ1 and OsJAZ2) in wild-type and Ossec3a plants.Fig. S6. qRT–PCR of jasmonic acid synthesis-related genes (Fig. S7. Evolutionary analysis of OsSEC3A.Fig. S8. Interaction of OsSEC3A with a group of known immunity- and defense-related protein factors.Fig. S9. BFA treatment of leaf epidermal cells co-expressing OsSEC3A–GFP and Man1–mRFP.Table S1. Sequences of DNA oligonucleotides used in this studySupplementary Figures and TableClick here for additional data file.


Mycobacterium tuberculosis infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.The variable outcome of  Mycobacterium tuberculosisinfection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-γ) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) × CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes (Tip1 to Tip4). These included QTL that were associated with bacterial burden, IFN-γ production following infection, and an IFN-γ-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L , which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The CC042Itgal genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross.Host genetics plays an important role in determining the outcome of  Mycobacterium tuberculosis, yet less than 10% of these exposures progress to clinical disease  strain, are able to restrict the replication of M. tuberculosis for over a year (+ T cell activation and the production of gamma interferon (IFN-γ) in the infected tissue  or B6 mice  mice are an outbred population of genetic mosaics based on eight inbred founders, including highly divergent wild-derived strains . M. tube strains . While t strains . In cont strains . We have strains . This pr B6 mice .Tip1 to Tip4), that were differentially associated with the bacterial burden and/or IFN-γ production. In this population, canonical IFN-γ-dependent immunity was controlled by a novel mutation in the Itgal gene, which disrupts expression of the αLβ2 adhesion molecule and prevents the recruitment of cytokine-expressing T cells to the site of infection. Other Tip loci were driven by wild-derived founder alleles that either reduce IFN-γ production or control IFN-γ-independent immunity. Together, these observations explain the extreme susceptibility of CC042 mice and indicate that the CC panel can be used to understand diverse mechanisms of protective immunity to M. tuberculosis.Here we investigated the basis of TB susceptibility in the CC042 strain using a parallel genetic and immunophenotyping approach. By producing an intercross population based on CC042 and the resistant CC001 strains, we identified multiple QTL, named tuberculosis immunophenotypes  (b). We crossed female CC001 mice with male CC042 mice to generate F1 progeny [(CC001 × CC042)F1 mice], which were then intercrossed to produce 201 F2 offspring. We infected male and female F1 and F2 progeny, along with parental strains, with M. tuberculosis (H37Rv) via low-dose aerosol. The mice were sacrificed at between 28 and 31 days postinfection, a time point that maximized phenotypic differences while minimizing morbidity. The phenotypes measured included the numbers of lung CFU, the numbers of spleen CFU, and lung IFN-γ levels.To investigate the genetic basis of CC042 mouse susceptibility, we created an FB6 mice)  and to m1 mice showed an intermediate phenotype and F2 mice displayed a distribution of values spanning the range for the parental strains  were genotyped with the MiniMUGA array , which was previously shown to underlie this trait in the pre-CC population  Coat color phenotypes of parent mice, CC001 (black), CC042 (agouti with white head blaze), F1 (agouti), and F2  mice. (B) Genetic map of the F2 population used in QTL studies. Vertical lines show chromosomes . Horizontal ticks show marker locations (in centimorgans). (C) Number of male (M) versus female (F) mice. (D) Number of mice with no head blaze (−) versus head blaze (+) in F2 mice. (E) Results of genome scan for blaze trait. (F) Bayes interval for the blaze trait on chromosome 10 (shaded), containing Kitl, previously shown to be associated with the WSBblaze phenotype    and B.Tip1 at 72 Mb and distal Tip2 at 125 Mb /EiJ (CAST) and WSB haplotypes exhibiting a phenotype intermediate to the phenotypes of both homozygotes    and B. A founder , a straipoxvirus , 45, 46.Tip1 and Tip2 are on chromosome 7 and are driven by the WSB parent haplotype, we tested the independence of these peaks by remapping the number of spleen CFU using the genotypes at Tip1 as a covariate. After removing the variation explained by the proximal Tip1 QTL, the distal Tip2 QTL still met the threshold for genome-wide significance  The number of spleen CFU trait was remapped using the genotype probabilities at Tip1 and Tip2 separately as covariates. The distal QTL (Tip2) reaches a significant LOD after the variation explained by Tip1 was removed and vice versa. (B) A multi-QTL model including Tip1, Tip2, and Tip3 was fit for the number of spleen CFU phenotype (batch and sex are included as covariates). The table shows the results from drop-one-term analysis of variance, where each QTL is dropped from the model, one at a time, and the submodel with that factor omitted is compared to the full model. The results provide substantial evidence for all QTL. Column headings: df, number of degrees of freedom; Type III SS, type III sum of squares; LOD, LOD score; %var, percentage of variance explained; Pvalue(Chi2), P value for chi square; Pvalue(F), P value for the F distribution. The values for the number of degrees of freedom, type III sum of squares, LOD score, and percentage of variance explained are those used to compare the full model to the submodel. Download FIG S2, PDF file, 0.1 MB.Tests for independence of Copyright © 2019 Smith et al.2019Smith et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + Gr1+ granulocytes . Using a standard intracellular cytokine staining (ICS) approach, we found fewer CD4 and CD8 T cells from CC042 mice that produced IFN-γ, tumor necrosis factor (TNF), interleukin-17a (IL-17a), IL-2, IL-10, and CD107a upon stimulation than T cells from B6 mice , and spleen at 28 days postinfection using flow cytometry. At the same time, we enumerated the number of antigen-specific CD4 and CD8 T cells using ESAT-6 (CD4) and TB10.4 (CD8) tetramers. CC042 mice had fewer total CD4 and CD8 T cells in the lung than B6 mice . This waigration , 48. We igration . However10.1128/mBio.02791-19.3FIG S3M. tuberculosis infection. Representative flow plots showing the frequencies of ESAT-6-specific CD4 T cells and TB10.4-specific CD8 T cells in the lung (A), mediastinal lymph node (mLN) (B), and spleen (C) of B6 (gray shading) and CC042  mice at 4 weeks after pulmonary M. tuberculosis infection. Bar plots show the mean ± SD for ESAT-6-specific CD4 T cells and TB10.4-specific CD8 T cells in the lung (D), mLN (E), and spleen (F) at the same time point. Sidak’s multiple-comparison test was used to determine significance. *, P < 0.05; ***, P < 0.001. Download FIG S3, PDF file, 0.5 MB.Frequency of antigen-specific T cells in B6 and CC042 mice following pulmonary Copyright © 2019 Smith et al.2019Smith et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Itgal, is located on chromosome 7, within the Tip2 locus identified in our intercross. The lack of CD11a expression on CC042 lymphocytes implicated Itgal variation as the basis for Tip2. To investigate this possibility, CD11a expression was assayed in the WSB and CC011 strains, which contain the susceptibility-associated WSB haplotype at Tip2  at the 3′ terminus of intron 1 . Although the resultant mutant sequence (TG) has been observed to function as a splice acceptor in certain transcripts  underlying Tip1 appears to be functionally distinct from Itgal, as the effects of Tip1 and Tip2 were additive, these traits differed in their mode of inheritance, and Tip1 was not associated with IFN-γ levels. These data indicate that IFN-γ-independent mechanisms, such as those underlying Tip1, can act in an additive fashion to increase the susceptibility of animals with a more canonical immunodeficiency that affects Th1 cell activity.While the disease-promoting effect of CD11a deficiency was generally consistent between the B6 background and the recombinant CC genotype, the loss of  B6 mice , likely M. tuberculosis infection, CAST mice do not produce detectable levels of IFN-γ in the lung  circulate in this population and can drive disease responses , IFN-γ-independent bacterial control (Tip1), and IFN-γ production (Tip3 and Tip4). The dissociation of bacterial control from IFN-γ production was likely facilitated by the presence of haplotypes that are absent from standard mouse strains, supporting the value of genetic diversity to understand highly variable traits, such as TB susceptibility.The immune response to 1 mice were generated from crossing CC001 females with CC042 males (CC001 × CC042). The F2 mice used for QTL mapping were obtained from crossing these F1 mice; e.g., all F2 animals were [(CC001 × CC042) × (CC001 × CC042)]F2. Two hundred one F2 mice were made and phenotyped, and 170 were successfully genotyped and used for QTL analysis . Both female and male mice were used throughout the study, as indicated throughout the text. All animals used for experiments were 8 to 12 weeks old.C57BL/6J (Jax stock number 0664) (B6) mice were purchased from The Jackson Laboratory. CC042/GeniUnc and CC001/Unc mice were obtained from the Systems Genetics Core Facility at the University of North Carolina  and bredM. tuberculosis strain H37Rv (phthiocerol dimycocerosate positive) was used for all studies. Prior to infection, the bacteria were cultured in 7H9 medium containing 10% oleic acid-albumin-dextrose-catalase (OADC) growth supplement enrichment  and 0.05% Tween 80. For aerosol infections, bacteria were resuspended in phosphate-buffered saline (PBS) containing Tween 80 (PBS-T). Prior to infection, the bacteria were sonicated and then delivered via the respiratory route using an aerosol generation device (Glas-Col). Groups of mice were sacrificed at 24 h postinfection to enumerate the infectious dose. The infectious dose for all experiments ranged from 50 to 150 CFU.Wild-type To determine the number of CFU, mice were anesthetized via inhalation with isoflurane  and euthanized via cervical dislocation. The organs were aseptically removed and individually homogenized, and viable bacteria were enumerated by plating 10-fold serial dilutions onto 7H10 agar plates. The plates were incubated at 37°C, and the colonies were counted after 21 days. Cytokine concentrations in cell-free lung homogenates were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer’s instructions.M. tuberculosis were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm. The sections were stained with hematoxylin and eosin (H&E). All sectioning and staining were done by the Diabetes and Endocrinology Research Center Morphology Core (DERC) at the University of Massachusetts Medical School. Images were captured on a TissueGnostics TissueFAXS Plus slide scanning microscope at ×2 and ×20 magnifications.Lung lobes from B6 and CC042 mice infected with Lung tissue was harvested in RPMI containing fetal bovine serum (FBS) and placed in C tubes (Miltenyi). Collagenase type IV/DNase I was added, and the tissues were dissociated for 10 s on a GentleMACS system (Miltenyi). The tissues were incubated for 30 min at 37°C with oscillations and then dissociated for an additional 30 s on a GentleMACS system. Lung homogenates were passed through a 70-μm-pore-size filter. Cell suspensions were washed in RPMI, passed through a 40-μm-pore-size filter, and aliquoted into 96-well plates for flow cytometry staining. Nonspecific antibody binding was first blocked using the Fc Block reagent, after which the cells were then stained with CD3-BV785 (clone 145-2CL1), CD8-allophycocyanin (APC)-Fire 750 (clone 53-8.7), CD44-peridinin chlorophyll protein-Cy5.5 (clone IM7), CD11a-BV711 (clone M17/4), CD69-phycoerythrin (PE)-Cy7 (clone H1.2F3), and CD62L-BV570 (clone MEL-14) from BioLegend and CD4-Alexa Fluor 700 (clone RM4-5) from BD Biosciences. In some experiments, intracellular cytokine staining was also performed. After surface marker staining, cells were subsequently permeabilized using Cytofix/Cytoperm solution (BD Biosciences) and stained with IFN-APC (clone XMG1.2), IL-2 PE-Cy7 (clone JES6-5H4), IL-17a-BV650 (clone TC11-18H10.1), TNF-BV421 (clone MP6-XT22), IL-10 (clone JES5-16E3), and CD107a-PE (clone 1D4B) from BioLegend and IL-4-Alexa Fluor 488 (clone 11B11) from Invitrogen. Live cells were identified using fixable Live/Dead Aqua stain (Life Technologies). The cells were stained for 30 min at room temperature and fixed in 1% paraformaldehyde for 60 min. All flow cytometry assays were run on either a MACSQuant Analyzer 10 (Miltenyi) or Aurora (Cytek) flow cytometer, and the results were analyzed using FlowJo (version 10) software (TreeStar).1 genotype was called heterozygous). After finding and removing misplaced markers, identified using the droponemarker function of the R package qtl (R/qtl), regions of dense marker coverage were thinned to a spacing of 0.1 cM. The final genetic map contained 1,806 markers. Genotype and phenotype data are available in DNA  was genotyped by Neogen Inc. using the MiniMUGA array. We filtered markers to those that were consistent within a previously published set of CC042 and CC001 mouse genotypes  and diag10.1128/mBio.02791-19.4TABLE S12 intercross mice. Download Table S1, XLSX file, 0.07 MB.Phenotype data for FCopyright © 2019 Smith et al.2019Smith et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02791-19.5TABLE S22 intercross mice. Download Table S2, XLSX file, 1.8 MB.MiniMUGA genotype data for CC001, CC042, and FCopyright © 2019 Smith et al.2019Smith et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Genotype and phenotype data were imported into R (version 3.4.3) and reformatted for R/qtl (version 1.42-8). Genotype probabilities were calculated at a 0.25-cM spacing, and QTL mapping was carried out using the scanone function and batch and sex as additive covariates. Significant LOD thresholds were established by a permutation test with 10,000 permutation replicates. Multi-QTL models were fit using R/qtl’s fitqtl function. LOD profiles and effect plots were generated using the plotting functions of the R/qtl package.l-glutamine, 10% fetal bovine serum , and 20% L929 conditioned medium. After 7 days, differentiated cells were lifted with PBS with 10 mM EDTA and seeded for subsequent experimentation. RNA was isolated by lysing the cells in the TRIzol reagent  and purified using a Direct-zol RNA Miniprep plus kit  per the manufacturer’s recommendations. Following RNA quantification with a NanoDrop spectrophotometer, samples were diluted to 5 ng/μl and used for quantitative reverse transcription-PCR (qRT-PCR) with a Luna One-Step Universal quantitative PCR kit . Gene-specific primers for target transcripts were used at a final concentration of 400 μM with 15 ng of RNA. Primer sequences for Itgal  were designed using publicly available genomic sequences for B6, WSB, and CC042 mice (Bone marrow-derived macrophages (BMDMs) from B6, WSB, and CC042 mice were generated. Briefly, marrow was isolated from the femurs and tibias of age- and sex-matched mice and cultured in high-glucose Dulbecco modified Eagle medium  supplemented with 042 mice . Itgal PStatistical tests were performed using GraphPad Prism (version 7) software. The correlation between measured traits was visualized using the chart correlation function in the PerformanceAnalytics package in R (version 3.2.4) software.All relevant data to support the findings of this study are located within the paper and supplemental files.


Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia, the use of primaquine is indicated for both transmission interruption and radical cure, respectively. However, the limited knowledge of the local prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its associated variants has hindered the use of primaquine.Building on the declining trend of malaria in Ethiopia, the Federal Ministry of Health aims to eliminate malaria by 2030. As Some 11,138 dried blood spot (DBS) samples were collected in 2011 as part of a national, household Malaria Indicator Survey, a multi-stage nationally representative survey of all malaria-endemic areas of Ethiopia. A randomly selected sub-set of 1414 DBS samples was successfully genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique. Considering the geographical position and ethnic mix of the country, three common variants: G6PD*A (A376G), G6PD*A− (G202A) and Mediterranean (C563T) were investigated.Of the 1998 randomly selected individuals, 1429 (71.5%) DBS samples were genotyped and merged to the database, of which 53.5% were from females. G6PD*A (A376G) was the only genotype detected. No sample was positive for either G6PD*A− (G202A) or Mediterranean (C563T) variants. The prevalence of G6PD*A (A376G) was 8.9% [95% confidence interval (CI) 6.7–11.2] ranging from 12.2% in the Southern Nations, Nationalities and Peoples’ (95% CI 5.7–18.7) to none in Dire Dawa/Harari Region.P. falciparum transmission interruption and radical cure of P. vivax in Ethiopia. As the presence of other clinically important, less common variants cannot be ruled out, the implementation of radical cure will be accompanied by active haematological and adverse events monitoring in Ethiopia.The common G6PD*A− (G202A) or Mediterranean (C563T) variants were not observed in this nationwide study. The observed G6PD*A (A376G) mutation has little or no clinical significance. These findings supported the adoption of primaquine for  Plasmodium falciparum  and Plasmodium vivax (36.3%) [P. falciparum malaria transmission and radical cure of P. vivax has the potential to accelerate malaria control and elimination efforts. Primaquine, an 8-aminoquinoline, is currently the only generally available drug for these indications in malaria endemic countries. The widespread use of primaquine has been limited due to its potential to induce haemolytic anaemia in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. Haemolytic anaemia in G6PD-deficient individuals can range from mild to life-threatening conditions [P. vivax infections [In Ethiopia, malaria is caused by both  36.3%) . Due to  (36.3%) . A drug 6.3% . Dunditions . AnotherG6PD deficiency is an X-linked genetic disorder and it is estimated to affect more than 400 million people worldwide . It is oP. falciparum transmission reduction and P. vivax radical cure without prior G6PD testing, which will be crucial in achieving their vision. This study aimed to evaluate the genotypic prevalence of common G6PD deficiency allelic types in the general population residing in malaria-endemic areas of Ethiopia.Because of substantial progress made in malaria control, Ethiopia’s vision is to be malaria-free by 2030 –14. DeteDried blood spot (DBS) samples collected during the national Malaria Indicator Survey in 2011 (MIS 2011) were used for the current study. The MIS 2011 was a cross-sectional, multi-stage, representative household survey that produced national and sub-national estimates for malaria-endemic and malaria prone areas of Ethiopia . Using aA total of 11,138 DBS samples were collected and stored at − 20 °C as part of the MIS 2011 . RepreseBased on a review of the literature, the country’s geographic proximity to the Arabian peninsula and ethnic mix, the following three G6PD mutations common in Africa and the Mediterranean Regions were selected for genotyping: G6PD*A A376G), G6PD*A− (G202A) and Mediterranean (C563T) , 7, 17.6G, G6PD*A polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) technique was used for genotyping G6PD based on previous publications , 20 modiThree restriction endonuclease enzymes, FokI (G6PD*A (A376G)), NlaIII (G6PD*A− (G202A)) and MboII (Mediterranean (C563T)) , were used for the digestion of the amplified products Table . SamplesKnown blood samples archived at CDC, collected from a cross-sectional survey conducted in Kenya and a malaria in pregnancy project conducted in Malawi served as positive controls for G6PD*A (A376G) and G6PD*A− (G202A) (African type mutations). However, there were no positive controls used for Mediterranean (C563T) type mutations. Results with faint and intermediate bands were repeated by using the concentrated DNA samples to achieve more distinct test results. The testing was conducted at the Malaria and other Parasitic Diseases Research Team at the Ethiopian Public Health Institute; a sub-set of samples (10%) was genotyped at CDC Atlanta for quality assurance testing and training.Of the 1998 randomly selected samples, only 1641 (82.1%) DBS were found. DNA was extracted and successfully amplified from 1548 samples (94.3%). An additional 119 samples were not able to be merged to the original database, resulting in 1429 merged genotype outcomes and 1414 with valid genotype results (15 samples with unsatisfactory gel results after repeated attempts). Of all the samples analysed and merged to the database, 53.5% (764/1429) were from females.P. falciparum and 4 P. vivax).Figure Table Although not statistically significant, there was regional variation in the prevalence of the G6PD*A (A376G) mutation: the highest prevalence was observed in Southern Nations, Nationalities, and Peoples’ and Tigray Regions , compared to Dire Dawa and Harari where no G6PD mutations were observed (P = 0.278). The prevalence of G6PD*A (A376G) was 9.2% (95% CI 6.7–11.8) in areas with altitude less than 2000 m, and 7.9% (95% CI 2.4–13.4) in areas with altitude between 2000 and 2500 m above sea level (P = 0.403).In this nationally representative study, 8.9% of the samples were noted to carry the G6PD*A (A376G) variant; the clinically relevant variants of G6PD*A− (G202A) and Mediterranean C563T) were not observed. Although the more clinically important type G6PD*A− (G202A) is found in a sub-set of individuals with the G6PD*A (A376G) variant in other studies 63T were , 23, nonThe results are consistent with prior evidence of low G6PD deficiency prevalence in Ethiopia –11, 25. There are several limitations to this evaluation. First, the large sample loss (29.2%) mainly due to not being able to locate the corresponding DBS, is a major limitation, although there is likely little or no systematic bias introduced in this random loss. Second, the current study only tested for three common variants of the hundreds of known G6PD variants . The curP. falciparum. In addition, they are adopting a policy for the first time in decades of primaquine use for radical cure of P. vivax infections without G6PD testing, starting in low transmission highland districts with directly observed therapy and close supervision [P. vivax malaria without G6PD testing. The safety of implementing this policy will be evaluated through active adverse events and haematological monitoring using a standardized approach with common endpoints [P. vivax case management landscape evolves to incorporate developing regions in the lowlands, new evidence around higher dose and shorter course of primaquine and as new point-of-care, quantitative G6PD diagnostics and tafenoquine become available [No common, G6PD deficiency variants (G6PD *A−) and Mediterranean (C563T) were found in this nationwide study. Following a review of the data from prior studies and the current study, the Federal Ministry of Health has adopted the addition of single low dose primaquine for transmission reduction of ervision . Ethiopindpoints . The risvailable –35.


The role of the influenza virus polymerase complex in host range restriction has been well-studied and several host range determinants, such as the polymerase PB2-E627K and PB2-D701N mutations, have been identified. However, there may be additional, currently unknown, human adaptation polymerase mutations. Here, we used a database search of influenza virus H5N1 clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated from 2008–2012 in Southern China, Vietnam and Cambodia to identify polymerase adaptation mutations that had been selected in infected patients. Several of these mutations acted either alone or together to increase viral polymerase activity in human airway cells to levels similar to the PB2-D701N and PB2-E627K single mutations and to increase progeny virus yields in infected mouse lungs to levels similar to the PB2-D701N single mutation. In particular, specific mutations acted synergistically with the PB2-D701N mutation and showed synergistic effects on viral replication both in human airway cells and mice compared with the corresponding single mutations. Thus, H5N1 viruses in infected patients were able to acquire multiple polymerase mutations that acted cooperatively for human adaptation. Our findings give new insight into the human adaptation of AI viruses and help in avian influenza virus risk assessment. Several adaptation mutations have been identified in the PB2 gene in seasonal human influenza viruses and in AI viruses that have caused occasional human infections. The best known human adaptation polymerase mutation is the PB2-E627K substitution, which has been identified in several human H5N1 and H7N9 isolates and in one human H7N7 isolate9. The PB2-E627K mutation is correlated with increased H5N1 replication and virulence in mammals and also allows the virus to replicate efficiently in the human upper respiratory tract, where the temperature is slightly lower than in the lower respiratory tract10. Other mutations in PB2, PB1 and PA also influence the host range of influenza viruses; e.g., PB2-D701N12, PB2-K526R8, PB2-Q591K13, PB2-E192K and -K702R14, PB2-I147T/K399T/A588T15, PB2-E158G16, PB1-L13P/S678N11, PB1-N105S14, PB1-D175N/K198R17 and PA-K356R18. The mechanism of mammalian adaptation due to these mutations remains largely unknown, although some of the mutations reportedly modified interaction between polymerase and host factors, contributing to host adaptation20.The influenza A virus genome consists of eight single-stranded, negative-sense, RNA segments. In the virion, each viral RNA (vRNA) segment is coated by viral nucleoprotein (NP) and has a viral polymerase complex bound at its 5′ and 3′ termini, to form a ribonucleoprotein (vRNP) for viral replication. The viral polymerase complex consists of three subunits, encoded by the viral PB2, PB1 and PA genes. The polymerase complex is one of the main determinants affecting the host range of influenza viruseshttp://www.who.int/). H5N1 viruses have become endemic in birds in some countries; e.g., China, Vietnam, Cambodia, Indonesia and Egypt. Continuous circulation has allowed H5N1 viruses to diverge genetically to form phylogenetically distinct clades (designated clades 0 to 9) in different geographic areas4. H5N1 viruses have moved across the borders of Southern China, Vietnam and Laos, with different H5N1 clades  circulating in the field23 and associated with a high number of H5N1 human infections. This geographic area is regarded as a hot-spot for H5N1 virus evolution.Since its emergence in China in 1996, highly pathogenic H5N1 AI virus has caused 860 confirmed human infections with 454 deaths  circulation of distinct H5N1 clades in the field in East and Southeast Asiawww.ncbi.nlm.nih.gov/genomes/FLU/). This yielded a total of 24 clade 1.1, clade 2.3.2.1 and clade 2.3.4 human virus PB2, PB1, PA and NP sequences, which included all the public database available sequences from human viruses in Asia during this period  and a total of 198 avian virus PB2, PB1, PA and NP sequences in the corresponding clades. Although a few sequences from human influenza viruses in these clades were isolated later and were available in the GISAID EpiFlu database (https://www.gisaid.org/) at the time of our database search, only sequences isolated in 2008–2012 were used in this study.To avoid such potential problems, we conducted a database search targeting a geographically and chronologically close group of PB2, PB1, and PA polymerase and NP nucleoprotein gene sequences in human and avian H5N1 strains isolated in Southern China, Vietnam and Cambodia during 2008–2012. The nucleotide sequences were downloaded from the National Center for Biotechnology Information (NCBI) Influenza Virus Resource  that were either only in human viruses or were more prevalent in human viruses than in avian viruses as putative human adaptation mutations in clade 1.1, clade 2.3.2.1, and clade 2.3.4 viruses Table . Therefo27 with no known human adaptation mutations  in its HA and polymerase genes. VN/HN was used as the genetic background in this study because most of the identified mutations (13/24) were in clade 2.3.4, clade 2.3.4.1 or clade 2.3.4.3 viruses  of the mutations on viral polymerase activity, we carried out minigenome assays of polymerases with these mutations in the genetic background of A/Vietnam/HN31432/2008 (VN/HN). VN/HN is a representative H5N1 clade 2.3.4 straines Table . Human 230  of the polymerase mutations on the replication kinetics of clade 2.3.4 viruses in human cells using recombinant viruses generated by reverse genetics in the VN/HN genetic background. Based on their polymerase activity in human cells, we selected seven of the mutations that produced significant increases in polymerase activity  for further study and rescued replicating viruses carrying these mutations. In addition, viruses carrying either a putative negative control mutation (PB2-G685R) that had little effect on polymerase activity or a representative human adaptation mutation (PB2-E627K) were included in this study for comparison.Human A549 cells and human Calu-3 cells were infected with each of the mutant viruses or with VN/HN (wt) at a multiplicity of infection (MOI) of 0.03, incubated at 33 or 37 °C, and virus titers were determined for 96 h post-infection. In human cells, most of the mutant viruses produced significantly higher progeny virus titers than VN/HN (wt) at all the time points. At 37 °C, the PB2-T81A/V344M/D701N, PB2-Y658H, PB2-Y658H/V344M and PA-M86V/A343T mutants produced progeny virus titers that were up to 4.0-fold higher in A549 cells and 8.3-fold higher in Calu-3 cells, compared to VN/HN (wt) Fig. . Progeny32. Most mutants produced significantly larger foci diameters than VN/HN (wt)  Fig. , and the50) was 1.47 FFU for the PB2-Y658H mutant, 2.05 FFU for the PB2-D701N mutant and 1.47 FFU for the PB2-T81A/V344M/D701N mutant, which were as much as 16.1-fold less than the MLD50 of 23.71 FFU for VN/HN (wt). In agreement with these results, the virus yield in the lungs of mice infected with 100 FFU of the mutants was up to over 100 times greater than with VN/HN (wt) at both 3 and 6 d post-infection  VN/HN (wt) during the 14 d observation period Fig. . In contion Fig. , with sy33. Our models showed that the mutations that produced increased H5N1 polymerase activity in human cells were located in several domains of the polymerase subunits other than the PB2-627 domain 31. In this study, we targeted a geographically and chronologically related group of polymerase mutants identified in human H5N1 strains isolated in Southern China, Vietnam and Cambodia during 2008–2012. This targeted bioinformatics approach enabled us to identify a number of polymerase gene mutations in H5N1 viruses that had not been previously reported to be human adaptation mutations, in addition to the previously reported PB2-D701N human adaptation mutation. These mutations increased the polymerase activity and replication of clade 2.3.4 viruses in both human cells and mouse lungs to levels similar to the PB2-D701N and PB2-E627K single mutations, which are known human adaptation polymerase mutations. The identification of previously reported and unreported human adaptation mutations in H5N1 viruses confirmed the applicability of our experimental approach and the design of this study. Although the effects of the mutations identified in this study on polymerase activity were relatively low  in our minigenome assay system compared with other studies31, this may have been due to differences in the genetic background used to generate recombinant viruses and to the assay conditions used in these studies.Previous studies to identify human adaptation mutations in influenza viruses have typically relied on comparison of viruses with high and low pathogenicity in mammals. High-throughput screening now allows analysis of large numbers of mutations and identification of previously unknown human adaptation mutations20. However, in most cases, these mutations were detected as single mutations, implying that they did not have to act synergistically with other mutations. In fact, the PB2-D701N and PB2-Q591R mutations alone, but not combined with the PB2-E627K mutation, produced a replication advantage for the avian H5N1 virus polymerase during human cell infection13. In contrast, in this study we found synergistic effects with the PB2-T81A/V344M/D701N and PA-M86V/A343T multiple mutations, producing an additional increase in viral replication in human cells and mouse lungs. The PB2-T81A, PB2-V344M and PB2-T81A/V344M mutations alone had little effect but increased polymerase activity when the two mutations were combined with the PB2-D701N mutation  underlying the differences in the location of human adaptation polymerase mutations remains unknown. However, the different locations may be due to the different genetic backgrounds of the Asian H5N1 virus clades and the Egyptian clade 2.2.1 viruses. In particular, it should be noted that all Egyptian clade 2.2.1 viruses have the PB2-E627K mutation, whereas all avian viruses in the Asian H5N1 clades, including clade 2.3.4, clade 2.3.2 and clade 1.1 viruses, have the PB2-E627 residue.In this study, we identified human adaptation mutations that acted to increase polymerase activity in the genetic background of both clade 2.3.4 and clade 2.2.1 viruses, although clade 2.2.1 viruses are geographically and phylogenetically far from Asian H5N1 clades. This implied that the mutations enabled viruses in different H5N1 genetic lineages to increase viral replication in human cells, although not all the mutations may have the same effects in other clades. However, most of the mutations that significantly increased clade 2.3.4 virus replication in human cells were in the PB2 and PA genes, which was in contrast to our previous study showing that human clinical isolates of clade 2.2.1 viruses had acquired multiple human adaptation polymerase mutations in the PB1 gene10, but the exact mode-of-action of the polymerase domains for host adaptation remains unknown. In contrast, our database search approach identified a number of human-adaptation mutations that were scattered over the entire polymerase complex, besides the PB2-627 domain. Although one of the human adaptation mutations identified in this study, PB2-Y658H, was located within the PB2-627 domain, it was at a substantial distance from PB2-627 and did not form a structural site with the PB2-627 residue. In addition, mutations that acted synergistically to increase viral replication were not located near one another in the PB2 structure. This was consistent with previous reports showing that H5N1 viruses could acquire human adaptation polymerase mutations at sites other than in the well-characterized PB2-627 domain17. The mutations identified in this study were also located on the surface of discrete domains of the polymerase complex or at the inter-subunit interface of the polymerase subunits. In addition, a number of the mutations in PB2 were clustered within the NLS and the Cap-binding domains. This suggested that the mutations may mediate the interaction of the polymerase complex with host factors or among the polymerase subunits. Previous studies reported that PB2-E627K in the 627 domain and PB2-D701N in the NLS domain affected the interaction between the viral polymerase and host importin-α isoforms to adapt mammalian machinery20. Also, some mutant residues may not make direct protein contacts but instead may affect protein flexibility, to help other protein regions maintain polymerase activity or to promote interactions with other protein domains or host factors. These considerations implied that the molecular basis underlying human adaptation by H5N1 polymerase mutations may be multifactorial. However, our structural models may be incomplete, because the viral polymerase complex has been found to be flexible, which allows it to adopt different conformations. Therefore, the exact mechanism(s) by which mutations affect human adaptation of AI viruses needs to be further investigated.The PB2-627 domain has been reported to be a frequent host-adaptation site in the AI polymerase38. The PB2-Y658H mutation has been reported in three human clade 2.3.4.1 virus strains  and in four avian clade 2.3.4.1 virus strains 39. The PB2-Y658H mutation has also been detected in one avian H9N2 virus strain (A/chicken/Jiangsu/DT0112/2012)40, one avian H5N8 virus strain 41 and seven avian H5N6 virus strains 42. Neither the PB2-T81A/V344M/D701N nor the PB2-Y658H mutation has been found in recent  isolates of human seasonal H1N1 and H3N2 viruses. However, recent studies have suggested the possible relevance of these mutations to the human adaptation of AI viruses43. In a high-throughput screening study of random mutagenesis libraries, the PB2-Y658S amino acid change was identified as a mutation that increased H5N1 replication in human cells14. PB2 residue 658 also forms a host-specific structural pocket along with residues 613 and 661 that may be involved in avian to human adaptation43. PB2-V344M has been reported to be one of the mutations in influenza virus A/Indonesia/UT3006/2005 (clade 2.1.3) that increased viral replication in human cells13. However, in these previous studies, the effect of these mutations on host adaptation was not assessed because of their relatively low prevalence in H5N1 viruses.The human adaptation mutations identified here have been detected sporadically in H5N1 viruses. The PB2-T81A/V344M/D701N triple mutation has only been detected in influenza virus A/Hunan/1/2008 (clade 2.3.4)44 and the NS gene46) to become pandemic. Future database searches should be done using larger sequence data sets, since larger numbers of AI sequences from human isolates have recently been deposited in multiple public databases and should be available for bioinformatics analyses.This study was limited by the relatively small number of sequences of clinical human clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated in 2008–2012 in databases, although all of the available sequences of human strains isolated during 2008–2012 were analyzed in this study. Furthermore, host adaptation of influenza viruses involves polygenetic traits, implying that AI viruses probably require adaptation mutations in addition to those in PB2  emerge in novel strains and perhaps facilitate virus adaptation to mammals. In fact, novel H5N6 viruses carrying clade 2.3.4 internal genes have emerged since 2013 and caused outbreaks in East Asia and Southeast AsiaAll animal experiments were conducted in compliance with Japanese legislation  and guidelines under the jurisdiction of the Ministry of Education, Culture, Sports, Science & Technology in Japan, and were approved by the Animal Experiment Committee of the Kyoto Prefectural University of Medicine . Mice that lost more than 30% of their original weight were humanely euthanized.All experiments with live H5N1 viruses were performed at enhanced Biosafety Level 3+ (BSL3+) in the Research Institute for Microbial Diseases, Osaka University and Kyoto Prefectural University. All studies with recombinant DNAs were approved by the Biological Safety Committee of Osaka University  and Kyoto Prefectural University of Medicine  after risk assessments were conducted by the Living Modified Organisms Committee of Osaka University, Kyoto Prefectural University of Medicine and, when required, by the appropriate minister of Japan.The BSL3+ facility of the Research Institute for Microbial Diseases, Osaka University, and Kyoto Prefectural University of Medicine consists of negative-pressure laboratories in which all experimental work is carried out in biosafety cabinets. Air exhausted from the class 3 units is filtered by HEPA filters and then leaves the facility via a second set of HEPA filters. The BSL3+ has a dedicated electrical generator in the event of power loss.Only authorized personnel that have received appropriate training can access the BSL3+ facility. All personnel working in the BSL3+ facility wear a disposable protective, FFP3 facemasks and multiple pairs of gloves. Furthermore, all personnel conducting this study were vaccinated against seasonal and H5N1 influenza viruses. Antiviral drugs are directly available to further mitigate risks upon incidents.In this study, all the mutations introduced into the recombinant VN/HN virus (clade 2.3.4) have been detected as single or multiple mutations in H5N1 viruses isolated from patients, except the PB2-E627K mutation. Also, the mouse infection study was performed after no increase was observed in the progeny virus titers of the selected viruses carrying the mutations in this study compared to previously published studies. The PB2-E627K mutant virus was excluded from the mouse infection experiments in this study.24. Briefly, sequences of PB2, PB1, PA and NP genes from 222 influenza A virus subtype H5N1 strains isolated in Southern China, Vietnam and Cambodia from 2008–2012 were obtained from the National Center for Biotechnology Information (NCBI) Influenza Virus Resource (www.ncbi.nlm.nih.gov/genomes/FLU/). Duplicate sequences from the same strain and sequences with questionable amino acid translation were removed. Nucleotide sequences of genes with more than 100 amino acids missing at either end were also excluded. These sequences were aligned using the MAFFT program48. Polymerase mutations in 24 human and 198 avian H5N1 virus strains were identified by comparing these sequences to a consensus sequence of each protein that was determined from the aligned sequences of all the H5N1 strains downloaded. The prevalence of the mutations in the human and avian virus strains was then calculated and compared between viruses isolated from human and avian hosts. To increase the likelihood of identification of relevant human-adaptation mutations, polymerase mutations that were either in human viruses or were more prevalent in human viruses than in avian viruses  were included.A database search was conducted as described previously2. Human bronchial epithelial Calu-3 cells and chicken fibroblast DF-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum at 37 °C in 5% CO2. Chicken embryo fibroblasts (CEFs) were prepared from 10-day-old embryonated eggs as described previously49.Human embryonic kidney 293T cells, human lung carcinoma A549 cells, canine kidney MDCK cells and quail fibroblast QT-6 cells were maintained in Dulbecco’s Modified Eagle’s Medium or Ham’s F-12K medium supplemented with 10% fetal calf serum at 37 °C in 5% CO50. Virus titers were assayed as FFU by focus-forming assays using MDCK cells as described below.Influenza viruses were grown in 10-day-old embryonated chicken eggs that had been purchased from Shimizu Laboratory Supplies, Japan. The allantoic fluids and culture supernatants were then harvested and stored as seed viruses at −80 °C. Virus purification to produce working stocks was described previouslyMDCK cells (90% confluent in 96-well plates) were infected and, after 1 h at 37 °C, the virus inoculum was removed and the cells were washed with PBS and overlaid with 1% methylcellulose in Modified Eagle’s Medium. At 16 h post-infection at 37 °C, the cells were fixed with 4% paraformaldehyde in PBS, incubated with anti-H5N1 virus polyclonal antibody for 1 h at 37 °C, washed 3 times with PBS, and reacted with Alexa Fluor 488 anti-rabbit antibody for 1 h at 37 °C. After 3 washes with PBS, the plates were coated with a 50% glycerol solution. Digital images were taken using an inverted fluorescence microscope ECLIPSE Ti2 system (Nikon) and foci sizes were measured using NIS-Elements software (Nikon).50. Briefly, 293T and QT-6 cells were transfected with plasmids each expressing the PB2, PB1, PA or NP gene of A/Vietnam/HN31432/2008 (VN/HN) or A/duck/Egypt/D1Br/2007 (EG/D1), and a human or chicken polymerase I-driven plasmid expressing firefly luciferase from a virus-like RNA. Cells were also transfected with a plasmid expressing Renilla luciferase to monitor transfection efficiencies. Cells were transfected with these plasmids using TransIT-LT1 (Mirus) and incubated at 33 or 37 °C. Firefly luciferase activity values at 48 h post-transfection were normalized relative to the Renilla luciferase activity.Minigenome assays based on a dual-luciferase system were performed as described previously52. All viruses were sequenced to ensure the absence of unwanted mutations.Recombinant viruses were generated with a plasmid-based reverse genetics system in the VN/HN (wt) virus genetic background as described previouslyA549 and Calu-3 cells were infected in triplicate with viruses at an MOI of 0.03, and DF-1 cells were infected in triplicate with viruses at an MOI of 0.01. After 1 h at 37 °C, the cells were washed with PBS, followed by further incubation at 33 or 37 °C. At the indicated times post-infection, virus titers in the cell culture supernatants were assayed by focus-forming assays.−1 to 104 FFU of viruses in PBS. The mice body weight and survival were monitored daily for 14 d. Mice that lost more than 30% of their original weight were euthanized. In addition, at 3 and 6 d after inoculation with 1 × 102 FFU virus, mouse lungs were collected and virus titers were assayed as FFU in MDCK cells.Five-week-old female BALB/C mice (Japan SLC), under mixed anesthesia (medetomidine-butorphanol-midazolam), were intranasally inoculated with 1033 as a template, as described previously17.Homology modeling of VN/HN polymerase used the crystal structure of the heterotrimeric polymerase complex of influenza virus A/little yellow-shouldered bat/Guatemala/060/2010 (H17N10) (Protein Data Bank ID code 4WSB)Statistical analysis was carried out using GraphPad Prism Version 6 software (GraphPad Software Inc.).Supplementary Information