Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse and hamster target cells using a different, yet unknown, receptor than NiV and HeV and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryo-electron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing previously unknown conformational landscapes and their distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs. NiV has spilled over to humans on a regular basis in Bangladesh as well as in India and the detection of HNVs in other continents underscore their global distribution7. Moreover, the recent discovery of a previously unknown HeV genotype in Australia is an urgent reminder of the pandemic threat HNVs represent9. To date, no approved vaccines or therapeutics exist for use in humans against any HNVs10.Nipah virus (NiV) and Hendra virus (HeV) are bat-borne zoonotic pathogens of the Henipavirus (HNV) genus causing severe encephalitis and respiratory symptoms in humans, with fatality rates between 50 and 100%11. The homotrimeric type I transmembrane protein F is the viral fusion protein which is cleaved into disulfide-linked F1 and F2 subunits by cathepsin L through an endosomal recycling process14. Upon G-mediated receptor engagement, F is triggered and undergoes large-scale conformational changes promoting membrane fusion and viral entry15. F and G are the targets of neutralizing antibodies which are correlates of protection against NiV and HeV23.HNV infections are facilitated by two viral surface glycoproteins, known as attachment glycoprotein (G) and fusion glycoprotein (F), coupling receptor engagement to fusion of the viral and host membranes. The homotetrameric type II transmembrane protein G is the receptor-binding protein and comprises an N-terminal cytoplasmic domain, a transmembrane domain, a stalk domain, a neck domain and a C-terminal receptor binding head domain28. Furthermore, although most HNVs are bat-borne viruses, including the recently discovered Angavokely virus (AngV)7, Mojiang virus (MojV) was discovered in rats29 whereas Gamak (GAKV) and Daeryong (DARV) HNVs were identified in shrews30. Most recently, Langya virus (LayV) was found in shrews and also isolated from febrile patients in China31, thereby representing the first confirmed human-infecting HNV from shrew origin. Phylogenetic analyzes indicate that LayV, MojV, GAKV and DARV cluster together and are distantly related to bat-borne HNVs32. Although receptor usage for MojV is known to differ from that of NiV, HeV, GhV and CedV, the specific host receptor(s) used by MojV, LayV, GAKV and DARV remain elusive32.NiV, HeV, Ghanaian virus (GhV) and Cedar virus (CedV) utilize a subset of ephrin receptors to gain entry into host cells32, including the newly emerged human-infecting LayV. The scarcity of structural and functional information for the LayV and MojV F and G glycoproteins combined with their weak sequence relatedness to NiV/HeV F and G limit our ability to assess conserved and divergent architectural traits for the development of countermeasures and antigen design strategy broadly applicable to the HNV genus34. To address this knowledge gap, we determined cryo-electron microscopy structures of LayV F in the prefusion and postfusion conformations, revealing the previously uncharacterized refolding events underlying HNV F-mediated membrane fusion. Although NiV/HeV-elicited polyclonal and monoclonal antibodies (mAbs) did not cross-react with LayV F and G, we identified a MojV F-directed monoclonal antibody (mAb) (4G5) binding to LayV F, and unveiled the molecular basis of recognition, as well as a MojV G-directed mAb (2B2) cross-reacting with LayV G. We determined a cryoEM structure of LayV G, which adopts a distinct conformation than that previously described for NiV G or other paramyxovirus attachment proteins, defining another snapshot of the host invasion process. We computationally designed stabilized LayV G constructs and demonstrate the broad applicability of an HNV F prefusion-stabilization strategy which will pave the way for vaccine development efforts. Collectively, our data provide molecular blueprints of the two key targets of LayV vaccine design and pave the way for designing countermeasures against this emerging pathogen.The LayV F and G glycoproteins are most closely related to MojV F and G, sharing 90% and 86% respective amino acid sequence identity, but distantly related to the better studied NiV and HeV counterparts. As a result, it is expected that countermeasures in development for NiV and HeV will not be effective against these divergent viruses35  with approximately similar ratio to that observed for other HNVs (36 and suggest that the LayV/MojV F proteolytic cleavage pathway is likely distinct from that described for NiV and HeV F.LayV F possesses a putative cleavage site at residue 104 (R104), as is the case for NiV (R109) and HeV (K109), but lacks the YSRL and YY motifs present in the NiV/HeV F C-terminal cytoplasmic domains to promote endosomal recycling and subsequent cathepsin L-mediated F cleavage35 . Neverthher HNVs –d. These36.We subsequently assayed the ability of LayV F and G to promote cell-cell fusion using a split luciferase system. Transient transfection of Neuro-2a effector cells with LayV F and G resulted in detectable fusion with target HEK293T or Neuro-2a cells –f. Furth17. Using electron microscopy (EM) imaging of negatively stained samples, we observed that LayV F formed well-folded compact homotrimers, which are typical of the prefusion state, but that the protein spontaneously refolded to the postfusion state over time , highlighting the evolutionary relationships among the fusion machinerysu of these distinct viruses45.Postfusion LayV F forms a ~150 Å-high and ~70 Å-wide conical trimeric structure with a prominent triple helical bundle assembled from the central helices and HR1, surrounded by three antiparallel HR2 helices yielding a six-helix bundle at one end of the trimer. The opposite end of the molecule forms a triangular base –l. The H2) and the postfusion F state , underscoring the irreversible nature of these structural rearrangements46.Although F refolding affects a large fraction of the trimer, the N-terminus, the β-rich domains comprising residues 281–420, and the upstream helix remain mostly unchanged, besides modification of their relative orientations, as is the case for the disulfide bonds ,l. This 50. Previous studies showed that immunization with prefusion NiV F or HeV F, but not postfusion F, allowed elicitation of neutralizing antibodies against these vaccine targets51, thereby motivating the identification of suitable strategies to stabilize F in the prefusion state. Hence, we evaluated the portability of several previously described NiV/HeV prefusion-stabilizing mutations to the LayV F trimer. We evaluated (i) the NiV L172F  and S191P  substitutions previously identified for NiV F51 and (ii) the engineered disulfide bond spanning the F2 and F1 subunits (NiV/HeV N100C/A119C corresponding to LayV F N95C/A114C), near the proteolytic F cleavage site, originally described for HeV F13 and subsequently ported to NiV F17, which appears to be compatible with our structure (and S1). Combining all four mutations, however, produced prefusion F trimers along with some aggregates , and provide post-exposure protection against lethal challenge in ferretso LayV F , likely o LayV F . Howevero LayV F , two antTo determine if 4G5 binding was specific for a given F conformation, we separately assessed binding to the prefusion-stabilized LayV F trimer (N95C/A114C) and to the postfusion F trimer (obtained through spontaneous refolding) using biolayer interferometry . We founand S6). We improved local map resolvability by carrying out local refinements of the 4G5-bound membrane-proximal region and of the membrane-distal conical base of the trimer yielding reconstructions at 3.6 and 3.3 Å, respectively  likely account for the lack of 4G5 binding to NiV and HeV F , and a formulation suitable for use in humans was evaluated in phase 1 clinical trials domains .56 were overall consistent with the NiV G stalk organization11 and suggested that substitutions of three aromatic residues present in the wildtype LayV G stalk  could improve core packing . The LayV G head domain conformation observed in our structure markedly differs from that found in NiV G as the latter glycoprotein adopts an asymmetric organization of its head domains with only two of them interacting with the stalk . Indeed, we could not detect binding of human ephrin B2 or B3 to LayV G using biolayer interferometry . Moreover, a vaccine based on the HeV G tetrameric ectodomain has been shown to protect against both NiV and HeV challenge in preclinical studies and is commercially available for use in horses in Australia 65. Although a similar immunogen adjuvanted with alum has recently entered phase 1 clinical trials (NCT04199169), we demonstrate here that NiV G vaccine-elicited antibodies, that are neutralizing NiV, HeV and the recently emerged divergent HeV-g252, did not appreciably cross-react with LayV G. Collectively, our data strongly indicate that countermeasures being advanced to the clinic will be ineffective against LayV G which has already been detected in several individuals and therefore represent a current threat to public health31. Our data urge for the development of vaccines and mAb therapies against LayV G and closely related HNVs .The cross-neutralizing NiV and HeV G-specific m102.4 mAb has been administered on an emergency compassionate use basis to 15 individuals with high-risk exposure to HeV or NiV and recently completed a phase 1 clinical trial in Australia. m102.4 is the sole antibody evaluated in the clinic against any HNV68. Prefusion-stabilization (2P) of coronavirus spike glycoproteins also led to the groundbreaking development of COVID-19 vaccines in a record time in 202071. We describe here the computational design of LayV G constructs, enabling high-yield recombinant production of the LayV G ectodomain tetramer, and show that a disulfide stapling HNV F trimers in the prefusion state17 is a broadly applicable prefusion-stabilization strategy yielding monodisperse LayV F and GhV F trimers. These protein design strategies will enable the preclinical evaluation of next-generation vaccine candidates against the newly emerged LayV.Structure-guided viral glycoprotein engineering has revolutionized vaccine design against a range of emerging or endemic pathogens. Stabilization of the respiratory syncytial virus fusion glycoprotein in its prefusion conformation (DS-Cav1) propelled the development of a safe and effective vaccine against this pathogen2 and 130 rpm. Vero 76 (ATCC CRL-1587) cells were maintained at 37°C, 5% CO2 in Dulbecco’s modified eagle media (DMEM)  supplemented with 10% cosmic calf serum (CCS) and 1% L-glutamine  (DMEM-10). CHO-K1 cells (ATCC CCL-61) were maintained in Ham’s F-12K (Kaighn’s) Medium (Gibco) with 1% penicillin–streptomycin and 10% CCS at 37°C with 5% CO2. HEK293T cells and Neuro-2a cells (ATCC CCL-131) were maintained in DMEM supplemented with 2 mM L-glutamine, 1% penicillin–streptomycin  and 10% CCS. HEK293T cells were maintained at 37°C with 5% CO2, while Neuro-2a cells were maintained at 37°C with 8% CO2.Expi293F is a derivative of the HEK293 cell line (Thermo Fisher). Expi293F cells were grown in Expi293 Expression Medium (Thermo Fisher), cultured at 37°C with 8% CO72) followed by a GSGGGS linker and a 6×His tag. GhV F wildtype is composed of residue 1–586 with a Cterminal GSGGGS linker followed by a 6×His tag. The LayV F wildtype, LayV F N95C/A114C, LayV F I167F/S186P, LayV F I167F/S186P/N95C/A114C, GhV F wildtype, GhV F S196C/A215C, GhV F I268F/Q287P, GhV F I268F/Q287P/S196C/A215C and GhV F I50–50A, were synthesized by Twist Bioscience. LayV F and GhV F ectodomains were produced in 100mL Expi293F cells grown in suspension using Expi293 Expression Medium (Thermo Fisher) at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. The cultures were transfected using ExpiFectamine™ 293 Transfection Kit (Thermo Fisher) with cells grown to a density of 3 million cells per mL and cultivated for 4 days. The supernatants were harvested, and proteins were purified from clarified supernatants using a 1mL HisTrap HP column (Cytiva), buffer exchanged, concentrated and flash frozen in either TBS . SDS-PAGE was run to check purity.The LayV F ectodomain construct used for all experiments in this study includes codon optimized LayV F (residue 1–482) fused to a C-terminal GCN4 followed by a factor Xa sequence, a linker (GSGGGS) and an hexa-histidine tag (HHHHHH) into pTwist CMV vector for transient expression using Expi293F cell line. The GhV F ectodomain construct used for all experiments in this study includes codon optimized GhV F used for structure determination comprise residue 1–586 and is C-terminally fused to the I53–50A component via an intervening 16 GS linker (as previously described2 incubator rotating at 130 rpm). Cells grown to a density of 3 million cells per mL were transfected with the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) and cultivated for four days at which point the supernatant was harvested. LayV G was purified from clarified supernatants using 2 mL of cobalt resin (Takara Bio TALON), washing with 200 column volumes of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 5 mM imidazole, and eluted with Tris-HCl pH 8.0, 150 mM NaCl, and 600 mM imidazole. Eluted protein was further purified by size exclusion chromatography using a Superose 6 increase 10/300 GL column (Cytiva) equilibrated in a buffer containing Tris-HCl pH 8.0 and 150 mM NaCl. Purified protein was concentrated using a 100 kDa centrifugal filter  to 2 mg/mL and flash frozen with liquid nitrogen. The LayV GSM ectodomain has a human azurocidin signal peptide (MTRLTVLALLAGLLASSRA) followed by an N-terminal His-tag (SGPHHHHHHHHGSSP) followed by residues 72 through 624 of LayV G. The LayV GSM ectodomain also has F89L, G98T, G117A, F126V, and Y130L mutations, unless otherwise denoted in the text. The LayV GSM ectodomain used for structure determination also had a designed tetramerization sequence (QAEELKAIKEELKAIKEELKAIKEELKAI) between the His-tag and residue 72 of LayV G for further stabilization; though this was subsequently found to be unnecessary as LayV GSM is stable on its own.LayV G ectodomains were produced in 25 mL culture of Expi293F Cells (ThermoFisher Scientific) grown in suspension using Expi293 Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 8% CO®  and subcloned into a pcDNA3.1 Hygro CMV mammalian expression vector. For construction of soluble and secreted versions of F glycoproteins, the transmembrane domain (486–520) and C-terminal cytoplasmic tail (521–545) of MojV F were replaced with a GCN4 trimerization motif (GCNt) (MKQIEDKIEEILSKIYHIENEIARIKKLIGE), followed by a factor Xa protease cleavage site (IEGR) and an S-tag to generate trimeric soluble construct of MojV sF. To produce tetrameric soluble MojV G (MojV sG), the N-terminal cytoplasmic tail (2–37) and transmembrane domain (38–70) were replaced with an Igκ leader sequence (METDTLLLWVLLLWVPGSTGD) and an S-tag followed by a factor Xa protease cleavage site (IEGR) and a GCN4 tetramerization motif (GCNtet) (MKQIEDKLEEIESKLKKIENELARIKK).Codon-optimized MojV (Genbank NC 025352.1) F and G open reading frames (ORFs) were synthesized by GenScript™ 293F cells were transfected with sF, and Neuro-2a cells with sG plasmid constructs. Their stable cell lines were obtained through two rounds of limiting dilutions. Stable expressed MojV sF and sG secreted into FreeStyle™ 293 Expression Medium were collected and firstly purified through a S-agarose affinity column (XK 26 column). For MojV sG, the column was washed with 3x bed volumes of Buffer I , followed by 6x bed volumes of Wash Buffer II . The protein was eluted with Elution buffer , then neutralized with buffer (1 M Tris-HCl pH 8.0 or 1 M HEPES buffer pH 9.0). The protein was buffer exchanged to PBS, pH7.4. For MojV sF, the column was washed with 3x bed volumes of Wash buffer I . The protein was eluted with Elution buffer  and neutralized with buffer (1 M HEPES buffer pH 9.0). The protein was buffer exchanged into PBS, 0.01% Triton® X-100, 0.1 M L-Arginine. S-tag was removed with Factor Xa Cleavage Capture kit (Millipore Sigma). The untagged sF or sG was further purified with size-exclusion column . The buffer PBS was used for sG and the PBS with 0.01% Triton X-100 was used for sF purification.For protein expression and purification,human FreeStyleGCNt or sGGCNtet glycoproteins were used to immunize BALB/c mice purchased from Jackson Labs . All animal studies were carried out under an approved protocol MIC-16–262 for animal experiments obtained from the Uniformed Services University Animal Care and Use Committee. All mice were immunized 4 times.The administered antigens (sF/sG) were formulated in a Sigma adjuvant system . Mouse splenic lymphocytes were isolated 4 days following a final immunization without adjuvant and fused with SP2/0 cells using polyethylene glycol by standard methods. Hybridoma supernatants producing MojV F (4G5) or G(2B2) -specific antibodies were identified by enzyme-linked immunosorbent assay (ELISA) using purified S-peptide-cleaved MojV sF or sG. The anti-F or G mAbs were prepared under serum-free conditions using HyClone SFM4MAb  and purified by protein G-Sepharose affinity chromatography.To generate F- or G-directed monoclonal antibodies, purified sF™ 293 Transfection Kit with cells grown to a density of 3 million cells per mL and cultivated for 6 days. The supernatants were harvested, and proteins were purified from clarified supernatants using a 1mL StrepTrap™ HP column (Sigma), buffer exchanged, concentrated and flash frozen in either TBS or PBS. SDS-PAGE was run to check purity.nAH1.3 heavy chain and light chain were codon optimized and synthesized by GeneArt (2019AAG4YC) with a signal sequence (MPMGSLQPLATLYLLGMLVASVLA) at the N-termini of both heavy and light chains, as well as a StrepII tag (WSHPQFEK) linked by a linker sequence (GSGGGS) at the C terminal of the heavy chain. The synthesized heavy and light chain genes were each subcloned into a pcDNA3_1(+)_MAr vector. nAH1.3 IgG was produced in 25mL Expi293F cells grown in suspension using Expi293 Expression Medium (Thermo Fisher) at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. The cultures were double transfected with 1:1 ratio of heavy chain and light chain plasmids using ExpiFectamineHENV-32, HENV-103, and HENV-117 IgG genes were codon-optimized for a mammalian cell expression system, synthesized, and cloned into a pCDNA3.1+ vector by GenScript. The HENV-32 light-chain sequence was obtained from the Fab sequence of crystal structure Protein Data Bank . nAH1.3 and HENV-32 heavy chains include a StrepII tag (WSHPQFEK) linked by a linker sequence (GSGGGS) at the C terminus. HENV-103 and HENV-117 light- and heavy-chain constructs have been produced by replacing the variable domain of HENV-32 constructs with the corresponding HENV-103 or HENV-117 sequence. The C-terminal StrepII tag and linker have been removed for HENV-103 and HENV-117 heavy-chain constructs.55. Briefly, CHO-K1 cells were transfected with a linearized m102.4 PDR12 construct and the antibody was purified with protein A from a selected stable m102.4-producing cell line55. The human IgG1 m102.4 mAb has been extensively characterized as a highly potent HeV and NiV cross-reactive mAb74.m102.4 was isolated from a recombinant human phage-displayed Fab library and prepared as previously described46.5C4, 5B3, 12B2, 6E5, 13D6, 10G2, hAH1.3 IgGs were produced from hybridomas and were previously described asAssays were performed on an Octet Red (ForteBio) instrument at 30°C with shaking at 1,000 RPM.For anti HNV mAbs screening, Anti-penta His (HIS1K) biosensors were hydrated in water for 10 min prior to a 120 s incubation in 10X kinetics buffer (undiluted). LayV F N95C/A114C or LayV G central stalk stabilized ectodomain was loaded at 10 μg/mL in 10X Kinetics Buffer for 360 s prior to baseline equilibration for 300 s in 10X kinetics buffer. Association of anti HNV IgGs in 10X kinetics buffer at 200nM concentration was carried out for 360 s prior to dissociation for 360 s.D determination of prefusion LayV F, anti-penta His (HIS1K) biosensors were hydrated in water for 10 min prior to a 120 s incubation in 10X kinetics buffer (undiluted). LayV F N95C/A114C was loaded at 20 μg/mL in 10X Kinetics Buffer for 300 s prior to baseline equilibration for 300 s in 10X kinetics buffer. Association of 4G5 Fab in 10X kinetics buffer at various concentrations in a two-fold dilution series from 100nM to 3.125nM was carried out for 100 s prior to dissociation for 100 s.For 4G5 KD determination of postfusion LayV F (spontaneous refolded over 4 months at 4°C), anti-penta His (HIS1K) biosensors were hydrated in water for 10 min prior to a 120 s incubation in 10X kinetics buffer (undiluted). LayV F spontaneous refolded was loaded at 20 μg/mL in 10X Kinetics Buffer for 300 s prior to baseline equilibration for 300 s in 10X kinetics buffer. Association of 4G5 Fab in 10X kinetics buffer at various concentrations in a two-fold dilution series from 40nM to 1.25nM was carried out for 360 s prior to dissociation for 360 s.For 4G5 KD determination, the data were baseline subtracted prior to fitting performed using a 1:1 binding model and the ForteBio data analysis software. Mean kon, koff values were determined with a global fit applied to all data. The experiments were done with two separate purification batches. Data was plotted and fit in Prism (GraphPad).For 4G5 K96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4°C with 2 μg/mL of NiV G or LayV G central stalk stabilized ectodomain in 50mM Tris and 150mM NaCl at pH 8. Plates were slapped dry, washed 3X in Tris Buffered Saline Tween (TBST) and blocked with Blocker Casein (ThermoFisher) for 1 h at 37°C. Plates were slapped dry and washed 4X in TBST. 1:4 serial dilutions of NHP sera were made in 50 μL TBST and incubated at 37°C for 1 h. Plates were slapped dry and washed 4X in TBST followed by addition of 50 μL 1:5000 Goat anti-Human IgG Fc Secondary Antibody with HRP (Invitrogen) for one hour at 37°C. Plates were slapped dry and washed 4X in TBST followed by addition of 50 μL TMB Microwell Peroxidase (Seracare). The reaction was quenched after 4 minutes with 1 N HCl and the A450 of each well was read using a BioTek Synergy Neo2 plate reader. Data was plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration).75. All data were processed using CryoSPARC76 following the pipeline described in 3 μl of purified sample of each F constructs was applied to a glow discharged 300 mesh copper grids (Ted Pella) coved with evaporated continuous carbon on top of collodion (Electron Microscope Science) layer for 1 min before blotting away excess liquid with Whatman no. 1 filter paper. Grids were stained with 3 μl of 2% (w/v) uranyl formate stain for 1 min twice. Grids were imaged using an FEI Tecnai Spirit 120 kV electron microscope equipped with a Gatan Ultrascan 4000 CCD camera. The pixel size at the specimen level was 1.60 Å. Data collection was performed using Leginon77. To enable detection, an S peptide tag (KETAAAKFERQHMDS) was inserted at the C-terminal end of LayV F and at the N-terminal end of LayV G. A peptide corresponding to the heptad repeat 2 (HR2) domain of LayV F  and the corresponding scrambled peptide  were synthesized by Biosynth .Codon optimized LayV F and G open reading frames were synthesized by Genscript and subcloned into a mammalian promoter modified expression vector pcDNA3.1-CMV36. At 48 hrs post transfection, cells were lysed with 1x RIPA (radioimmunoprecipitation assay) Lysis and Extraction Buffer containing a protein inhibitor cocktail and incubated with S protein agarose beads at 4°C overnight. Proteins were resolved by SDS-PAGE and detected with an anti-S-peptide-HRP conjugated antibody.CHO-K1, HEK293T and Neuro-2a cells were transfected with pcDNA3.1-LayV F, pcDNA3.1-LayV G, pcDNA3.1-LayV F and pcDNA3.1-LayV G encoding S-tagged constructs. As a reference, cells were transfected with pcDNA3.1-MojV F, pcDNA3.1-MojV G or pcDNA3.1-MojV F and pcDNA3.1-MojV G encoding S-tagged constructs78. Briefly, the effector cells (Neuro-2a cells) were transfected with pcDNA3.1-LayV F, or pcDNA3.1-LayV G or pcDNA3.1-LayV F and pcDNA3.1-LayV G together with one half of the dual-split-reporter luciferase expression plasmid (DSP1–7). As a control, Neuro-2a cells were co-transfected with pcDNA3.1-MojV F, pcDNA3.1-MojV G and DSP 1–7. Each target cell  were transfected with the other half of the dual-split reporter plasmid (DSP 8–11). After 48 hours post-transfection, the effector cells were gently detached and applied onto the target cells in equal numbers. At 48 hrs post mixing, EnduRen (Live cell substrate for the luciferase) was added to the plate. Content mixing between the effector cells and the target cells as a result of cell-cell fusion was measured using a luminometer . Each assay was done for three replicates. Cell-cell fusion inhibition was assessed by LayV HR2 and scrambled LayV HR2 peptides.We used a quantitative dual-split-reporter luciferase-based cell-cell fusion assay as previously described75.To prepare the CryoEM grids of LayV F for solving prefusion state, 3 μL of the 0.14 mg/mL LayV F wildtype were loaded onto holey carbon grids covered with graphene oxide, prior to plunge freezing using a Vitrobot MarkIV (ThermoFisher Scientific) with a blot force of −1 and 3 second blot time at 100 % humidity and 22°C. Data were acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K3 direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. The dose rate was adjusted to 15 counts/pixel/s, and each movie was fractionated in 75 frames of 40 ms. Automated data collection was carried out using Leginon at a nominal magnification of 105,000x with a pixel size of 0.843 Å in defocus range between −0.7 and −1.7 μm79 grid (200 mesh), plunge frozen using a vitrobot MarkIV (ThermoFisher Scientific) using a blot force of 0 and 6 second blot time at 100 % humidity and 22°C. Data were acquired using the SerialEM to control a Glacios transmission electron microscope equipped with a Gatan K3 Summit direct detector and operated at 200 kV. The dose rate was adjusted to 7.5 counts/pixel/s, and each movie was acquired in 100 frames of 50 ms with a defocus range between −0.7 and −1.7 μm and a pixel size of 0.89 Å81.To prepare the CryoEM grids of LayV F for solving postfusion state, 3 μL of 2.7 mg/ml LayV F wildtype (spontaneous refolded to postfusion over 3 months at 4°C) with 10mM OG detergent were applied onto a freshly glow discharged 2.0/2.0 UltraFoil79 grid (200 mesh), plunge frozen using a vitrobot MarkIV (ThermoFisher Scientific) using a blot force of 0 and 6 second blot time at 100 % humidity and 22°C. Data were acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K3 direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. The dose rate was adjusted to 15 counts/pixel/s, and each movie was fractionated in 75 frames of 40 ms. Automated data collection was carried out using SerialEM at a nominal magnification of 105,000x with a pixel size of 0.843 Å in defocus range between −1.3 and −1.7 μm81.To prepare the CryoEM grids of LayV F in complex with 4G5 Fab, 3 μL of 1 mg/ml LayV F wildtype (spontaneous refolded to postfusion over 4 months at 4°C) with 10mM OG detergent were applied onto a freshly glow discharged 2.0/2.0 UltraFoil79 grid (200 mesh), plunge frozen using a vitrobot MarkIV (ThermoFisher Scientific) using a blot force of −1 and 6 second blot time at 100% humidity and 23°C. Data were acquired using the Leginon software75 to control a Glacios transmission electron microscope equipped with a Gatan K3 Summit direct detector and operated at 200 kV. The dose rate was adjusted to 7.5 counts/pixel/s, and each movie was acquired in 100 frames of 50 ms. For LayV G with and without Fluorinated Octyl Maltoside detergent, 4894 and 4086 micrographs, respectively, were collected in a single session with a defocus range between −0.5 and −2.4 μm and a pixel size of 0.89 Å.To prepare the CryoEM grids of LayV G, 3 μL of 0.6 mg/mL LayV G or 2 mg/ml LayV G with 0.01% fluorinated octyl-maltoside (Anatrace) detergent were applied onto a freshly glow discharged 2.0/2.0 UltraFoil75.To prepare the CryoEM grids of GhV F I53–50A, 3 μL of the 0.05 mg/mL GhV F I53–50A were loaded onto holey carbon grids covered with graphene oxide, prior to plunge freezing using a Vitrobot MarkIV (ThermoFisher Scientific) with a blot force of −1 and 3 second blot time at 100 % humidity and 22°C. For the data set of GhV F, data were acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K3 direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. The dose rate was adjusted to 15 counts/pixel/s, and each movie was fractionated in 75 frames of 40 ms. Automated data collection was carried out using Leginon at a nominal magnification of 105,000x with a pixel size of 0.843 Å in defocus range between −0.5 and −2.5 μm76. Two rounds of reference-free 2D classification were performed using CryoSPARC76. 3D refinements were carried out using non-uniform refinement82 with reference map from previously solved HNV F.For structure of LayV F prefusion, movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking, and extraction were carried out using CryoSPARC76. Reference-free 2D classification was performed using CryoSPARC76. Ab initio structure reconstruction was performed in cryoSPARC using three classes76. Subset of good postfusion LayV F particles were used for homologous refinement followed by non-uniform refinement. Particles were extracted with original pixel size followed by another round of non-uniform refinement.For structure of LayV F postfusion, movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking, and extraction with 2X binning were carried out using CryoSPARC83. Two rounds of reference-free 2D classification were performed using CryoSPARC76. Heterogeneous refinement was used to sort good particles followed by homologous refinement and non-uniform refinement with per-particle defocus refinement in CryoSPARC82. Selected particle images were subjected to the Bayesian polishing procedure84 implemented in Relion before performing another round of homologous refinement and non-uniform refinement in cryoSPARC. Subsequently, local non-uniform refinements were performed for the proximal and distal parts of the complex yielding reconstructions at 3.6Å and 3.3Å resolution, respectively.For structure of LayV F postfusion in complex with 4G5 Fab, movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking, and extraction were carried out using Warp83, estimation of the microscope contrast-transfer function parameters, particle picking, and extraction (with a box size of 440 pixels2) was carried out in cryoSPARC76. Reference-free 2D classification was performed using cryoSPARC to select well-defined particle images. Ab initio structure reconstruction and non-uniform refinement in cryoSPARC were then performed. 3D classification with 50 iterations each  were carried out using Relion85 without imposing symmetry to select well-defined particle classes. Particle images were then subjected to Bayesian polishing using Relion84 during which the box size was adjusted to 440 Å with a pixel size of 0.89 Å. Another round of non-uniform refinement in cryoSPARC was performed concomitantly with global and per-particle defocus refinement as well as beam tilt refinement85. The particles from this refinement were used with 3DFlex59 in cryoSPARC to determine the motions of LayV G, using the default setting for Flex Data Prep, Flex Mesh Prep, Flex Train, and Flex Generate, with the exception that the training box size was set to 220 pixels.For the structure of LayV G, movie frame alignment and binning to 1.78 Å was carried out using Warp83. Two rounds of reference-free 2D classification were performed using CryoSPARC76 to select well-defined particle images. 3D refinements were carried out using non-uniform refinement82 along with per-particle defocus refinement in CryoSPARC. Selected particle images were subjected to the Bayesian polishing procedure84 implemented in Relion before performing another round of non-uniform refinement in cryoSPARC followed by per-particle defocus refinement. Local resolution estimation, filtering, and sharpening were carried out using CryoSPARC.For the structure of GhV F, movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking, and extraction were carried out using Warp87.Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) of 0.143 criterion and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution88. UCSF ChimeraX89 and Coot91 were used for docking and manually building into the cryoEM maps. All models were refined into the cryoEM maps using Rosetta94. The LayV G model was further refined with Phenix95 and ISOLDE96 Validation used MolProbity97, EMringer98, Phenix95 and Privateer99. Figures were generated using UCSF ChimeraX89 and UCSF Chimera100.Initial models of both prefusion and postfusion LayV F were built automatically using ModelAngeloSupplement 1


In addition, the WGS analyses showed that the nucleotide and amino acid similarities of the coding regions of the two strains obtained in this study with the prototype of the E5 strain Noyce were 80.2% and 97.2%, respectively. Recombination analysis showed that the two E5 strains may have intraspecific recombination with E6, EV-B85, and CVA9 serotypes in the nonstructural region. This study reports the isolation of E5 from SARI cases for the first time, and genetic characterization and evolutionary analysis provide valuable information on global E5 molecular epidemiology.Echovirus 5 (E5) belongs to the enterovirus B (EV-B) species, which is one of the pathogens causing respiratory diseases. Only two whole genome sequences (WGSs) of E5 are available in GenBank, and few studies on E5 have been reported worldwide. In this study, human rhabdomyosarcoma (RD) cells were used to isolate E5 from respiratory samples associated with severe acute respiratory infection (SARI). The nucleotide sequence of the VP1 region and the WGSs of two E5 strains were sequenced using Sanger sequencing and high-throughput sequencing technology, respectively. Together with 31 E5 VP1 sequences and 2 WGSs downloaded from GenBank, a total of 33 complete VP1 sequences and 4 WGSs were used for genetic characterization and evolutionary analysis. Phylogenetic analysis revealed five genotypes (A–E) based on complete VP1 sequences. Evolutionary analysis showed that the time to the most recent common ancestor of E5 was approximately 1952 , with a mean substitution rate of 7.74 × 10This study is the first report of echovirus 5 (E5) associated with severe acute respiratory infection and obtained the first E5 whole-genome sequence in China. Combined with the sequences available in the GenBank database, the first genotyping, phylogenetic characteristics, recombination, and genetic evolutionary analysis of E5 was performed in this study. Our findings providing valuable information on global E5 molecular epidemiology. Enterovirus and the family Picornaviridae. The International Committee on Taxonomy of Viruses (ICTV) classified EVs into four species (enterovirus A–D) based on nucleotide (nt) variability in the VP1 region (Enterovirus B (EV-B) species. The genome is approximately 7.5 kb and contains a long open reading frame (ORF) ﬂanked by a 5′ untranslated region (UTR) and a 3′UTR. The ORF is 6,588 nt and can be translated into 2,196 amino acids, which can be cleaved into three polyprotein precursors, P1, P2, and P3. P1 encodes four structural proteins, VP1–VP4, while P2 and P3 encode nonstructural proteins 2A–2C and 3A–3D, respectively (5––Human enteroviruses (EVs) are RNA viruses belonging to the genus tively 5––13. The ively 5––. Subsequively 5––, 16. Howively 5––, 18, andFour hundred pharyngeal swab samples from SARI patients in Luohe Central Hospital, Henan Province from October 2017 to May 2021 were tested, and two E5-positive samples were detected, of which one sample (SA18-334/E5i/Luohe-henan-China/2018) was successfully isolated and the other sample (SA19-378/E5s/Luohe-henan-China/2019) failed.The two E5 sequences obtained in this study together with the complete VP1 coding region of 31 global E5 sequences were used to construct a maximum likelihood (ML) phylogenetic tree. The results showed that the 33 sequences could be classified into five genotypes: A to E . The meaAnalysis of nucleotide polymorphisms in the complete VP1 region of 33 strains revealed that E5 had a nucleotide polymorphism Pi(Π) of 0.14, with a total of 373 mutation sites, mainly synonymous mutations (350). Meanwhile, 57 polymorphic sites were found, and S3T was the common amino acid mutation site compared to the prototype strain. Most E5 VP1 sequences belonging to genotypes C–E displayed specific amino acid substitutions. Genotype C sequences predominantly displayed amino acid changes at Q271E and Y292F/S. Genotype D contained two specific mutations, V11M and I48M, whereas genotype E contained six specific mutations, namely S71A, I268A, E274T, N288S, H290L, and Y292H .−3 subs/site/year , with a predicted time to most recent common ancestor (tMRCA) of 1952 . Genotype D was suggested to have diverged in approximately 1977 , and the tMRCAs of genotypes B, C, and E could be traced back to 1969 , 1998 , and 2014 (95% HPD 2013–2016), respectively.The maximum clade credibility (MCC) trees based on the 33 complete E5 VP1 sequences were generated using the Markov chain Monte Carlo (MCMC) method . The evode novo assembly results were 1118 bp and 877 bp, respectively. Using the contig splicing results as reference sequences, the numbers of reads mapped to the reference sequences were 18,057,493 and 4,313,464, respectively. Almost the complete genome  could be assembled by de novo with an average sequencing depth of 140,369× and 100% coverage  were downloaded from GenBank together with the two E5 strains obtained in this study to construct the VP1, P1, P2, and P3 regions and the complete genome phylogenomic tree . The phyBased on the results of the phylogenetic analysis, the sequences that were more similar to E5 were selected for recombination analysis using SimPlot . The resAccording to the U.S. National Enterovirus Surveillance System (NESS), from 1970 to 2005 E5 frequently observed in the 15 most common EVs, with a high rank of fourth in 1982. Children aged <1 year were the most common source for E5 detection, and CSF was the most common specimen. Fatal outcomes were reported in 3.8% of E5 infections with known outcomes . InformaAccurate classification of genotypes helps discover the differences among genotypes and further understand the genetic evolution of viruses. We determined five genotypes in this study according to the genotype classification criteria of EVs. Currently, the prevalent E5 strains found in China are mainly genotype E. The E5 strains obtained in this study were clustered with isolates from Jinan and Liaocheng in Shandong Province, China, suggesting that the Henan E5 strains may have originated in Shandong Province. Given that the VP1 region can specifically neutralize antigens and affect neurotoxicity sites  based on complete VP1 region sequences was used for EV serotyping.According to the manufacturer’s instructions, viral RNA was directly extracted from clinical specimens using the Tianlong RNA Extraction Kit , and samples were screened using real-time RT‒PCR . All thede novo assembled into contigs using CLC Genomics Workbench 22.0  with default parameters , and RNA quantification was performed using a Qubit 4 Fluorometer . Next-generation sequencing libraries were prepared using the VAHTS Universal V8 RNA-seq Library Prep Kit for MGI , and MiSeq sequencing using a 2 × 150 bp paired-end reads method was performed by Deep GenePlus-Shenzhen Clinical Laboratory. Clean reads from the viral database were rameters , 31. Con8 generations was implemented with a sampling frequency of 1 × 104 generations. The output from BEAST was analyzed using TRACER (v1.7.1). A MCC tree was generated using TreeAnnotator, and the results were subsequently visualized using FigTree (v1.4.3). Nucleotide and amino acid similarity analysis and polymorphism analysis were performed using BioEdit (v7.0.4.1) and DnaSP6, respectively , 39de no results . The seq


The pediatric genetic white matter disorders are characterized by a broad disease spectrum. Genetic testing is valuable in the diagnosis. However, there are few studies on the clinical and genetic spectrum of Chinese pediatric genetic white matter disorders.The participants were enrolled from the cohort of Peking Union Medical College Hospital. They all received history collection, brain MRI and gene sequencing. Their neurologic complaints which were related to white matter disorders occurred before 18. Brain MRI indicated periventricular and/or deep white matter lesions, fazekas grade 2–3.ABCD1 showed the greatest mutation frequency . The EIF2B3 A151fs, EIF2B4 c.885 + 2T > G, EIF2B5 R129X and MPV17 Q142X were novel pathogenic/likely pathogenic variants. 100% (4/4) ABCD1 carriers were accompanied by visual impairment, whereas 100% (3/3) EIF2B carriers developed dysuria. 100% (4/4) ABCD1 carriers exhibited diffuse white matter hyperintensities mainly in the posterior cortical regions, while the EIF2B4 and EIF2B5 carriers were accompanied by cystic degeneration.Among the 13 subjects, there were 11 males and two females. The average age of onset was 10.0 ± 5.5 years old. The potential genetic variants were found in 84.6% (11/13) subjects. The There is genotypic and phenotypic heterogeneity among Chinese subjects with pediatric genetic white matter disorders. The knowledge of these clinical and genetic characteristics facilitates an accurate diagnosis of these diseases.The online version contains supplementary material available at 10.1186/s13052-023-01555-z. The genetic white matter disorders are a group of heterogeneous diseases that predominantly affect the white matter of central nervous system. The clinical onset usually occurs in childhood. The motor impairment is the most prevalent, followed by cognitive deficit, behavioral abnormality, seizure, etc. . On MRI,According to Vanderver’s definition and classification, the genetic white matter disorders involve leukodystrophies and genetic leukoencephalopathies. Leukodystrophies include X-linked adrenoleukodystrophy, metachromatic leukodystrophy and Krabbe leukodystrophy, etc. And genetic leukoencephalopathies include Fabry disease, mitochondrial disorders, etc. .These diseases are heterogeneous and overlapping in phenotype. Some have characteristic enzymatic alterations, such as the elevated very long chain fatty acids (VLCFA) in X-linked adrenoleukodystrophy . Some doABCD1, EIF2B, PLP1, ARSA, MLC1, GALC, ASPA and GFAP genes, etc. [Many studies focused on the clinical and genetic spectrum of these diseases. In a UK pediatric cohort with leukodystrophies and genetic leukoencephalopathies (n = 803), the clinical spectrum involved mucopolysaccharidoses 12.5%, 100), GM1/GM2 gangliosidoses , metachromatic leukodystrophy 9.5%, 76), adrenoleukodystrophy , Krabbe leukodystrophy , etc. [%, 100, G, adrenolHowever, there are few studies on the clinical and genetic spectrum of Chinese pediatric genetic white matter disorders. This is a retrospective study from the cohort of Peking Union Medical College Hospital (PUMCH). Herein, we will describe the clinical and genetic characteristics of Chinese pediatric patients with genetic white matter disorders.The participants were enrolled from PUMCH cohort. The inclusion criteria were as following: ① Intact data on clinical history, brain MRI and gene sequencing. The MRI sequences included T1/T2 weighted, fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted images (DWI). ② The neurologic complaints which were related to white matter disorders occurred before 18. The neurologic symptoms or signs could be developmental delay, intellectual disability, speech difficulty, psychiatric disturbance, motor regression, gait ataxia, spasticity, hearing or visual loss, autonomic dysfunction, seizure, etc. ③ Brain MRI indicated periventricular and/or deep white matter lesions, fazekas grade 2–3. The white matter lesions presented with T2/FLAIR hyperintensity with T1 hyper-, iso- or hypointensity.The subjects were excluded if they were better explained by acquired white matter disorders due to infection, toxicity, ischemia with large or medium vessel occlusion, nutritional deficiency, trauma, neoplasm or systemic autoimmune disease.As illustrated in Supplement Fig. Three cases received whole exome sequencing. Ten subjects had targeted exome sequencing of 278 genes which were related to dementia and white matter disorders. The next-generation sequencing were performed on the Illumina platform . The pathogenicity of the variants was interpreted using the standards of American College of Medical Genetics and Genomics (ACMG) [APOE-ε4 non-carriers, and one was ε4ε4 genotype. The elder brother of Case 1 was diagnosed with adrenoleukodystrophy at eight and died at 10. As for the other 12 subjects, no similar clinical manifestations were shown among their first-degree or second-degree family members (Table As shown in Tables ABCD1 (n = 4), EIF2B3 (n = 2), EIF2B4 (n = 2), EIF2B5 (n = 2), ARSA (n = 2), GFAP (n = 1), NDUFS1 (n = 2) and MPV17 variants (n = 3) . Of them, the EIF2B3 E44K, EIF2B4 R446H and MPV17 K88M were reported before [ABCD1 G343D, EIF2B5 R211S, ARSA P150S, G81D, NDUFS1 V89E, I537L and MPV17 M89L were novel.According to the ACMG criteria, there were eight pathogenic or likely pathogenic variants. Of them, the d before –11. The ABCD1 variants. They all had elevated VLCFA. Three of them were accompanied by adrenocortical insufficiency. Brain MRI mainly showed bilateral posterior white matter hyperintensities (WMH) , folic acid, vitamin B12 and organic acids. The brain MRI mainly indicated bilateral WMH in the centrum semiovale and corona radiata  and A151fs  were found in a 17-year-old male. He presented with intellectual impairment and dysuria after a syncope at 16. The EIF2B4 R446H  and c.885 + 2T > G (de novo) were in a 29-year-old male. He presented with progressive ataxia since seven. He could run and climb, but fell frequently. After an accidental trauma at 28, he showed unsteadiness while standing. Gradually, he developed memory deficit, disorientation, dyscalculia, delusion, spastic tetraplegia and dysuria. The EIF2B5 R211S  and R129X (de novo) were detected in a 22-year-old female. She started with seizure attack at 12. Gradually, she showed poor academic performance at school, accompanied by spastic tetraplegia, dysarthria and dysuria. At 20, she could not vocalize, eat or move.The ARSA, GFAP, NDUFS1 and MPV17. They had normal serum VLCFA, HCY, folic acid, vitamin B12 and organic acids.Four subjects carried the variants in the ARSA P150S  and G81D  were found in a two-year-old boy. At the age of one, he was able to walk and call mom, dad. Four months later, he showed gait instability and dysarthria. Gradually he could not speak, eat or move. Physical examination indicated spastic tetraplegia. Brain MRI showed bilateral diffuse WMH in the centrum semiovale, corona radiata, splenium of corpus callosum with stripe-like pattern , followed by the EIF2B  and mitochondrial genes , which suggest the diagnosis of adrenoleukodystrophy, vanishing white matter disease and mitochondrial disease, respectively. These three diseases are also the main components of pediatric leukoencephalopathies in other countries. In a Finnish pediatric cohort with genetic white matter disorders, the most common diseases are mitochondrial  and adrenoleukodystrophy  [This is a group of subjects with pediatric genetic white matter disorders. The potential genetic variants are found in 84.6% (11/13) subjects. The al genes .4%, 2/13l genes 1.4%, 2/13EIF2B3 A151fs, EIF2B4 c.885 + 2T > G, EIF2B5 R129X, MPV17 Q142X are frameshift, splicing or stopgain variants, They are rare or absent in the 1000genome, ESP6500, GnomAD databases. The EIF2B4 c.885 + 2T > G and EIF2B5 R129X are de novo based on the pedigree analysis. The EIF2B3 A151fs and EIF2B5 R129X are determined as likely pathogenic in the Clinvar database (www.clinvar.com). Taken together, these variants are pathogenic/likely pathogenic according to the ACMG criteria.11 novel variants are found in this report. Of them, the ABCD1 G343D, EIF2B5 R211S, ARSA P150S and G81D, NDUFS1 V89E/I537L and MPV17 M89L. They are rare or absent in the 1000genome, ESP6500, GnomAD databases. They are deleterious from SIFT, Polyphen2 and Mutationtaster predictions. The ARSA P150S is novel, while the ARSA P150L is reported before [www.clinvar.com). The subjects harboring the ABCD1 G343D and the ARSA P150S/G81D demonstrate increased VLCFA and decreased Arylsulfatase A levels, respectively. Taken together, these variants are VUS according to the ACMG criteria.Seven novel VUS are detected, including the d before . The latThere are some common phenotypic features among the 13 subjects. For instance, 76.9% (10/13) cases are characterized by insidious onset and gradual progression. The most prevalent symptoms are cognitive impairment  and motor disorder 69.2%, 9/13). 100% (13/13) subjects have diffuse WMH in the supratentorial subcortical or periventricular regions. These are almost consistent with previous research [%, 9/13. EIF2B variant carriers exhibit a typical pattern of insidious onset and gradual progression. The EIF2B5 carrier presents with an acute onset of seizure attack. The EIF2B3 and EIF2B4 carriers experience a rapid progression of neurological regression following syncope and trauma. Previous reports reveal that patients with vanishing white matter diseases can worsen rapidly under stress [Moreover, the subjects with different gene mutations have some characteristic phenotypes. For instance, none of the three ABCD1 carriers are accompanied by visual impairment. This might be related to their occipital involvement. The MPV17 carrier has peripheral neuropathy. El-Hattab pointed out that peripheral neuropathy occurred in 18.7% (17/91) MPV17 carriers [EIF2B carriers develop dysuria. Kar found that the mRNAs encoding the EIF2B2 were present in the axons of rat sympathetic neurons [EIF2B genes might exert effects on autonomic nerves.In terms of clinical symptom, 100% (4/4) ABCD1 carriers exhibit diffuse WMH mainly in the posterior cortical regions. The EIF2B4 and EIF2B5 carriers are accompanied by cystic degeneration. The ARSA carrier shows stripe-like pattern, indicating remaining tissue strands within the WMH. The T2 hyperintensity of the MPV17 carrier is primarily limited in the subcortical regions. These are similar to previous findings [On brain MRI, 100% 4/4) /4 ABCD1 findings , 20. HowNDUFS1 carrier presents with motor development delay, intellectual disability, seizure with diffuse cerebral and cerebellar WMH. According to Björkman’s finding, these clinical and imaging features are common in the NDUFS1 carriers except for the cerebellar involvement [NDUFS1 gene and cerebellum could be further investigated. Fortunately, the MPV17 carrier has a late onset without hepatic impairment. El-Hattab summarized the clinical characteristics of 100 MPV17 mutation carriers. He found that almost 96% individuals had an poor prognosis during infancy or early childhood due to hepatic failure. However, 4% subjects with later onset during late childhood or adulthood had no or minimal liver injury [There are two subjects carrying the variants related to mitochondrial diseases. The olvement . The assABCD1, EIF2B and mitochondrial genes have high mutation frequencies. The subjects with different gene mutations exhibit characteristic manifestations, which have suggestive implications for the underlying genetic basis. The knowledge of these clinical and genetic characteristics facilitates an accurate diagnosis of these diseases. The functional verification of the novel variants should be performed in the following studies.There is genotypic and phenotypic heterogeneity among Chinese pediatric genetic white matter disorders. The Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Cancer cachexia, occurring in ~ 80% pancreatic cancer (PC) patients overall, is a paraneoplastic syndrome mediated by cancer-induced systemic inflammation and characterized by weight loss and skeletal muscle wasting. Identifying clinically relevant PC-derived pro-inflammatory factors with cachexigenic potential may provide novel insights and therapeutic strategies.Pro-inflammatory factors with cachexigenic potential in PC were identified by bioinformatic analysis. The abilities of selected candidate factors in inducing skeletal muscle atrophy were investigated. Expression levels of candidate factors in tumors and sera was compared between PC patients with and without cachexia. Associations between serum levels of the candidates and weight loss were assessed in PC patients.S100A8, S100A9, and S100A8/A9 were identified and shown to induce C2C12 myotube atrophy. Tumors of PC patients with cachexia had markedly elevated expression of S100A8 (P = 0.003) and S100A9 (P < 0.001). PC patients with cachexia had significantly higher serum levels of S100A8, S100A9 and S100A8/A9. Serum levels of these factors positively correlated with percentage of weight loss  and independently predicted the occurrence of cachexia .Atrophic effects of S100A8, S100A9, and S100A8/A9 indicated them as potential pathogenic factors of PC-induced cachexia. In addition, the correlation with the degree of weight loss and prediction of cachexia in PC patients implicated their potential utility in the diagnosis of PC-induced cachexia.The online version contains supplementary material available at 10.1186/s12885-023-11009-8. Pancreatic cancer (PC) is the 7th leading cancer deaths worldwide and projected to become the second leading cancer deaths in US by 2030 , 2. BesiCancer cachexia is a paraneoplastic syndrome mediated by cancer-induced systemic inflammation and characterized by refractory weight loss and skeletal muscle wasting , 7. NotaIn this study, we utilized bioinformatic approach to discover PC-derived pro-inflammatory factors with cachexigenic potential. The abilities of the selected candidate secreted proteins in inducing skeletal muscle atrophy were investigated. To ascertain the clinical relevance of those factors, expression of the potential cachexigenic factors was compared between patients with and without cachexia at the time of PC diagnosis, and the associations between those factors and the risk of cachexia and cachexia-related parameters were further analyzed in PC patients.http://gepia.cancer-pku.cn)  + [(% of 1+) x 1] + [(% of 2+) x 2] + [(% of 3+) x 3] .Normally distributed variables were summarized as We first assessed whether the CM of MIA PaCa-2 cells could induce atrophy of skeletal muscle cells. As shown in Fig. To determine whether the selected candidate proteins can induce cachexia, we assessed their effects on skeletal muscle cells in vitro. Treatment with S100A8 and S100A9 significantly reduced the diameter of C2C12 myotubes in all indicated concentrations  showed markedly higher expression of S100A8 (P = 0.003) and S100A9 (P < 0.001) compared with tumors of PC patients without cachexia  had cachexia at cancer diagnosis. The prevalence of cachexia was 50% in stage I, 59.4% in stage II, 57.5% in stage III, and 54.4% in stage IV, without significant differences across stages  1.11 , 1.02–1.21; P = 0.014) per 1 ng/mL increase in serum S100A8, 1.10  per 1 ng/mL increase in serum S100A9, 1.04  per 1 µg/mL increase in serum S100A8/A9]  and PC-induced cachexia are distinct PC-induced aberration of host metabolism to supply nutrients such as amino acids and glucose for tumor growth . We prevBesides collecting information on weight loss, we also accurately measured skeletal muscle mass in the study, enabling accurate determination of the cachexia status in PC patients. We focused on cachexia at the time of diagnosis which was mainly attributed to PC-derived cachexigenic factors, reducing confounding by factors that may induce or exacerbate cachexia later in the disease course, such as anti-cancer treatment and gastrointestinal obstruction. However, information on skeletal muscle mass before the onset of PC was not available; therefore, we could not examine whether blood levels of the identified cachexigenic factors were also correlated with the degree of muscle mass loss. Given that measurement of skeletal muscle mass is seldom performed for healthy individuals, information on usual muscle mass is unlikely to be available. However, we did note that blood levels of the identified cachexigenic factors positively correlated with the degree of weight loss for which muscle wasting accounts a major portion in cancer cachexia , 9, suggIn conclusion, in vitro atrophic effects of S100A8, S100A9, and S100A8/A9 suggest their potential as pathogenic factors of PC-induced cachexia. Moreover, the positive correlation with the degree of weight loss and their ability to predict cachexia in PC patients using clinical specimens imply their potential as diagnostic markers for PC-induced cachexia. Further exploration of the significance and application of S100A8, S100A9 and S100A8/A9, as well as elucidation of their mechanistic roles, could make a promising contribution to the advances of PC detection and clinical management.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Penaeus monodon (PmFortilin) and Fortilin Binding Protein 1 (FBP1) have recently been shown to interact and to offer protection against the widespread White Spot Syndrome Virus infection. However, the mechanism is yet unknown. We investigated this interaction in detail by a number of in silico and in vitro analyses, including prediction of a binding site between PmFortilin/FBP1 and docking simulations. The basis of the modeling analyses was well-conserved PmFortilin orthologs, containing a Ca2+-binding domain at residues 76–110 representing a section of the helical domain, the translationally controlled tumor protein signature 1 and 2  at residues 45–55 and 123–145, respectively. We found the pairs Cys59 and Cys76 formed a disulfide bond in the C-terminus of FBP1, which is a common structural feature in many exported proteins and the “x–G–K–K” pattern of the amidation site at the end of the C-terminus. This coincided with our previous work, where we found the “x–P–P–x” patterns of an antiviral peptide also to be located in the C-terminus of FBP1. The combined bioinformatics and in vitro results indicate that FBP1 is a transmembrane protein and FBP1 interact with N-terminal region of PmFortilin.The Fortilin  in  A WSSV infection typically causes lethargic behavior, cessation of feeding, a pink to reddish-brown discoloration, and white inclusion of calcium salts embedded in the cuticle especially inside the surface of the carapace. The moribund shrimp swim near the surface at the edge of ponds. The populations of shrimp showing these signs have high mortality rates with cumulative mortalities reaching up to 100% within three to ten days. The transmission of disease is typically via cannibalism of the sick or dying prawns and directly through contaminated water. The virus can persist and retain infectivity in seawater for four to seven days. The histopathology of WSSV infected shrimp shows severe nuclear hypertrophy, chromatin margination, and eosinophilic to large basophilic intranuclear inclusions with variable multifocal necrosis in most tissues of ectodermal and mesodermal origin Penaeus monodon, Penaeus indicus, and Penaeus penicillatus. Type II is latent.The White Spot Syndrome Virus (WSSV) is a common pathogen that causes significant production losses in the shrimp industry around the world. The virus has a wide host range in a variety of crustaceans, both freshwater and seawater species, including Pacifastacus leniusculusPenaeus monodonMarsupenaeus japonicasBased on the morphology and the genomic structures, WSSV has been classified to the genus Whispovirus and the family Nimaviridae, The virions are enveloped, ovoid to bacilliform 276±26 nm long with a diameter of 121±9 nm, containing double-stranded circular DNA with the size ranging from 290 kbp to 305 kbp, which covers approximately 185 open reading frames, ORFs Over the years, a lot of effort has been put into attempts to protect against WSSV infection in shrimp, such as the usage of random library peptides 2+2+ levels from increasing and activating Ca2+-dependent apoptosis pathways −ATPase and regulates the function of Na, K−ATPase in cooperation with Nexin6 (SNX6) Fortilin  has attracted a lot of attention due to its implication in a variety of functions, i.e., control of cell cycle, cell growth and cell division Penaeus monodon, named PmFortilin (Accession No: AY186580.1) and shown it to have anti-apoptotic properties. This protein is well conserved, contains a Ca2+-binding domain, TCTP signatures similar to that of the Fortilin or TCTP genes reported in plants and animals P. monodon and based on a yeast two-hybrid screening assay and GST-pulldown, we further found Fortilin to interact with a previously unknown protein, named Fortilin Binding Protein 1 (FBP1) (Accession No: EU435133.1) PmFortilin with FBP1 and its function. From our results we suggest that FBP1 is a transmembrane protein and its likely function is to facilitate transport of PmFortilin across the cell membrane.Our group has previously identified Fortilin in PmFortilin or FBP1. For this reason, we have predicted the structures by homology modeling. The best scoring 3-D structure of PmFortilin contains three sheets, six beta hairpins, two beta bulges, three helices and one helix-helix interaction. The model displays one helical domain, stretching from residue 77 to 126 and one right handed hook (RHH) of the disulphide type  residues were in the most favored regions, additional allowed regions contained 33 (22.10%) residues, generously allowed regions contained seven (4.70%) residues and disallowed regions contained only a single (0.70%) residue  mutant E12V, the chain A with the crystal structure of translationally controlled tumor associated protein from Plasmodium knowlesi and with the Fortilin structure of Drosophila melanogaster . A further comparison of multiple sequences reveals that PmFortilin contains conserved regions that correspond to the helical domain in the predicted 3-D structure. The amino acid residues 76–110, part of the helical domain of PmFortilin are contained within a Ca2+-binding domain, forming a helical domain between the H2-helix (residues 81–99) and the H3-helix (residues 106–122) producing an EF-hand structure (PmFortilin contains two signature patterns. The TCTP_1 and TCTP_2 at the residues 45–55 and 123–145 with the signatures “[IFAED]–[GA]–[GASF]–N–[PAK]–S–[GTA]–E–[GDEVCF]–[PAGEQV]–[DEQGAV]” and “[FLIV]–x(4)–[FLVH]–[FY]–[MIVCT]–G–E–x–[DENP]–[GAST]–x–[LIVM]–[GAVI]–x(3)–[FYWQ]”, respectively (tructure . Importaectively .http://myhits.isb-sib.ch/cgi-bin/motif_scan) to detect signatures. The search revealed some interesting signatures: Amino acid residues 4–6 at the N–terminal represented by “[ST]–x–[RK]” pattern at protein kinase C phosphorylation site and by “x–G–[RK]–[RK]”, which is a known amidation pattern, were found to be located at the residues 90–93 at the C-terminal end of FBP1 (http://smart.embl-heidelberg.de) . It was of some interest that FBP1 contains a number of “x–P–P–x” signature sequences found in antiviral peptides. The longest such pattern is located at residues 59–76 http://agknapp.chemie.fu-berlin.de/gplus)   and S2, e/gplus) .http://www.enzim.hu/hmmtop) and TMHMM2 servers (http://www.cbs.dtu.dk/services/TMHMM). According to these predictions, the helix residues 7–26 are transmembrane segments , including the PIPER docking software based on the Fast Fourier Transform (FTT) method PmFortilin as a ligand. The models were predicted using four separate modes: Balance, Electrostatic, Hydrophobic and VdW+Elec mode. The short interacting elements were extracted from the best scoring interaction complex and collected to produce a refined model –, 5.26 and 5.60 for (PmFortilin_Asn48)– and 5.51 for the interaction of (PmFortilin_Ala51)–(FBP1_Pro79). (2) The predicted Ca2+-binding domain (PmFortilin_Thr77)–(FBP1_Pro48), 4.72 and 4.70 for (PmFortilin_Gly78)–. (3) The TCTP_2 on the C-terminal of PmFortilin (PmFortilin_Gln127)–(FBP1_Ala51), 9.96 for (PmFortilin_Phe128)–(FBP1_Pro48), 9.74 for (PmFortilin_Phe129)–(FBP1_Pro48), and 11.59 for (PmFortilin_Met134)–(FBP1_Pro44), according to the balance mode (These interactions coincide with three domains: (1) the TCTP_1 on the flexible loop of nce mode .PmFortilin-FBP1. For this reason, we performed additional docking simulations, this time simultaneously using two FBP1s as receptors. In the trimer structure, the Ca2+-binding domains and the flexible loop of the PmFortilin structure interacted as in the dimer (FBP1-PmFortilin) simulations. The trimer docking simulation based on the balance mode gave the lowest docking energy score of −1,162.00 Kcal/mol and the highest −888.70 Kcal/mol, the average of docking energy being −986.43 Kcal/mol , PmFT2 (residues 71–120) and PmFT3 (residues 121–168). The interaction of FBP1 with each of the three fragments was tested by the yeast two-hybrid assay. The yeast growth in YPDA was monitored to ensure that there were no growth defects in all recombinant clones  and PROCHECK software. The 3-D template structures were brought from the PDB database (http://swissmodel.expasy.org).The homology modeling for the 2-D and 3-D structures were performed with SWISS-MODEL PmFortilin and FBP1. Motif Scan PmFortilin and FBP1 with the default parameters, including outlier homologs and homologs of known structures, Pfam domains, signal peptides, internal repeats and intrinsic protein disorder databases.The GANGSTA and GANGSTA+ The ProFunc server was used to identify the potential functions within the 3-D structures. The HMMTOP http://www.bioslax.com).The molecular interaction and docking simulations were performed using the ClusPro 2.0 server 5′-GCTAGCATGAAGTTCTCATGT; containing NheI site and reverse primer FBP1-R: 5′-CCCGGGCTTCTTGCCCTTACT; containing the XmaI site without the stop codon at its C-terminal. The fragments were blunt-end inserted into phMGFP amplified by transformation into the high efficiency Escherichia coli Top10F'competent (Invitrogen) by the heat shock method. The plasmids were extracted and purified with QIAprep Spin Miniprep Kit (Qiagen). The Sf9 cells were seeded at a density of 5×105 cells onto the cover slips in Sf9-S2 medium (PPA). The phMGFP and phMGFP-FBP1 plasmids were transfected into Sf9 cells using Transfast™ Transfection reagent (Invitrogen) according to the product instructions and cultured in a 28°C incubator. After 48 h of transfection, the cells were washed by PBS twice. The plasma membrane was stained with 2 mM DiI (Invitrogen) for 15 min at 4°C and the Sf9 cells were observed directly with the confocal laser scanning microscope.FBP1 was cloned into the phMGFP vector  to generate an FBP1-GFP fusion protein. PCR was performed with forward primer FBP1-F: 6 cells, were grown on glass coverslips in Sf9-S2 medium (PPA), then cotransfected with phMGFP-FBP1 and pCDNA-PmFortilin using Transfast™ Transfection reagent (Invitrogen) according to the product instructions. Cells were cultured in a 28°C incubator. 48 h post-transfection, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min followed by washing with 0.1% Triton x-100 in PBS for 3 min at 4°C. Fixed cells were washed again with PBS before blocking with 5% skim milk at 4°C for 1 h. After incubating, cells were washed and incubated with specific rabbit anti-PmFortilin antibody (dilution of 1∶500 in PBS) at 4°C for 16–18 h. The cells were washed 3 times with PBST and incubated with Alexa flour 647-conjugated goat anti-rabbit IgG antibody  at room temperature for 2 h. After washing 3 times with PBST, the cover glasses were mounted with 10% glycerol in PBS. The fluorescence signal was detected using a confocal laser scanning microscope (Olympus FV300).Sf9 cells, at a density of 5×10PmFortilin was divided into 3 fragments: PmFT1 (residues 1–70), PmFT2 (residues 71–120) and PmFT3 (residues 121–168). Plasmids pGBKT7 (named BD vectors) were used for construction of BD-PmFortilin. The plasmids were constructed by inserting into EcoRI and SalI sites on a BD-bait vector. Each fragment was amplified by PCR using the following primers;The yeast two-hybrid screens were performed with the Clontech Matchmaker GAL4 Two-Hybrid System 3 . PmFT1:BD-PmFT1-F: 5′-CGGAATTCATGAAGGTCTTCAAG,BD-PmFT1-R: 5′-GCGTCGACTCATATAACTACATCAACACBD-PmFT2:BD-PmFT2-F: 5′-GGAATTCTATATGCGTCTGCAGGAAAC,BD-PmFT2-R: 5′-GCGTCGACTCACAAAAGGTCTGTCABD-PmFT3:and BD-PmFT3-F: 5′-CGGAATTCAAGAAGTTCAAGGACTTGCA,BD-PmFT3-R: 5′-GCGTCGACTTATAGCTTCTCCTCTGT.BD-BamHI and XhoI sites;The plasmid pGADT7 (named AD vector) was used for construction of the AD-FBP1 and contained the AD-FBP1:5′-GGATCCCGATGAAGTTCTCATGTAAAGT,AD-FBP1-F: 5′-CCCTCGAGTTACTTCTTGCCCTTACTAT.AD-FBP1-R: Saccharomyces cerevisiae strain AH109 were co-transformed either with BD-PmFT1 and AD-FBP1, BD-PmFT2 and AD-FBP1, BD-PmFT3 and AD-FBP1. Cells were then spread on SD selective medium lacking tryptophan, leucine, histidine and adenine (SD-TLHA). The plates were incubated at 30°C until positive colonies were visible after 2–4 days. The transformations were confirmed by PCR. The positive clones were in addition assayed by a filter lift β-galactosidase assay for LacZ activity as described in the manufacturer's protocols.All plasmids constructed were verified by sequencing. Competent yeast cells, Figure S1Penaeus monodon. The number of residues in most favored regions  is 108 (72.50%), the number of residues in additional allowed regions  is 33 (22.10%), the number of residues in generously allowed regions  is 7 (4.70%) and one (0.70%) residue in disallowed regions. The number of non-glycine and non-proline residues is 149 (88.69%), the number of end-residues (excl. Gly and Pro) is 2 (1.19%), the number of glycine residues (shown as triangles) is 11 (6.55%) and the number of proline residues is 6 (3.57%).The ramachandran analysis of the Fortilin model from (TIF)Click here for additional data file.Figure S2The ramachandran analysis of the FBP1 model. The number of residues in most favoured regions  is 31 (49.20%), the number of residues in additional allowed regions  is 21 (33.30%), the number of residues in generously allowed regions  is 6 (9.50%), the number of residues in disallowed regions is 5 (7.90%). The number of non-glycine and non-proline residues is 63 (67.74%), the number of end-residues (excl. Gly and Pro) is 2 (2.15%), the number of glycine residues (shown as triangles) is 3 (3.23%) and the number of proline residues is 25 (26.88%).(TIF)Click here for additional data file.Figure S3PmFortilin/FBP1 interaction model, based on balance mode, FBP1 as the receptor and PmFortilin as the ligand. (A) The simulation results of the FBP1 monomer combined with two PmFortilin molecules (1∶2), yields the lowest docking energy of −913.00 Kcal/mol. (B) FBP1 monomer combined with three PmFortilins (1∶3), yields the lowest docking energy of −1,244.80 Kcal/mol. (C) Two FBP1 molecules combined with two PmFortilins (2∶2), yields the lowest docking energy of −1,015.80 Kcal/mol. (D) Two FBP1 molecules and three PmFortilins (2∶3), yields the lowest docking energy of −1,015.80 Kcal/mol. The Ca2+-binding domain orange color and the TCTP signature 1 of the flexible loop is green.Multimer simulations of (TIF)Click here for additional data file.Table S1PmFortilin and FBP1.The SMART and Motif Scan analysis for (DOCX)Click here for additional data file.Table S2The SMART analysis result of FBP1 on the SMART database.(DOCX)Click here for additional data file.Table S3The 3D structure alignment of FBP1 on ASTRAL40 database (version 1.75).(DOCX)Click here for additional data file.Table S4PmFortilin/FBP1.List of the balance mode docking simulation of (DOCX)Click here for additional data file.Table S5PmFortilin/FBP1.List of the electrostatic mode docking simulation of (DOCX)Click here for additional data file.Table S6PmFortilin/FBP1.List of the hydrophobic mode docking simulation of (DOCX)Click here for additional data file.Table S7PmFortilin/FBP1.List of the VdW+Elec mode docking simulation of (DOCX)Click here for additional data file.


Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral ‘seeds’ for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six ‘internal’ genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth The genomes of 14 wt parental viruses, 23 HYRs  and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry.Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA.in ovo may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine ‘seed’ viruses.In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth  OrthomyxoviridaeInfluenza A viruses (IAV) are single stranded, negative sense RNA viruses belonging to the family Vaccination is the primary method of influenza prevention and control in ovo, namely a wt virus and a hy donor virus (A/Puerto Rico/8/1934 (PR8) or an H3N2 HYR with six PR8 genes encoding the ‘internal’ proteins). HYRs must contain genes for the two surface glycoproteins, HA and NA, of the wt virus and one or more of the ‘internal’ genes from PR8 or its derivatives. The classical reassortment method is based on the selection of HYRs from a potential pool of 254 reassortants  via negative selection using antibodies and/or antisera against the hy donor HA and NA; thus reducing the pool to 64 candidates in ovo, out-replicating lower growing reassortants. HYR candidates are then cloned by limiting dilution Currently there are two techniques to generate HYRs or HGRs (High Growth Reassortants); the ‘classical’ reassortment method and the reverse genetics approach. In the classical method, HYRs are generated by co-inoculation of two viruses Most genetic characterization studies of influenza wt and reassortant viruses have focused on the importance of HA coding changes in ovo occurring in the genes for the surface glycoproteins and/or the internal (non HA and non NA) proteins in ovo, the complete genomes of a total of 23 HYRs prepared as candidate vaccine seeds between 1957 and 2009, from 14 different wt viruses belonging to H1N1, 1976 H1N1-SOIV, 2009 H1N1pdm, H2N2 and H3N2 subtypes were sequenced and analyzed. The mutations observed in this analysis will be of considerable interest to influenza vaccine researchers and can be applied to modification of the sequences of the wt virus and/or the hy donor virus, PR8, during the development of influenza A vaccine candidate viruses with improved growth properties in ovo.Our laboratory at New York Medical College (NYMC) specializes in producing HYR seed viruses for both seasonal and pandemic influenza vaccines by the classical reassortment method. Earlier, we reported the gene constellation analysis of a panel of HYRs prepared as candidate vaccine viruses and demonstrated that the gene ratio and gene constellation yielding a reassortant with hy phenotype is dependent on the individual wt virus and its interaction with the hy donor virus HYRs designated with an X, for example, X-31b were developed by E.D. Kilbourne and his associates; HYRs designated as NYMC X-  was used to generate H3N2 HYRs. To develop the H1N1 HYRs, H3N2 hy reassortants were used to have a clear antigenic distinction between wt (H1N1) and donor surface glycoproteins (H3N2) to facilitate antibody selection during reassortment. Hy H3N2 donor viruses included X-31b  and NYMC X-157 . In the case of the 1976 swine influenza vaccine, PR8 was used to generate the 1976 H1N1-SOIV reassortants X-53 and X-53a.The HYRs with more than one in the same series  are derived from independent co-infection experiments performed in parallel using the same wt viruses. A total of 38 viruses including 23 HYRs, 14 wt viruses and the hy donor virus PR8 were sequenced and analyzed.The quality of the extracted RNA for sequencing purposes was checked by amplifying the M gene of all 38 samples by RT-PCR. Viral RNA was isolated from 280 µl of allantoic fluid using QIAmp® Viral RNA Mini Kit  per the manufacturer’s recommendations. RNA preparations were stored at −20°C until further use. RT-PCR was carried out using Takara One Step RNA PCR Kit  according to the manufacturer’s recommendations. The RT-PCR reaction mixture and the thermal conditions used were as previously published The complete genome sequencing of the 38 RNA preparations was carried out at J. Craig Venter Institute  under the NIAID Influenza Genome Sequencing Project (NIGSP). Whole-genome sequencing of isolates was performed using the high-throughput sequencing pipeline. The protocol was as follows. Briefly described, an M13 sequence tag was added to the 5′ end of each degenerate primer used for RT-PCR. Primers were designed to produce approximately 500-nucleotide (nt) overlapping amplicons and provide 2X coverage of each genomic segment. Each amplicon pair overlaps with its neighboring amplicon by approximately 100 nt. Additionally a second set of primers was designed to produce 500-nt amplicons offset by about 250 nt from the original primer pair, providing at least 4X sequence coverage of each segment. Primers were arranged in a 96-well plate format; all RT-PCRs for each sample were performed in one plate. Genomic RNA was amplified directly by RT-PCR and sequenced. Ninety-six RT-PCRs reactions were performed per RNA sample using a One-Step RT-PCR kit . Sequencing reactions were performed using Big Dye Terminator chemistry  with 2 µl of template cDNA. Each amplicon was sequenced from each end using M13 primers, and sequencing reactions were analyzed on a 3730 ABI sequencer .The sequences as determined for our laboratory PR8 internal gene sequences had nucleotides missing at both ends. Therefore, the PR8 gene sequences were compared with the PR8 sequences from Schickli et al The complete genome sequence of all 23 HYRs, 14 wt viruses and PR8 were submitted to GenBank via JCVI. The GenBank accession numbers of influenza A wt viruses and HYRs analyzed in this study are given as supporting information . The seqStatistical significance of the nucleotide changes in terms of fold increase in HA titer was analyzed using Student’s t-test. A value of p<0.05 was considered statistically significant.in ovo. The frequency of origin of ‘internal’ genes in the HYRs is shown in All HYRs mentioned in this study were generated by classical reassortment H1N1 HYRs were generated using an H3N2 hy donor virus, either X-31b or X-157 for delivery of PR8 internal genes during reassortment of HYRs .  developed for 2009 H1N1pdm, no silent changes were seen; X-179 had two coding changes in PA, Gln564Leu and Glu621Lys.All four HYRs obtained the PR8 NP gene. X-179A had the coding change, Ile131Met; this change was also found in the H1N1 X-163 series .No nt changes were seen in the PR8 derived M gene for any of the four H1N1-SOIV HYRs.All four HYRs derived the NS gene from PR8. Both X-53 and X-53a had the recurrent silent change, T144C in NS1  and 6. XTwo H2N2 HYRs were developed using hy donor virus PR8 . PR8xKO/X-135 had a silent change C1540A in PR8 PB2 gene. No change was seen in wt PB2 gene of PR8xKO/68 HYR.Both H2N2 HYRs derived wt PB1 and had no change.Both H2N2 HYRs derived PR8 PA; PR8xKO/68 HYR had a silent change A1218T; X-135 had a coding change, Asn578Asp.Both H2N2 HYRs had wt NP gene; only PR8xKO/68 had a coding change, Tyr76Cys.No silent or coding changes were found in M genes for either HYR.Both HYRs derived PR8 NS. X-135 had three silent changes A123G, C147T and G549A and a coding change Glu55Lys in NS1. In addition X-135 also had a coding change Gly26Glu in NS2. PR8xKO/68 had no changes.Twelve H3N2 HYRs were sequenced and compared with parental sequences; all were generated using the H1N1 hy donor virus PR8 .Nine H3N2 HYRs obtained the PB2 gene from PR8. Of which, seven HYRs had silent changes, including a recurrent silent change, A49C in X-31b, X-117 and X-157; C1827T in X-157A, X171A, X-171B, and A2025G in X-157A and X171A. Non-recurrent changes include, G594A, G1128A, G1950A, C2073T and G2307A (X-171B), G1056A and A1953G (X-117), C1068A (X-157B) . A codinThe PR8 derived PB1 for X-157 had a single silent change G243T, while the PB1 gene derived from wt virus (NY/04) for X-157 was found to have 13 silent changes . Six H3NNine of the 12 H3N2 HYRs had the PR8 PA gene. A silent change, G1504A was seen in the PR8 PA gene of X-157A. A total of four coding changes were found in the PR8 PA; one in X-157B  and three in X-175C . The remaining HYRs had no changes in the PA gene regardless of parental origin .Ten HYRs obtained the PR8 NP gene. Three HYRs with PR8 NP had silent changes, X-149C6 and X-157B had the same changes, G210A, C369T and G924A. X-117 had three silent changes, C369T, C426T and G924A. These recurrent silent changes are not found in HYRs of other subtypes. Coding changes, Gly117Arg and Lys46Glu, were seen in X-171 and X-175C, respectively. No changes were found in seven H3N2 HYRs whether the NP gene was derived from PR8 or wt.No silent or coding changes were found for the PR8 derived M gene in any of the 12 H3N2 HYRs.Ten H3N2 HYRs contained the PR8 NS gene and each had at least one change. Six HYRs, X-157, X-157A, X-157B, X-161B, X-171B and X-175C had a recurrent silent change, T144C in NS1. Another group of four HYRs, X-31b, X-117, X-149C6 and X-171 had recurrent silent changes C147T and G549A in NS1 and a recurrent coding change Gly26Glu in NS2  and 8. FA total of 10 HYRs derived PB1 from PR8 and retained the 87 aa long PB1-F2. The HIN1 HYR, X-127 with BJ/95 PB1 had a truncated 57aa long PB1-F2. The H1N1 SOIV HYRs, X-53a had truncated 52 aa long PB1-F2; X-179 and X-179A had a truncated PB1-F2 at 12 aa. The H2N2 HYRs (X-135 and PR8xKO/68) and five out of 12 H3N2 HYRs  with wt derived PB1 had a PB1-F2 protein size of 90 aa. X-157 which was a mixture had PB1 from both PR8 and wt viruses  had PB1-F2 proteins of 87 and 90 aa, respectively. The HYRs deriving PB1 (encoding PB1-F2) from PR8 (except X-117 and X-163 series) had a single recurrent silent nt change, A57G. Whereas, the X-163 series of HYRs had a recurrent coding change, Cys42Phe, which is the only coding change in the PB1 gene associated with PB1-F2.The HYR seed viruses must derive HA and NA genes from their respective wt viruses. Among the HYRs analyzed, three silent changes and 30 coding changes were found in the HA genes , 3 and 4A silent nt change was found in X-53 (G525A). X-53 and X-127, each had two coding changes in the HA; Glu153Lys and Ala178Thr (X-53), and Arg202Gly and Asp203Val (X-127). X-113 had a single coding change, Gly239Asp. X-53a had a coding change, Gly155Glu which was also found in X-179 (a 2009 H1N1pdm HYR). In addition, X-179 and X-179A shared three coding changes, Lys209Thr, Gly/Asp222Asp and Gln223Arg.The Lys209Thr change seen in X-179 and X-179A was also found in an H2N2 HYR (PR8xKO/68) .X-171A had two silent nt changes, C54T and A198G. Coding changes were found in the X-157 series of HYRs including the same recurrent coding change, Ser219Tyr which was also found in X-171B . X-157B Four coding changes were found in the HA2 region, Lys401Asn ; Arg487Thr and Val510Gly  and Asp489Asn  in the amino terminus of the HA2. These changes are unique to this study.www.pymol.org)  .In our study, no silent nt changes were found in the NA gene for any of the 23 HYRs. In contrast to HA with 31 coding changes, only two HYRs had coding changes in the NA genes, both were N2 subtype, Thr31Ala in X-171A (H3N2) and Thr32Ile in X-175C (H3N2). Both are located in the amino terminal end which anchors NA in the viral envelope.Both recurrent and individual silent changes were observed in all the internal genes of the HYRs derived from PR8 except the M gene. The A49C change found in the PB2 gene of six HYRs  and 6 isThe statistical significance of the recurrent changes was determined using Student’s ‘t’ test and found the recurrent coding changes in NS1 (p<0.0331), NS2 (p<0.0012), NP (p<0.026) and PB1-F2 (p<0.0075)  are statOut of 23 HYRs analyzed in this study, the mutations found in 9 HYR vaccine seed viruses used in vaccine preparation since X-31b are given in The first inactivated IAV vaccine was produced in chicken eggs in the 1940s using wt virus. Since clinical isolates grow to low titer in embryonated chicken eggs, influenza HYRs were developed by E.D. Kilbourne for use as the ‘seed’ virus for influenza vaccines and continue to be used for vaccine production to the present. The hy property of reassortants depends on the natural selection of variants with mutations favorable to growth. HYRs which derive PR8 ‘internal’ genes generally grow better than the original wt virus. However, the egg adaptive changes which occur in the internal genes of the HYRs are not well defined. Hence, in this study, the complete genome sequences of 23 HYRs developed from 14 different wt viruses and the hy donor PR8 were analyzed.in ovo. The only coding change in PB2, Pro121Ser, was found in X-157B and this change may alter secondary structure.The polymerase complex of influenza virus is comprised of PB2, PB1 and PA. The PB2 gene is one of the major components of the replication complex. HYRs with either PR8 or wt PB2 gene retained a lysine residue at position 627 which has been associated with host adaptation and virulence in ovoPB1 plays a key role in cross-species infection and pathogenesis in ovo replication, has not been reported.PB1-F2, the second protein encoded in the PB1 gene has been implicated in pathogenicity in ovo. Other individual coding changes observed in HYRs deriving PA from PR8, involving residues which may affect the structure and polarity of the PA protein may allow more efficient interaction with the other polymerase subunits. Of interest, in three H3N2 HYRs , PA, PB2 and PB1 gene segments are incorporated from wt viruses. This may permit better co-operativity of the polymerase subunits for these reassortants.The PA subunit of the polymerase complex is reported to induce generalized proteolysis, an activity found in the amino-terminal third of the PA protein, 247 residues Conserved regions of IAV polymerase genes critical for efficient vRNA packaging have been reported to play a role in RNA structure, RNA-RNA interactions and RNA-protein interactions Our analysis of HYRs also showed some correlation between coding mutations in the polymerase subunits. Although PB2, PB1 and PA interact with each other in a specific manner in controlling virus replication, the PB1-PA interaction unlike the PB2-PA interaction, has been well-characterized in ovoThe NP and its interaction with the polymerase subunits is well documented. Earlier studies showed that the presence of PR8 NP was associated with hy phenotype in ovoin ovo, even though few coding changes were found , X-31b  and X-171A (Asp489Asn). Although the number of changes are fewer than seen for HA1, it is interesting to note that three of four changes in HA2 involve loss of charged aa residues  which will lead to a reduction in overall protein charge. As the amino terminus region of HA2 is involved in virus entry 2 terminus of NA, which is inserted in the membrane in ovo. No changes are seen at the active site of the NA in contrast to the many changes seen in the RBS in HA. Evidently, wt NA is well-suited to replicate in ovo or readily accommodates the changes in HA for optimal viral replication.The NA of influenza virus is a homotetramer and functions in the release of progeny virus from the infected host cell in ovo prepared HYRs also showed a majority of replacement of third codon G and C by A and T, thereby reducing the overall GC content.The importance of silent mutations on virus replication and evolution has been shown in ovo. When two distinct influenza viruses replicate in embryonated chicken eggs the interactions between the virus and the host cell proteins are crucial for virus replication, assembly and trafficking in ovo, thus providing the maximal amount of virus for preparation of the inactivated vaccine. The gene constellation differences (PR8:wt genes) are equally important for the high growth phenotype exhibited by the HYRs in ovo. Apart from the gene composition, based on the analysis of the HYRs, we hypothesize that in addition to the recurrent mutations, the individual changes found in all eight genes and their encoded proteins of HYRs prepared as candidate seed viruses may augment growth by playing important roles in in ovo virus replication. Hence, further identification of mutations in additional HYRs as they are developed may help in the design of high growth seed viruses with improved growth in ovo.Influenza viruses undergo Darwinian selection resulting in mutants that efficiently replicate Table S1GenBank Accession number of influenza A wt viruses and HYRs analyzed in this study.(DOCX)Click here for additional data file.