The pressureless sintered B4C/C(graphite) composites exhibited extremely low mechanical characteristics. The liquid silicon infiltration technique was employed for enhancing the mechanical property of B4C/C(graphite) composites. Since the porosity of the B4C/C(graphite) composites was about 25–38%, the liquid silicon was able to infiltrate into the interior composites, thereby reacting with B4C and graphite to generate silicon carbide. Thus, boron carbide, silicon carbide, and residual silicon were sintered together forming B4C-SiC-Si composites. The pressureless sintered B4C/C(graphite) composites were transformed into the B4C-SiC-Si composites following the silicon infiltration process. This work comprises an investigation of the microstructure, phase composition, and mechanical characteristics of the pressureless sintered B4C/C(graphite) composites and B4C-SiC-Si composites. The XRD data demonstrated that the pressureless sintered bulks were composed of the B4C phase and graphite phase. The pressureless sintered B4C/C(graphite) composites exhibited a porous microstructure, an extremely low mechanical property, and low wear resistance. The XRD data of the B4C-SiC-Si specimens showed that silicon infiltrated specimens comprised a B4C phase, SiC phase, and residual Si. The B4C-SiC-Si composites manifested a compact and homogenous microstructure. The mechanical property of the B4C-SiC-Si composites was substantially enhanced in comparison to the pressureless sintered B4C/C(graphite) composites. The density, relative density, fracture strength, fracture toughness, elastic modulus, and Vickers hardness of the B4C-SiC-Si composites were notably enhanced as compared to the pressureless sintered B4C/C(graphite) composites. The B4C-SiC-Si composites also manifested outstanding resistance to wear as a consequence of silicon infiltration. The B4C-SiC-Si composites demonstrated excellent wear resistance and superior mechanical characteristics.The B The research findings revealed that the pressureless sintered B4C/C(graphite) composites exhibited excellent machinability, while their mechanical characteristics decreased steadily with the rise in graphite content. The surface hardness and wear resistance of the pressureless sintered B4C/C(graphite) composites decreased remarkably. Thus, the lack of wear resistance, low surface hardness, and poor mechanical property of the pressureless sintered B4C/C(graphite) composites are downsides to their utilization for practical applications. The porosity of the composites was found to be 25–38%. Hence, in an attempt to bring about an improvement in the surface hardness, wear-resistance, and mechanical property of the pressureless sintered B4C/C(graphite) composites, a high-temperature liquid silicon infiltration process was employed. Following machining, the pressureless sintered B4C/C(graphite) composites were processed via a silicon infiltration process. Following liquid silicon infiltration, the boron carbide and graphite react with the liquid silicon, producing silicon carbide [4C/C(graphite) composites have a porosity of about 25–38%, the liquid silicon could be conveniently infiltrated into the interior of the composites [4C and graphite to produce the silicon carbide [4C-SiC-Si composites [4C/C(graphite) composites are thus transformed into the B4C-SiC-Si composites via the silicon infiltration process [4C/C(graphite) composites as well as B4C-SiC-Si composites fabricated by the silicon infiltration process were then investigated and compared. We also looked into the mechanism of the reaction between B4C/C(graphite) composites and liquid silicon. The density and relative density, fracture strength, fracture toughness, Vickers hardness, wear-resistance, and elastic modulus of the silicon infiltered B4C-SiC-Si composites were evaluated and subsequently compared with the pressureless sintered B4C/C(graphite) composites. B4C-SiC-Si composites, upon silicon infiltration, exhibited a homogenous and compact microstructure, an extremely high relative density, a low porosity, outstanding mechanical characteristics, high surface hardness, and superb wear resistance when compared with the pressureless sintered B4C/C(graphite) composites.In one of our previous works, the mechanical property and machinability of the pressureless sintered B carbide ,18,19,20mposites ,18,19,20 carbide ,28,29,30 carbide ,38,39,40 carbide ,48,49,50 carbide ,48,49,50mposites ,53,54,55 process ,53,54,55In particular, there has been much work using first-principles approaches to understand the strength of these and other materials . In part4C/C(graphite) composites. The starting materials included B4C powder and graphite powder having average particle dimensions of 3.5 μm and 10–15 μm, respectively. The graphite powder contents of the B4C/C(graphite) composite powders were kept as 10 wt%, 20 wt%, 30 wt%, and 40 wt%. In total, 10 wt% sintering aids comprising Y2O3 and Al2O3 were then mixed with B4C and graphite powders. Subsequently, ethanol was added to the powder mixture and the mixture was ball milled and wet mechanically milled for 24 h and allowed to dry slowly. The B4C/C(graphite) composite powders were thus prepared. The powders were then put into the steel moulds and fabricated into the strip samples under 200 MPa pressure, with the holding time being 2 min. The strip sample size was about 5 mm × 6 mm × 50 mm. The strip specimens were put into a graphite crucible and set inside a vacuum sintering furnace. A pressureless sintering process was carried out at 1800 °C for 2 h in vacuum conditions yielding the composite’s bulks based on B4C/C(graphite). The B4C/C(graphite) composite’s bulks thus produced were cut into strip specimens, with the size of the sample being 5 mm × 6 mm × 50 mm, which were subsequently processed by silicon infiltration.A pressureless sintering method was used for fabricating B4C monolith was fabricated by a pressureless sintering process. For fabricating B4C monolith, the raw materials included B4C powders having an average particle dimension of 3.5 μm. In total, 10 wt% sintering aids of Al2O3 and Y2O3 powder were mixed with B4C powders. Ethanol was added into the powdered mixture which was subsequently balled milled and wet mechanically milled for 24 h. Following milling, the powder was subjected to slow drying yielding B4C, Al2O3, and Y2O3 composite powders. The composite powders were then poured into a steel mould and fabricated into the stripe samples under 200 MPa. The holding time was 2 min, and the strip sample size was about 5 mm × 6 mm × 50 mm. These stripe samples were then put into a graphite crucible and set within a vacuum sintering furnace. The fabrication of B4C monolith bulks was achieved using pressureless sintering for 2 h at 1800 °C under vacuum conditions.The B4C monolith specimens fabricated by pressureless sintering were cut into the strip samples. These strip specimens were coated with silicon powders, placed within a graphite crucible, and set inside a vacuum sintering furnace. The silicon infiltration process was performed for 2 h at 1550 °C under a vacuum. The solid silicon was completely transformed into liquid silicon at 1550 °C (MP of silicon = 1410 °C) during the silicon infiltration process. Thus, the stripe samples were completely immersed in the liquid silicon. Since the porosity of pressureless sintered specimens was about 20–30%, the liquid silicon thoroughly infiltrated into the interior of the specimens. In the process of infiltration, the liquid silicon underwent a reaction with B4C, producing silicon carbide and the boron carbide partially reacted with the liquid silicon generating silicon carbide. The residual boron carbide and silicon were found within the matrix of the composite. The boron carbide, silicon carbide, and residual silicon were sintered together and formed the B4C-SiC-Si composites. Following the silicon infiltration process, the silicon infiltrated specimens were ground, polished, and fabricated into strip samples of 5 mm × 6 mm × 50 mm. The strip silicon infiltrated specimens were then processed and fabricated into smaller strips with a size of 3 mm × 4 mm × 30 mm. The microstructure and phase composition of the silicon infiltrated specimens were subsequently examined.The B4C/C(graphite) composite samples were fabricated by a pressureless sintering process and then cut into stripe samples. The B4C/C(graphite) composites fabricated by a pressureless sintering process have a graphite content of 10 wt%, 20 wt%, 30 wt%, and 40 wt% and were then subjected to a silicon infiltration process by being placed into a vacuum sintering furnace in a graphite crucible. The specimens were covered with silicon powder and the silicon infiltration process was performed for 2 h at 1550 °C under vacuum conditions. The solid silicon was completely transformed into liquid silicon at 1550 °C (MP of silicon = 1410 °C) during the silicon infiltration process. In this way, the stripe samples were completely immersed in the liquid silicon. Since the porosity of pressureless sintered specimens was about 20–30%, the liquid silicon thoroughly infiltrated the interior of the composites. The liquid silicon reacted with B4C and graphite producing silicon carbide, the boron carbide partially reacted with the liquid silicon to produce silicon carbide, and the graphite was completely combined with the liquid silicon to produce silicon carbide. The residual boron carbide and silicon, however, were present in the matrix of the composite and sintered together with silicon carbide forming the B4C-SiC-Si composites. Following silicon infiltration, the silicon infiltrated specimens were ground, polished, and fabricated first into strip samples of about 5 mm × 6 mm × 50 mm and then cut into smaller sized specimens of 3 mm × 4 mm × 30 mm. The phase composition and microstructure of the B4C-SiC-Si composites specimens yielded via silicon infiltration were then investigated.The B4C/C(graphite) composites fabricated via a pressureless sintering process and that of silicon infiltrated specimens (B4C-SiC-Si composites) was ascertained using X-ray diffraction analysis . The microstructural features of the B4C/C(graphite) composites obtained via the pressureless sintering process and that of the silicon infiltrated specimens (B4C-SiC-Si composites) were examined and analyzed by a scanning electron microscope . An energy-dispersive spectroscopic analysis  of the silicon infiltrated specimens (B4C-SiC-Si composites) was conducted using the scanning electron microscope .The phase composition of the B4C/C(graphite) composites fabricated by pressureless sintering and the B4C-SiC-Si composites by silicon infiltration process were evaluated for their mechanical property, density, relative density, fracture toughness, fracture strength, Vickers hardness, elastic modulus, and wear resistance. Archimedes’ methods were used to estimate the density and relative density of the composites. The composites were evaluated for their fracture strength by a three-point bending test utilizing specimens of sizes 3 mm × 4 mm × 30 mm. The span was kept at 16 mm and the speed of the crosshead was 0.5 mm/min (Testing machine: Instron1195). The fracture toughness of the composites was estimated using a single edge notch beam (SENB) using samples of 3 mm × 4 mm × 30 mm. The respective depth and width of the notch were 1.5 mm and 0.2 mm, the span was 16 mm, and the crosshead speed was 0.05 mm/min (Testing machine: Instron1195). The elastic modulus of the pressureless sintered B4C/C(graphite) composites, as well as B4C-SiC-Si composites obtained via silicon infiltration, was estimated using a three-point bending test with the dimensions of the specimen being 3 mm × 4 mm × 30 mm, the span and the crosshead speed being 16 mm and 0.5 mm/min (Testing machine: Instron1195), respectively. A Vickers hardness meter equipped with a load force of 49 N and a holding time of 20 s was employed to test the composites for their Vickers’s hardness (HV-5 Vickers hardness meter). The wear experiments were performed at MM-W1 B friction and wear tester. Friction wear tests were carried out to measure the wear resistance of the pressureless sintered B4C/C(graphite) composites and B4C-SiC-Si composites obtained via silicon infiltration, wherein the load force was 20 N and the rotational speed was 220 rpm. The wear loss rates of pressureless sintered specimens were also measured. The wear loss rate (R) was the ratio of wear weight loss(M) to wear time(t), thus the wear loss rate is computed using the following equation: R = M/t. The mechanical property and wear resistance of the pressureless sintered B4C monolith were measured similarly using the methods employed for the pressureless sintered B4C/C(graphite) composites.The strip samples of the B4C monolith bulks produced by pressureless sintering have been shown in 4C monolith, 4C phase were visible in the observed XRD data, at 19.71°, 22.02°, 23.50°, 31.90°, 34.95°, and 37.82°. The sintering aids, i.e., Al2O3 and Y2O3 reacted and produced the Y3Al5O12 (YAG) during the pressureless sintering process. The corresponding peaks were also evident in the XRD patterns.The XRD patterns of the B4C/C(graphite) composite’s bulks produced by pressureless sintering have been shown in 4C/C(graphite) composites sintered bulks. The diffraction peaks of the B4C phase and graphite phase are apparent in the XRD patterns. Whereas the diffractive peaks of the B4C phase were at 19.71°, 22.02°, 23.50°, 31.90°, 34.95°, and 37.82°, the peaks at 26.60°, 42.71°, 43.45°, 44.67°, 46.31°, 54.79°, 56.66°, 60.02°, and 77.69° arise from the graphite phase. The sintering aids, i.e., Al2O3 and Y2O3 upon reaction produced Y3Al5O12 (YAG) during the pressureless sintering process. The diffractive peaks of the low content of Y3Al5O12 (YAG) are also obvious in the XRD patterns.The characteristic XRD pattern of the B4C monolith bulks fabricated by pressureless sintering. The pressureless sintered B4C monolith manifested a porous microstructure, with porosity as high as 20–30%. The B4C particles were disseminated within the composite’s matrix and the average particle dimension of boron carbide was approximately 3–5 µm. The porous microstructure of the B4C monolith bulks was presumably because of their fabrication from the pressureless sintering process. The pressureless sintered B4C monolith also exhibited an extremely low relative density; precisely about 70–80%.4C/C(graphite) composite’s bulks. 4C/C(graphite) composites, respectively. It is obvious from the micrographs that the pressureless sintered B4C/C(graphite) composites have a porous microstructure, ranging between 25 and 38%. The B4C and graphite particles are distributed within the composite matrix and the graphite particles generated a laminated structure. The average particle dimensions of boron carbide are about 3–5 µm, whereas the mean particle dimensions of graphite were about 10–15 µm. It has also been observed that with the increase in the amount of graphite, the microstructure of pressureless sintered B4C/C(graphite) composites became increasingly porous. The relative density of the pressureless sintered B4C/C(graphite) composites was found to be extremely low at approximately 62–75%, and their porosity was extremely high.4C monolith (B4C-SiC-Si composites). The pressureless sintered B4C monolith was transformed into the B4C-SiC-Si composites through the silicon infiltration process. 4C phase disappeared partially. Besides, some new diffraction peaks were apparent in the XRD pattern, which presumably arise due to the existence of silicon carbide and silicon phase. As seen before, the diffraction peaks of the B4C phase were observed at 19.71°, 22.02°, 23.50°, 31.90°, 34.95°, and 37.82°. The diffraction peaks of the SiC phase and Si phase were observed, respectively, at 20.78°, 26.67°, 35.60°, 41.38°, 59.97°, 71.77°, 75.49°, and 28.44°, 47.30°, 56.12°, 69.13°, 76.37°. The XRD results have also demonstrated the existence of a boron carbide phase, silicon carbide phase, and residual silicon phase on the surface of the specimen obtained after silicon infiltration. These XRD analysis results also confirmed our assumption that the liquid silicon upon reaction with the boron carbide produced silicon carbide, leaving the residual silicon on the surface of silicon infiltrated specimens. The residual boron carbide also existed in the matrix of the composite. The boron carbide, silicon carbide, and residual silicon were sintered together yielding composites of the B4C-SiC-Si type. This way the pressureless sintered B4C monolith was transformed into the B4C-SiC-Si composites following the silicon infiltration process. The porosity of the pressureless sintered B4C monolith was about 20–30%. The apparent decrease in porosity can be ascribed to the silicon carbide and residual silicon (generated following silicon infiltration), filling in the pores of the matrix of the composite.4C/C(graphite) composites (B4C-SiC-Si composites). 4C/C(graphite) composites. The pressureless sintered B4C/C(graphite) composites transformed into the B4C-SiC-Si composites following silicon infiltration. The silicon infiltrated B4C/C(graphite) composites have a precise chemical composition of the type B4C-SiC-Si. 4C phase and graphite phase vanished following the infiltration of silicon. While the diffraction peaks of the B4C phase disappeared only partially, those of graphite vanished completely. The appearance of some new diffraction peaks in the data corresponds to the existence of the silicon carbide phase and silicon phase. The diffraction peaks of the B4C phase appear at 19.71°, 22.02°, 23.50°, 31.90°, 34.95°, and 37.82°, the diffraction peaks of the SiC phase can be observed at 20.78°, 26.67°, 35.60°, 41.38°, 59.97°, 71.77°, 75.49°, whereas those of the Si phase are apparent at 28.44°, 47.30°, 56.12°, 69.13°, 76.37°. The XRD data point towards the existence of the boron carbide phase, silicon carbide phase, and residual silicon phase upon the surface of the specimens obtained after silicon infiltration. The XRD analysis suggests the infiltration of liquid silicon into the inner composite matrix and subsequent reaction with boron carbide and graphite producing silicon carbide. Some residual silicon stayed on the specimen surface following silicon infiltration. The boron carbide, silicon carbide, and residual silicon were sintered together forming the B4C-SiC-Si composites. The porosity of the pressureless sintered B4C/C(graphite) composites was about 25–38%. The apparent decrease in the porosity is because produced silicon carbide and residual silicon filled the pores in the matrix of the composite during the silicon infiltration process.4C monolith (B4C-SiC-Si composites). The pressureless sintered B4C monolith was transformed into the B4C-SiC-Si composites through the silicon infiltration process. These SEM images depict that the silicon infiltrated products B4C-SiC-Si composites possessed a compact and homogenous microstructure. The B4C particles and SiC particles were homogenously dispersed within the composite matrix. The mean particle size of B4C particles and SiC particles was about 4–5 µm. The existence of some residual silicon in the matrix of the composite is also evident. Because the pressureless sintered B4C monolith was a porous material; the porosity being 20–30%, the liquid silicon could infiltrate into the interior of the composite matrix during the silicon infiltration process. Boron carbide reacted with liquid silicon producing silicon carbide particles, and the residual boron carbide, as well as silicon, existed in the composite’s matrix. B4C-SiC-Si composites exhibited an exceptionally high relative density (98–99%), whereas their porosity was only about 1–2%. The silicon carbide was produced following silicon infiltration and the residual silicon filled the pores of the composites, so the porosity of the silicon infiltrated specimens decreased remarkably, and the B4C-SiC-Si composites became more compact and denser.4C/C(graphite) composites (B4C-SiC-Si composites). 4C/C(graphite) composites. These SEM data present a compact and homogenous microstructure of the silicon infiltrated B4C-SiC-Si composites. The B4C particles and SiC particles were distributed homogeneously in the composite’s matrix, and some residual silicon also existed in the composite’s matrix. The mean particle size of B4C particles and SiC particles was about 4–5 µm. Because the pressureless sintered B4C/C(graphite) composites were porous (the porosity of the pressureless sintered B4C/C(graphite) composites was about 25–38%), the liquid silicon could infiltrate into the inner composite’s matrix during silicon infiltration. The reaction between boron carbide/graphite and liquid silicon produced silicon carbide particles, and the residual boron carbide and silicon existed in the composite’s matrix. The relative density of the B4C-SiC-Si composites was extremely high (98–99%), whereas the porosity of the B4C-SiC-Si composites was about 1–2%. The produced silicon carbide and residual silicon filled the pores, so the porosity of the silicon infiltrated specimens decreased remarkably, and the B4C-SiC-Si composites became more compact and denser.4C-SiC-Si composites fabricated by the silicon infiltration process. As already discussed, the B4C-SiC-Si composites manifested compact and homogenous microstructural features as shown in 4C-SiC-Si composites fabricated by the silicon infiltration process. The EDS spectroscopic analysis depicted that the silicon infiltrated composites were composed primarily of boron, silicon, and carbon, thereby demonstrating that the composites obtained following silicon infiltration comprised boron carbide and silicon carbide along with some content of silicon. It can therefore be assumed that the liquid silicon infiltrated into the interior of the composite and produce silicon carbide upon reaction with boron carbide, according to the following reaction equations [quations ,53,54,55The liquid silicon underwent a chemical reaction with graphite producing silicon carbide as shown by the following chemical reactions ,53,54,554C-SiC-Si composites. The residual silicon and silicon carbide produced during the process filled the pores in the composite’s matrix, hence the porosity of the specimens obtained following silicon infiltration decreased remarkably. Consequently, the B4C-SiC-Si composites exhibited an extremely high relative density and low porosity. The relative density of the B4C-SiC-Si composites was about 98–99%, and the porosity of the B4C-SiC-Si composites was about 1–2%.The residual boron carbide and silicon sintered together with the product silicon carbide to give rise to B4C/C(graphite) composites and the B4C-SiC-Si composites. The density of the pressureless sintered B4C/C(graphite) composites demonstrated a gradual decrease with the increase of graphite content. The density of the pressureless sintered B4C monolith was relatively high, but the density of the pressureless sintered B4C/C(graphite) composites was rather low. The underlying reason for this observation is that B4C materials have a density of 2.52 g/cm3, whereas graphite has a density of about 2.26 g/cm3, hence with the increase in the amount of graphite, the density of the pressureless sintered B4C/C(graphite) composites demonstrated a gradual decline. The porous microstructure of the pressureless sintered specimens also contributed to the extremely low density. The density of the pressureless sintered B4C/C(graphite) composites was about 1.5–1.9 g/cm3.4C-SiC-Si composites was phenomenally improved as compared to the pressureless sintered B4C/C(graphite) composites as depicted in 4C-SiC-Si composites progressively increased with the increase in the amount of added graphite in the B4C/C(graphite) composites. This observation was mainly because the liquid silicon infiltrated into the interior of the pressureless sintered B4C/C(graphite) composites and reacted with B4C and graphite to produce silicon carbide. Thus, with the increment in the amount of graphite in the B4C/C(graphite) composites, the graphite completely reacted with liquid silicon to produce silicon carbide, thus markedly increasing the content of produced silicon carbide in the B4C-SiC-Si composites. Since the density of silicon carbide is 3.21 g/cm3 and that of silicon is 2.32 g/cm3, the density of the produced B4C-SiC-Si composites increased progressively with the increase in the amount of silicon carbide produced. On the whole, the density of the B4C-SiC-Si composites was higher than the density of the corresponding B4C/C(graphite) composites. B4C-SiC-Si composites demonstrated a density value of 2.5855–2.9288 g/cm3 which is substantially higher than the pressureless sintered B4C/C(graphite) composites.On the contrary, the density of the B4C/C(graphite) composites and the B4C-SiC-Si composites. 4C/C(graphite) composites exhibited a gradual decline with the increase of graphite content. The relative density of the pressureless sintered B4C monolith was relatively high; however, the relative density of the pressureless sintered B4C/C(graphite) composites was rather low. This is presumably because the B4C/C(graphite) composites were produced via pressureless sintering and therefore exhibited a porous microstructure, causing their relative density to become extremely low, precisely 62–75%. The porosity of the pressureless sintered B4C/C(graphite) composites was about 25–38%.4C-SiC-Si composites demonstrated a remarkable improvement in comparison with the pressureless sintered B4C/C(graphite) composites. In the B4C/C(graphite) composites, a gradual increase in the relative density was observed with the increase of graphite content. With the infiltration of liquid silicon into the interior of pressureless sintered B4C/C(graphite) composites, the B4C and graphite reacted with liquid silicon to produce the silicon carbide. The produced silicon carbide as well as the residual silicon filled the pores in the composite’s matrix, thereby resulting in a remarkable decrease in the porosity of the silicon infiltrated specimens and a consequent increase in their relative density. The residual silicon, boron carbide, and silicon carbide were sintered into dense and compact bulks. The density of silicon carbide is 3.21 g/cm3, hence the relative density of the composites based on B4C-SiC-Si became higher than the relative density of the B4C/C(graphite) composites obtained through pressureless sintering. The silicon infiltrated products B4C-SiC-Si composites presented compact and homogenous microstructural features. The relative density of the B4C-SiC-Si composites was about 98–99%. The density and relative density of the B4C-SiC-Si composites showed a significant improvement in comparison to the pressureless sintered B4C/C(graphite) composites.4C/C(graphite) composites and the B4C-SiC-Si composites has been shown in 4C/C(graphite) composites was observed with the increase in the amount of graphite. While the fracture strength of the pressureless sintered B4C monolith was quite high, the pressureless sintered B4C/C(graphite) composites demonstrated a rather low fracture strength value. This is because the B4C/C(graphite) composites were fabricated by a pressureless sintering process, which imparted a porous microstructure, a low relative density (62–75%), and porosity of around 25–38% leaving them with an extremely low fracture strength. The fracture strength of the pressureless sintered B4C/C(graphite) composites lay within the 102–155 MPa range and reduced remarkably with the increase of graphite content.The impact of the amount of graphite on the fracture strength of the pressureless sintered B4C-SiC-Si composites demonstrated a remarkable improvement in their fracture strength as compared to the pressureless sintered B4C/C(graphite) composites. The fracture strength of the B4C-SiC-Si composites increased progressively with the rise in graphite content. It occurs because liquid silicon infiltrated into the interior of pressureless sintered B4C/C(graphite) composites, and B4C and graphite reacted with liquid silicon producing silicon carbide. The resultant silicon carbide and the residual silicon and boron carbide were sintered together fabricating B4C-SiC-Si composites. The silicon carbide and residual silicon settled within the pores in the composite’s matrix, thereby causing a remarkable decrease in the porosity of the silicon infiltrated specimens and a corresponding increase in the relative density of the silicon infiltrated specimens. The relative density of the B4C-SiC-Si composites was found to be higher than 98%, the B4C-SiC-Si composites thus became denser and more compact and their fracture strength was higher than that of the pressureless sintered B4C/C(graphite) composites. B4C-SiC-Si composites have a fracture strength equivalent to 385–438 MPa. In addition, the produced silicon carbide could also improve the fracture strength of the B4C-SiC-Si composites, with the fracture strength increasing gradually with the increase of silicon carbide content. As an obvious fact, silicon carbide content underwent a gradual increase with the increase of graphite content in the B4C/C(graphite) composites. All these factors contribute to a remarkable improvement in the fracture strength of the B4C-SiC-Si composites as compared to the pressureless sintered B4C/C(graphite) composites.4C/C(graphite) composites and the B4C-SiC-Si composite is also affected by the amount of graphite as shown in 4C/C(graphite) composites with the increment in the amount of graphite. The fracture toughness of the pressureless sintered B4C/C(graphite) composites was rather low as compared to that of the pressureless sintered B4C monolith, presumably because the B4C/C(graphite) composites were produced via pressureless sintering and exhibited a porous microstructure. Additionally, the B4C/C(graphite) composites obtained via pressureless sintering had a rather low relative density and the porosity was 25–38%. It was also observed that the pressureless sintered B4C/C(graphite) composites demonstrated a remarkable decrease in their fracture toughness with the increase of graphite content. This is an outcome of the inherently low fracture toughness of graphitic materials. The observed fracture toughness of the pressureless sintered B4C/C(graphite) composites was about 1.568–2.35 MPa·m1/2.The fracture toughness of the pressureless sintered B4C-SiC-Si composites underwent a marked improvement when compared with the pressureless sintered B4C/C(graphite) composites, demonstrating a gradual increase with the increase in the amount of graphite. This can be attributed to the infiltration of liquid silicon into the interior pressureless sintered B4C/C(graphite) composites and the subsequent reaction with B4C and graphite producing silicon carbide. Silicon carbide and residual silicon settled into the pores of the composite’s matrix, thereby decreasing its porosity and increasing the relative density up to >98%, rendering the B4C-SiC-Si composites even more dense and compact. Consequently, the fracture toughness of the B4C-SiC-Si composites was recorded as 6.05–7.55 MPa·m1/2 which is much higher in comparison to the fracture toughness of the pressureless sintered B4C/C(graphite) composites.It is also evident from 4C-SiC-Si composites, which underwent a progressive increase progressively with the increase of the silicon carbide content. The content of produced silicon carbide in turn gradually increased with the increase of the amount of graphite in the B4C/C(graphite) composites. Additionally, the relative density of the B4C-SiC-Si composites was extremely high, about 98–99%. Thus, the fracture toughness and fracture strength of the B4C-SiC-Si composites were significantly enhanced as compared to the pressureless sintered B4C/C(graphite) composites. While the pressureless sintered B4C/C(graphite) composites were soft and brittle and could be easily broken, the B4C-SiC-Si composites were compact and hard and exhibited extremely high fracture strength and high fracture toughness.The silicon carbide produced following silicon infiltration is also assumed to improve the fracture toughness of the B4C/C(graphite) composites and the B4C-SiC-Si composites has been shown in 4C/C(graphite) composites demonstrated a progressive decline with the increase of graphite content. While the Vickers hardness of the pressureless sintered B4C monolith was relatively high, its value was rather low for the pressureless sintered B4C/C(graphite) composites. Owing to their fabrication from the pressureless sintering process, the B4C/C(graphite) composites exhibited porous microstructures and have a low relative density (62–75%) and a porosity of about 25–38%. It has been known that the Vickers hardness of graphite is rather low. the laminated structured graphite materials have a softening effect on the ceramic composites. Consequently, the Vickers hardness of the pressureless sintered B4C/C(graphite) composites exhibited a gradual decrease with the increase in the amount of graphite. The pressureless sintered B4C/C(graphite) composites were soft and very brittle and could be easily broken. The Vickers hardness of the pressureless sintered B4C/C(graphite) composites was about 2.52–4.88 GPa.The impact of the graphite content on the Vickers hardness of the pressureless sintered B4C-SiC-Si composites was markedly enhanced when compared to the pressureless sintered B4C/C(graphite) composites and increased progressively with the increase in the amount of graphite. This can be ascribed to the infiltration of liquid silicon into the interior pressureless sintered B4C/C(graphite) composites, and the subsequent reaction with B4C and graphite, producing silicon carbide. Silicon carbide and residual silicon settle into the pores of the composite’s matrix, thereby decreasing its porosity and increasing the relative density up to >98%, rendering the B4C-SiC-Si composites even more dense and compact and subsequently increasing their Vickers’s hardness.4C-SiC-Si composites primarily comprises boron carbide, silicon carbide, and residual silicon. Consequently, the Vickers hardness of the B4C-SiC-Si composites following silicon infiltration is observed to be higher than pressureless sintered B4C/C(graphite) composites. B4C-SiC-Si composites demonstrated a Vickers hardness value as high as 22.885–26.768 GPa.Besides, the Vickers hardness of boron carbide and silicon carbide is exceptionally high, 20 GPa to be specific. The specimen surface of the B4C-SiC-Si composites demonstrated a gradual increase with the increase in the amount of silicon carbide. The content of silicon carbide increased steadily with the increment in graphite content in the B4C/C(graphite) composites, thereby enhancing the Vickers hardness of the B4C-SiC-Si composites to a remarkable extent. A high value of the Vickers hardness, in turn, guarantees an enhancement in the resistance of the B4C-SiC-Si composites to wear. Thus, the B4C-SiC-Si composites exhibit an outstanding wear resistance.Moreover, the Vickers hardness of the B4C/C(graphite) composites and the B4C-SiC-Si composites as a function of graphite content. The elastic modulus of the pressureless sintered B4C/C(graphite) composites reduced gradually with the increase in the amount of graphite. While the elastic modulus of the pressureless sintered B4C monolith was relatively high, that of the pressureless sintered B4C/C(graphite) composites was rather low. This is a consequence of the porous microstructure, low relative density (62–75%), and high porosity (25–38%) of the pressureless sintered B4C/C(graphite) composites.4C/C(graphite) composites. The pressureless sintered B4C/C(graphite) composites are highly porous and exhibit a low elastic modulus, with the elastic modulus gradually declining with the increment in graphite content. The exact value of elastic modulus of the pressureless sintered B4C/C(graphite) composites was about 106–145 GPa as illustrated in The elastic modulus of boron carbide is high, and the elastic modulus of graphite was rather low. This leads to the introduction of softness and brittleness into the pressureless sintered B4C/C(graphite) composites, the B4C-SiC-Si composites demonstrate an improved value of the elastic modulus. There was a gradual increase in the elastic modulus of the B4C-SiC-Si composites with the increase in the amount of graphite in the B4C/C(graphite) composites as shown in 4C/C(graphite) composites. Thus, the B4C and graphite undergo a chemical reaction with liquid silicon, generating silicon carbide. The silicon carbide thus generated takes up the porous spaces in the composite’s matrix along with the residual silicon, decreasing its porosity and increasing the relative density. The B4C-SiC-Si composites became denser and more compact with a relative density as high as >98%. Due to their compact and homogenous microstructure, B4C-SiC-Si composites have an elastic modulus in the range of 385–410 GPa, which is considerably higher than the elastic modulus of the pressureless sintered B4C/C(graphite) composites.When compared with the pressureless sintered B4C-SiC-Si composites in terms of their elastic modulus. A gradual increase in the elastic modulus of the B4C-SiC-Si composites was evident with the increase of the content of silicon carbide, which increases linearly with the increase in graphite content. B4C-SiC-Si composites thus demonstrated an extremely high elastic modulus which was substantially enhanced as opposed to the pressureless sintered B4C/C(graphite) composites.Both boron carbide and silicon carbide have an exceptionally high value of elastic modulus. Thus, the silicon carbide generated following silicon infiltration could also contribute to improving the B4C/C(graphite) composites and the B4C-SiC-Si composites were examined for their wear resistance with the help of wear experiments. The vertical coordinate was wear weight loss, whereas the wear time represents the horizontal coordinate. 4C/C(graphite) composites due to wear. The wear weight loss of the pressureless sintered B4C/C(graphite) composites manifested a progressive increase with the increment in wear time. Additionally, the extent of wear weight loss increased with the gradual rise in graphite content. The pressureless sintered B4C monolith gave a rather low wear weight loss curve owing to the relatively high inherent Vickers hardness of B4C ceramics. The composites with the graphite content of 10 wt%, 20 wt%, 30 wt%, and 40 wt% exhibited higher wear weight loss curves which may be attributed to the low value of Vickers hardness of the pressureless sintered B4C/C(graphite) composites. The weight loss curves in 4C/C(40 wt% graphite) composites while the lowest was observed for the B4C monolith. The findings suggest that for the pressureless sintered B4C/C(graphite) composites, the wear resistance was very poor, and the wear weight loss was high. The wear resistance of the pressureless sintered B4C/C(graphite) composites reduced gradually with the increase in the amount of graphite presumably on account of the low Vickers hardness of the pressureless sintered B4C/C(graphite) composites.The pressureless sintered B4C-SiC-Si composites has been shown in 4C/C(graphite) composites, the B4C-SiC-Si composites demonstrated an increase in wear weight loss with the increase in wear time. The wear weight loss of the B4C-SiC-Si composites manifested a gradual decrease with the increment in graphite content in the corresponding B4C/C(graphite) composites. The B4C-SiC-Si composites with the graphite content of 10 wt%, 20 wt%, 30 wt%, and 40 wt% in the B4C/C(graphite) composites gave lower wear weight loss curves presumably on account of the high Vickers hardness of the B4C-SiC-Si composites. As shown in 4C monolith, whereas the lowest was observed for the silicon infiltrated B4C/C(40 wt%graphite) composites. When the wear weight loss was low, the wear resistance was very high. Thus, the wear resistance of the B4C-SiC-Si composites was quite high on account of their high Vickers hardness which is equivalent to 22.885–26.768 GPa.The influence of wear time on the wear weight loss of the B4C-SiC-Si composites was relatively lower in comparison to the wear weight loss of the pressureless sintered B4C/C(graphite) composites. This implies that the wear resistance of the B4C-SiC-Si composites demonstrated a substantial improvement as compared to pressureless sintered B4C/C(graphite) composites which may be attributed to their high Vickers hardness.From The wear weight loss curve was created using line fitness in 4C/C(graphite) composites and the B4C-SiC-Si composites. It can be seen that the wear loss rates of the pressureless sintered B4C/C(graphite) composites gradually increased with the increase in the amount of graphite. This indicated that the wear resistance of the pressureless sintered B4C/C(graphite) composites underwent a gradual decrease with the increase in the amount of graphite. A possible explanation for this observation is that the B4C/C(graphite) composites were fabricated via a pressureless sintering method and have a low relative density and a porosity ranging between 25–38%. The Vickers hardness of the pressureless sintered B4C/C(graphite) composites reduced progressively with the increase in the amount of graphite. The surface hardness of the pressureless sintered B4C/C(graphite) composites was also exceptionally low. All these factors contribute to the reduced wear resistance of the pressureless sintered B4C/C(graphite) composites, which manifested a noticeable decline with the increase in the amount of graphite.The wear resistance of pressureless sintered specimens and silicon infiltrated specimens were measured by friction wear experiments. A ratio of wear weight loss to wear time is referred to as the wear loss rate. 4C-SiC-Si composites, their wear loss rates underwent a gradual decrease with the rise in graphite content as depicted in 4C-SiC-Si composites specimen’s weight demonstrated a steady decrease with the increase in the amount of graphite in the B4C/C(graphite) composites. 4C-SiC-Si composites was substantially enhanced when compared to the pressureless sintered B4C/C(graphite) composites, indicating that the silicon infiltrated specimen’s weight underwent a gradual decrement with the increment in the amount of graphite in the B4C/C(graphite) composites. The loss in weight observed for the silicon infiltrated specimens was exceptionally minimal. The B4C-SiC-Si composites manifested remarkable resistance to wear. The underlying phenomenon, in this case, is the infiltration of liquid silicon into the interior of the composites and subsequent reaction with B4C and graphite yielding silicon carbide. Silicon carbide and residual silicon ultimately occupy the pores in the composite’s matrix, decreasing its porosity and increasing its relative density. B4C-SiC-Si composites are thus rendered denser and more compact and their surface hardness and Vickers hardness were exceptionally high; hence, the wear resistance of the B4C-SiC-Si composites was also extremely high. Silicon carbide and boron carbide along with the residual silicon existing on the silicon infiltrated specimen’s surface also contribute towards the remarkable improvement in the resistance of the B4C-SiC-Si composites towards wear.As regards the BDuring the silicon infiltration, the liquid silicon infiltrated into the interior of the composite’s specimens, and subsequently reacted chemically with boron carbide producing silicon carbide according to the following chemical reactions ,53,54,55The liquid silicon combined with graphite to produce the silicon carbide according to the following chemical reactions ,53,54,554C-SiC-Si composites were primarily composed of boron carbide, resultant silicon carbide, and only a little content of silicon; the net content of boron carbide and silicon carbide was >90%, whereas the residual silicon was found to be <10%.The B4C/C(graphite) composites were produced via a pressureless sintering process. At an elevated temperature, liquid silicon infiltration was conducted for improving the mechanical property of the pressureless sintered B4C/C(graphite) composites. Since the porosity of pressureless sintered B4C/C(graphite) composites was about 25–38%, the liquid silicon could infiltrate into the interior of B4C/C(graphite) composites, reacting with B4C and graphite to produce silicon carbide. The boron carbide, silicon carbide, and residual silicon are then sintered together giving rise to B4C-SiC-Si composites. This way the pressureless sintered B4C/C(graphite) composites transformed into the B4C-SiC-Si composites following silicon infiltration.The B4C/C(graphite) composites obtained via a pressureless sintering process. The XRD data demonstrated that the pressureless sintered bulks were primarily made up of the B4C phase and graphite phase. Besides, they have a porous microstructure, with the porosity lying in the range of 25–38%. The relative density of the pressureless sintered B4C/C(graphite) composites was about 62–75% and their mechanical property underwent a gradual decline with the increase in the amount of graphite. The density, relative density, fracture toughness, fracture strength, Vickers hardness, elastic modulus, and wear resistance of the pressureless sintered B4C/C(graphite) composites also manifested a progressive decrease with the increase in the amount of graphite.This work is based on an investigation of the microstructure, phase composition, and mechanical properties of the B4C/C(graphite) composites reacted with liquid silicon and transformed into B4C-SiC-Si composites. The microstructure, phase composition, and mechanical characteristics of the B4C-SiC-Si composites fabricated via the silicon infiltration process were then thoroughly investigated. XRD data suggest the existence of a B4C phase, a SiC phase, and a residual Si phase on the silicon infiltrated specimen’s surface. The silicon infiltrated products, i.e., B4C-SiC-Si composites exhibited a homogenous and compact microstructure, an extremely high relative density, and remarkably low porosity. The microstructure investigation results showed that the silicon infiltrated specimens were primarily made up of boron carbide, silicon carbide, and residual silicon. There was a substantial improvement in the B4C-SiC-Si composites in terms of their mechanical property when compared with the pressureless sintered B4C/C(graphite) composites. The density and relative density, fracture strength and fracture toughness, elastic modulus, Vickers hardness, and wear resistance of the B4C-SiC-Si composites were also significantly improved and exhibited a gradual increase with the increase in the amount of graphite in the composites. It can thus be concluded that the silicon infiltration process efficiently improves the mechanical property and wear resistance of the pressureless sintered B4C/C(graphite) composites, and the B4C-SiC-Si composites thus generated can find potentially useful applications due to their superior physical attributes.Upon silicon infiltration, the pressureless sintered B


To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. The addition of a large cytoplasmic ring next to the C terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses. CryoEM structure of CHIKV replication complex reveals multicomponent RNA synthesis nanomachine stuck in host cell plasma membrane. Alphavirus, family Togaviridae) is a mosquito-borne pathogen that has spread globally and caused a substantial health burden of acute febrile illness progressing to debilitating, often chronic polyarthritis in millions of people. In addition, CHIKV can also induce rare but lethal encephalitis  and cryo–electron tomography (cryo-ET) methods to determine the molecular architecture of the alphavirus RC. The molecular structure of the reconstituted RC core (nsP1 + 2 + 4) fits nicely into part of the active RC density obtained from subtomogram averaging of the necks of the alphaviral spherules. Together, these data provide unprecedented molecular basis of the alphavirus genome replication process, likely applicable to other (+) RNA viruses, such as coronaviruses and flaviviruses.To reconstitute a functional alphavirus RC , we prep2, indicating that the nsP1:nsP4 interface is highly stable (fig. S3A). Intriguingly, the nsP1 ring acts as a chaperone to interact with and stabilize the otherwise highly dynamic nsP4  and the palm subdomain of nsP4 with a buried area of 906 Å2 . A flexiThe nsP4 RdRp resides at the center of the RC core, well stabilized by the surrounding nsP1 and nsP2. Almost all 611 residues of the nsP4 are well resolved in the cryo-EM density map, which includes the intrinsically disordered NTD (amino acids 1 to 105) and those disordered regions from the fingers subdomain  not captured in the crystal structures of the alphavirus nsP4 RdRps from Ross River virus (PDB 7F0S) and Sindbis virus (PDB 7VB4)  and reveN = 101 spherules), respectively. Proximal to the spherules, dense condensates in the cytosol are often observed proximal to RCs at the neck . At this time point, all RCs should be matured with fully processed nsPs and dsRNA replication intermediate . The swithe neck . Such deSubtomogram averaging of the spherule neck regions revealed a 12-fold symmetric complex at 7.3 Å resolution, consisting of a PM-associated crown ring connected to a larger, smooth ring on the cytoplasmic side . TogetheWe then determined an asymmetric reconstruction of the RC, revealing that the plug density in the center of the crown is positioned asymmetrically with a small gap on one side  and 2. AIn addition to the RCs positioned at the neck of spherules, we observed many ring-like complexes smaller than RCs docked to the inner leaflet of the PM in the absence of RNA or membrane invaginations. These macromolecular complexes were especially numerous on thin cell extensions with underlying bundled cytoskeleton filaments  10, 11,. NoticeaIn this study, we performed a multiscale study for the structural and functional characterization of the CHIKV replication machinery in vitro and in the infected cell using combinatory cryo-EM methods. Our work provides an integrated molecular view of the structure of a central core portion of the alphavirus nsP assembly within the RCs. The nsP1 dodecameric ring of nsP1 + 2 + 4 RC core architecture did not deviate much from the previously published nsP1 ring structure (PDB 7DOP)  RNA replication . The sphg after 5 days. The protein expression was boosted by 10 mM sodium butyrate 16 to 20 hours after transfection. The cell pellet was subsequently lysed in lysis buffer WI  with 1 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Sigma-Aldrich), 1% n-dodecyl-β-d-maltoside (Exceedbio), and BioLock solution  on a rotator at 4°C for 1.5 hours and pulse-sonicated for 5 min. The lysate was clarified with 100,000g centrifugation at 4°C in XPN-100 ultracentrifuge (Beckman Coulter). The Strep-Tactin Sepharose slurry beads  were incubated in Econo-Pac column (Bio-Rad) with the clarified supernatant. The nsP1-bound strep-bead was equilibrated to assembly buffer WII  in 5 column volume (CV) wash for downstream RC assemblies and RNA polymerase assay (refer to below).The preparation of nsP1 was adapted from (6-SUMO) tag and recombinantly purified as described in  and nsP2 helicase domain  were both subcloned into pSUMO-LIC expression vector carrying N-terminal hexa-histidine tag fused with cleavable SUMO  supplemented with 2.5% glycerol and then induced at 0.5 mM isopropyl-β-d-thiogalactoside (Gold Biotechnology) after a measured OD600  of ~0.8 for 18 hours at 18°C. The following purification process for nsP3MZ was maintained throughout at 4°C. The purification started with harvesting the bacterial pellet at 4000 rpm followed by lysis with buffer A  supplemented with 1 mM PMSF in a PANDAPLUS2000 (GEA) homogenizer. The supernatant obtained from 100,000g ultracentrifugation was incubated with Ni-NTA slurry beads (Bio Basic) for 1 hour. The nsP3MZ-bound Ni-nitrilotriacetic acid (NTA) was washed for 20 CV of buffer A and eluted in buffer B (buffer A with 300 mM imidazole). The eluant was cleaved with SUMO protease (at 10:1 w/w ratio) for 16 hours in 0.5× buffer B. Then, the tag-removed nsP3MZ was purified subsequently in HiLoad 16/600 Superdex 200 pg column (Cytiva) in buffer C  as a monomer. The monomeric peak fractions were concentrated to desired concentration in buffer C to flash freeze for long-term storage at −80°C till required.The nsP3MZ gene  was cloned from the same CHIKV cDNA described in  was sourced from ONNV  and cloned into pSUMO-LIC expression vector with N-His3COO)2, 2.5 mM MnCl2, 5 mM TCEP, 0.01% GDN, NEB murine RNase inhibitor (0.5 U/μl), and 1.25% sucrose (~pH 8)] prepared in nuclease-free water (HyClone). The reactions were initiated with 2 mM adenosine triphosphate (ATP) and quenched in a discontinuous time point manner for 0.2 to 18 hours after incubating at ~25°C in dark. The reactions were quenched with equal volume of denaturing RNA loading dye  to run on 17.5% denaturing urea-PAGE at 220 V for 1.5 to 2 hours. The urea-PAGE was imaged via Bio-Rad Gel-Doc XR+ Imager using Fluorescence Blot ALEX488 for analysis.The RNA polymerase assay is optimized and uses the same T1 RNA template from , a DNA template with T7 promoter (5′TAATACGACTCACTATA 3′) was ordered from Integrated DNA Technologies to perform IVT according to manufacturer recommendation from Hiscribe T7 High Yield RNA Synthesis Kit (NEB). The IVT product was purified via the NEB-recommended phenol-chloroform extraction method and Monarch RNA Cleanup Kits (NEB).T2 RNA sequences:Sense strand: 5′GGGAUAAUGCAAGCAUACCGAUCUUCCAACGUUUCUCCGAACCCACAGGGACGUAGGAGAUGUUAUUUUGUUUUUAAUAUUUC3′Antisense strand: 5′GGGAUAAUGCAAGCAUACCGAUCUUCCAACGUUGAAAUAUUAAAAACAAAAUAACAUCUCCUACGUCCCUGUGGGUUCGGAGA3′g in Amicon Ultra (Merck) and/or Pierce (Thermo Fisher Scientific) protein concentrators of 100 kDa molecular weight cutoff (MWCO) for the abovementioned RNA polymerase assay and structural studies. The RCs assembled at affinity pulldown and anion-exchange chromatography were fractionated and sampled for negative-staining transmission electron microscopy (TEM) on carbon-coated copper 300-mesh grids  for screening and optimization of RC reconstitutions.All in vitro reconstitutions and sample preparations were done at 4°C unless otherwise mentioned. The CHIKV-nsP1–immobilized strep-bead was incubated with the desired combinations of nsPs  to produce RC assemblies: nsP1 + 2 + 4 and nsP1 + 4. The strep-beads consisting of RC assemblies were eluted in the abovementioned buffer WII supplemented with 10 mM desthiobiotin at ~pH 8 and loaded directly to a HiTrap Q-FF column (Cytiva) for linear elution from 0.15 M NaCl to 1 M NaCl. The eluants were concentrated at 3000 to 7000The nsP1 + 2 + 4 RC assembly was mixed with T2 RNA and NTP in 1× buffer Z at 1:10:100 molar ratio for 1 hour at ~25°C before concentrating down to ~30 μl. The cryo-EM grid preparation (including single-layer graphene grid fabrication) was adapted from  supplemented with penicillin and streptomycin, 10 mM Hepes, nonessential amino acids, and 10% fetal bovine serum (HyClone). CHIKV vaccine strain 181/clone 25 (CHIKV-181) was amplified in BHK21 cells. CHIKV vaccine strain 181/clone 25 (CHIKV-181)  was obtained from BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and amplified in BHK21 cells.The human bone epithelial cell line U2OS  is a female cell line purchased from the American Type Culture Collection (ATCC). Hamster fibroblast cell line BHK21 cells  were purchased from ATCC. Cells were maintained at 37°C with 5% humidified COU2OS cells grown on fibronectin-coated gold 200 mesh R2/2 grids (Quantifoil) were infected with CHIKV-181 at a multiplicity of infection of 50 for an incubation period of 8 hours. The grids were then washed with PBS and a solution of 10 nm of bovine serum albumin gold tracer  was added directly before vitrification. Grids were blotted and plunged into liquid ethane using the LEICA EMGP plunge freezer device. Grids were stored under liquid nitrogen conditions until TEM data collection.−/A2, distributed over 51 tilt images, covering an angular range of −50° to +50° with an angular increment of 2°. Motion between frames of each tilt image in the tilt series was corrected using patch-tracking in MotionCor2 software  operated at 200 kV with a post-column energy filter (20 eV) and a K2 Summit detector with a calibrated pixel size of 3.54 Å. Single-axis, bidirectional tilt series were collected using SerialEM software  particles in different orientations were picked from tomograms at ×4 binning. These RC particles were input to the EMAN2 initial model generation program, performed in two steps. First, particles were iteratively aligned with C1 symmetry for three iterations and aligned to the symmetry axis. Then, an additional five iterations were performed, applying C12 symmetry to produce an initial model with low-resolution features of the spherule base, PM, and RC. This initial model was filtered to 40 Å resolution and used for subsequent 3D subtomogram refinement of the full dataset. Subtomogram averaging was then performed with 724 RC particles at ×1 binning (3.54 Å per pixel) from 49 tomograms while applying C12 symmetry with a loose mask around RC protein density to exclude the surrounding membrane from alignment. Afterward, subtomogram refinement and additional sub-tilt refinement of particle translation and rotation produced a final RC map at 7.3 Å resolution  with C12 symmetry applied. A soft mask was applied to focus alignment of particles on the protein ring density rather than the membrane. Subtomogram refinement and sub-tilt refinement of translation and rotation, again with C12 symmetry applied, produced a final map at 9.9 Å resolution (


During 2014–2016, clade 2.3.4.4 H5N8 high pathogenicity avian influenza virus (HPAIV) caused the largest known avian influenza epidemic in South Korea. Based on data from earlier H5N8 outbreaks, primitive H5N8 virus in South Korea was classified into five subgroups: C1, C2, C3, C4, and C5. The present study investigated the pathogenic and molecular epidemiologic characteristics of H5N8 viruses obtained from 388 cases of poultry farms and 85 cases of wild bird infections in South Korea during 2014–2016. Representative viruses of subgroups C1, C2, and C4 showed significant pathobiological differences in specific pathogen-free (SPF) chickens, with the H1731 (C1) virus showing substantially lower infectivity, transmissibility, and pathogenicity than the H2102 (C2) and H1924 (C4) viruses. Full genome sequence analysis showed the number of mutations that significantly increased in domestic duck-origin H5N8 HPAIVs compared to the viruses from gallinaceous poultry. These differences may have been due to the long-term circulation of viruses in domestic duck farms. The same mutations, at positions 219 and 757 of PB1, independently evolving in the C0, C1, and C2 subgroups may have been positively selected, resulting in convergent evolution at the amino acid level. Bayesian discrete trait phylodynamic analysis (DTA) indicated multiple introductions of H5N8 HPAIV from wild birds into domestic poultry in various regions in South Korea. Following initial viral introduction into domestic duck farms in the western part of Korea, domestic ducks played a major role in viral transmission and maintenance. These findings highlight the need for continued genomic surveillance and pathobiological characterization of HPAIV in birds. Enhanced biosecurity in poultry farms should be implemented to prevent the introduction, maintenance, and spread of HPAIV. The H5 subtype of high pathogenicity avian influenza viruses (HPAIVs) belonging to the Goose/Guangdong (Gs/Gd) lineage have been circulating since 1996 and evolving into genetically diverse clades and subclades . The H5NIn South Korea, four outbreaks of H5N1 viruses have been recorded, in 2003–2004 (clade 2.5), 2006–2007 (clade 2.2), 2008 (clade 2.3.2), and 2010–2011 (clade 2.3.2.1), with the lengths of these outbreaks ranging from 42 (2008) to 139 (2010–2011) days . During Little is currently understood about the evolution and spread of H5N8 HPAIV during the 2014–2016 outbreak in South Korea. Previous studies investigating the origin and transmission of H5N8 HPAIV in South Korea included limited datasets, such as partial genome sequences of early isolates or selected viruses . The preFecal samples, bird carcasses, and oropharyngeal and cloacal swabs were collected from poultry farms and wild bird habitats in South Korea through active and passive surveillance from January 2014 to June 2016. Viruses were isolated by inoculating samples into 9–11-day-old specific-pathogen-free (SPF) embryonated chicken eggs after incubation for 4 days at 37°C.Viral RNA was extracted from the infected allantoic fluids of SPF chicken eggs using Maxwell® RSC simplyRNA Tissue Kits . Complementary DNA was synthesized by reverse transcription reaction using Superscript III First-Strand Synthesis System  and eight segments of each virus were amplified by PCR using AccuPrime Pfx DNA Polymerase , as described . The comhttp://www.gisaid.org). Furthermore, our data set was added 20 sequences of the H5N8 viruses isolated from poultry and wild waterfowl in Japan. Maximum-likelihood (ML) phylogenies of the 493 concatenated complete genome sequences were generated using the Randomized Axelerated Maximum Likelihood (RAxML 8.2.12)   method, 50), five 4-week-old SPF white leghorn chickens each were inoculated intranasally with serial 10-fold dilutions (103 to 106 mean egg infectious dose [EID50]) of each virus. Viral transmissibility was evaluated by housing three virus-naïve chickens with the chickens challenged with 106 EID50/0.1 ml of each virus 8 h after infection. The chickens were monitored daily for clinical signs for 14 days. Oropharyngeal and cloacal swabs were collected at 1–7, 10, and 14 days post-infection (dpi). Serum was collected from surviving chickens at 14 dpi to determine seroconversion by hemagglutination inhibition test (HI)  were collected for viral titration. Virus titers were measured in DF-1 cells incubated with 10% tissue homogenates. Experiments in animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Animal and Plant Quarantine Agency (APQA) . All procedures were carried out in a biosafety level three facility at the APQA.The objective of this study was to evaluate the infectivity, transmissibility, and pathogenicity of the first detected viruses (index virus) of three subgroups  of H5N8 HPAIVs in chickens. Viruses evaluated in chickens included A/broiler duck/Korea/H1731/2014 , A/mallard/Korea/H2102/2015 , and A/mallard/Korea/H1924/2014 . To determine the mean bird lethal dose (BLDest (HI) . The tishttps://www.hiv.lanl.gov/content/sequence/ENTROPY/entropy.html). Shannon entropy differences (Hdiff) were calculated for each gene segment, with differences in variability considered significant when p < 0.05.Genomic variability between H5N8 HPAIVs derived from ducks and other hosts was evaluated by genome-wide Shannon entropy analysis using the entropy tool in the HIV sequence database .Gene- and site-specific selection pressures for all segments of the H5N8 viruses isolated in South Korea during 2014–2016 were measured as the ratio of non-synonymous substitutions (dN) to synonymous substitutions (dS) per site. These analyses were performed using a combination of four methods: single-likelihood ancestor counting (SLAC), fixed-effects likelihood (FEL), fast unconstrained Bayesian approximation (FUBAR), and a mixed effects model of evolution (MEME) available at the Datamonkey online version of the Hy-Phy package –21. The http://www.gisaid.org). An ancestral state reconstruction approach and Bayesian stochastic search variable selection (BSSVS) were utilized to determine the most probable transmission history. Geographically discrete nominal categories were assigned at the province level for viruses collected in Korea; these included “Wild Bird” (WB) (ntax = 115 sequences), “Jeollanam-do” (JN) (ntax = 115 sequences), “Jeollabuk-do” (JB) (ntax = 63 sequences), “Gyeonggi-do” (GG) (ntax = 77 sequences), ‘Gyeongsang-do' (GS) (ntax = 22 sequences), “Chungcheongnam-do” (CN) (ntax = 43 sequences), “Chungcheongbuk-do” (CB) (ntax = 69 sequences), “Gangwon-do” (GW) (ntax = 2 sequences), and “Jeju-do” (JJ) (ntax= 1 sequence). To account for potential sampling biases, sequences were subsampled to preserve diversity relative to geographic locations and host species. The WB, JN, JB, GG, and CB datasets were subsampled based on nucleotide sequence identities of 99.98, 99.9, 99.95, 99.95, and 99.95%, respectively, using CD-HIT (Hemagglutinin (HA) gene sequences were subjected to Bayesian discrete trait phylodynamic analysis (DTA) to investigate virus transmission history among regions and host species. These analyses included all 62 available complete HA genome sequences of clade 2.3.4.4A H5N8 HPAIVs identified in wild waterfowl in East Asia during 2013–2016 that had been downloaded from the GISAID EpiFlu database (g CD-HIT . The GW http://tree.bio.ed.ac.uk/software/tracer/). A maximum clade credibility (MCC) tree was generated using TreeAnnotator and visualized using FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). The number of transition events and the discrete state proportion over time in monthly intervals were calculated based on analyses of posterior trees using the program PACT (http://www.trevorbedford.com/pact).DTA was also performed to determine the transmission dynamics between host species, including “Wild Bird” (ntax = 115 sequences), “Duck” (ntax = 294 sequences), “Chicken” (ntax = 71 sequences), and “minor poultry” (ntax =27 sequences). The Wild Bird, Duck, and Chicken datasets were subsampled using CD-HIT, based on nucleotide identities of 99.95, 99.87, and 100%, respectively . This suThe impact of viral source/sink sample sizes on Bayesian discrete inference datasets was determined using generalized linear model (GLM) analysis as an extension of DTA . GLM calA ML phylogenetic tree of concatenated whole genome sequences of 493 H5N8 HPAIVs isolated in South Korea and Japan during 2014–2016 was generated to determine the genetic relationship among isolates . The virn = 82), CC (n = 72), GG (n = 41), GS (n = 17), GW (n = 2), and JJ (n = 1) provinces (n = 101) was the predominant H5N8 HPAIV isolated from poultry from September 2014 to November 2015, especially in domestic ducks (n = 74), with 57, 25, 12, and 3 obtained from ducks in JL, CC, GG, and GS provinces, respectively  of 5 days. By contrast, all chickens inoculated with 106 EID50/0.1 ml of the H2102 and H1924 viruses died in less than 4 days with MDTs of 3.8 and 3.7 days, respectively. The BLD50 of the H1731, H2102, and H1924 viruses were 104.4, 104.5, and 103.5 EID50/0.1 ml, respectively. Viral shedding of H1731 into the oropharynx and cloaca was detected from 2 to 6 dpi, whereas viral shedding of H2102 and H1924 were detected from 1 to 4 dpi (10 median tissue culture infectious dose [TCID50/0.1 ml]) than in chickens inoculated with H2102 (5.25 log10TCID50/0.1 ml) and H1924 (4.3 log10TCID50/0.1 ml). None of the surviving SPF chickens shed virus or showed an HI response.To investigate the differences in pathobiology among viral subgroups, chickens were intranasally inoculated with H1731, H2102, or H1924 virus, defined as the index viruses of the C1, C2, and C4 subgroups, respectively . All chito 4 dpi . The pea10TCID50/0.1 ml) into the oropharynx (10TCID50/0.1 ml) and cloaca (1.0–2.5 log10TCID50/0.1 ml). Chickens cohoused with the H1731- and H2102-inoculated chickens survived longer, with MDTs of 8 and 7.9 days, respectively, than chickens cohoused with H1924-inoculated chickens, with an MDT of 6.1 days.Analysis of viral transmission showed that one of the three chickens cohoused with the H1731-inoculated chickens died and shed virus (1.3–3.3 logopharynx . By contp < 0.001 each). In addition, viral titers in the kidney, lung, and cecal tonsil were significantly lower in H1731- than in H1924-inoculated chickens (p < 0.05 each), and viral titers in the thymus of H1731-inoculated chickens were significantly lower than titers in the thymus of chickens inoculated with H1924 (p < 0.01) and H2102 (p < 0.05) viruses.All viral strains replicated in all collected tissues, including brain, heart, kidney, lung, cecal tonsil, pancreas, proventriculus, spleen, muscle, liver, thymus, and trachea . Viral tCalculations of Shannon entropy for all positions of the coding regions identified 53 dominant mutations in duck-derived H5N8 HPAIVs when compared with other host-derived H5N8 viruses . H1731 .Analyses of the selection profiles of the H5N8 HPAIVs identified individual codons under positive selection pressure . Nine am−3 substitutions/site/year (95% BCI: 5.53 x 10−3 to 8.80 x 10−3), within the global range for AIV substitution rate  of all viruses identified in Korea was estimated to have arisen on July 2, 2013 , indicating that the H5N8 viruses of wild bird origin in South Korea most likely emerged during the breeding season of wild waterfowl in 2013 . The meaion rate .Source sink dynamics of the H5N8 HPAIVs identified in South Korea during 2014–2016 were analyzed by estimating the transition rate (TR), Markov jump count, and Markov rewards. Based on our DTA, well-supported transitions from wild birds to domestic ducks , chickens , and minor poultry , indicate that multiple virus introductions from wild birds to poultry species occurred mainly from August 2013 to March 2014 and from October 2014 to January 2015 . The welThe DTA among provinces in South Korea involved seven discrete nominal categories: CB, CN, GG, GS, JB, JN, and WB. GLM analysis showed that sample size had a negligible effect on potential bias toward the origin or destination state in this analysis . The DTA indicated multiple introductions of H5N8 HPAIV from wild birds into domestic poultry in various regions in South Korea ; Table 4AIVs evolve through the gradual accumulation of mutations and genome reassortment. H5N8 HPAIVs likely had opportunities to reassort with various AIV gene pools during the 28 months of the H5N8 HPAI outbreak in 2014–2016 in South Korea, which could result in the generation of novel genotypes. However, there was no evidence of reassortment with other AIVs. ML phylogenetic analysis showed that the C0 subgroup had diverged into multiple subgroups within South Korea. The detection of four distinct subgroups, C1, C2, C4, and C5, diverging from C0 viruses in the phylogenetic tree suggests that each subgroup had evolved independently within South Korea during 2014–2016 without any reassortment. These findings indicate that the H5N8 HPAIV genotype introduced into South Korea was a fixed genome constellation with a high fitness produced in the Eurasian AIV gene pool. Interestingly, the H5N8 HPAIV spread into North America in late 2014 and reassorted with North American low-pathogenicity AIVs to produce the novel Eurasian-North American reassortant H5N1, H5N2, and H5N8 viruses (1.5 to 103.4 (TCID50/0.1 ml) and from 101.5 to 102.8 (TCID50/0.1 ml), respectively, whereas the amounts of Buan2 virus shed into these tissues ranged from 104.2 to 106.3 (TCID50/0.1 ml) and from 103.1 to 107.5 (TCID50/0.1 ml), respectively. Viral titers in tissue samples were also lower in chickens inoculated with H1731 than with buan2 virus  had a longer MDT, significantly lower viral titers in tissues, and lower transmissibility in chickens compared with H2102 (C2) and H1924 (C4). The virulence of H1731 in chickens was also lower than that of the buan2 (the index virus of C0) . In partn2 virus . These fn2 virus . ShannonLong-term circulation of AIVs in a certain host species allows viruses to adapt to that species, affecting their virulence and host specificity via genetic evolution. Two amino acid residues (positions 219 and 757 in PB1) in C0, C1, and C2 were found to be under positive selection pressure, with Shannon entropy analysis showing significant mutations at these positions in H5N8 HPAIVs from ducks. Because viruses of the C0, C1, and C2 subgroups mainly circulated in domestic duck farms during the 2014–2015 outbreak, these results suggest that these viruses may have undergone positive selection in domestic ducks. Amino acid substitutions that occurred during the adaptation of H5 HPAIVs to a particular host were found to be related to viral pathogenicity in that host . H5N8 HPThe estimated mean tMRCA of all H5N8 HPAIVs identified in Korea was July 2013, indicating that the H5N8 HPAIVs of wild bird origin detected in South Korea most likely emerged during the breeding season of wild waterfowl in 2013. These findings were consistent with other studies on the estimated tMRCA of H5N8 HPAIV , 37. PhyThe western region of South Korea contains abundant habitats for wild waterfowl and a high density of domestic duck farms , creatinViral phylodynamic analysis also showed exchanges of HPAIVs between minor poultry populations and domestic ducks in South Korea, indicating that minor poultry may serve as reservoirs to maintain and disseminate HPAIV. Minor poultry are generally raised in small outdoor operations and backyards, and are usually marketed in live bird markets (LBMs), highlighting their importance as intermediary hosts in virus transmission under poor biosecurity conditions . LBMs inin vivo experiments support that the possible adaptation of H5N8 HPAIV in domestic ducks likely reduced its virulence in chickens. Enhanced genomic surveillance and pathobiological characterization of the viruses are essential for better understanding of HPAI epidemiology and the design of prevention strategies.Collectively, the present study presents unique molecular epidemiology and pathobiology of the H5N8 HPAIV outbreak in 2014–2016 in South Korea. Our data showed multiple introductions of the H5N8 HPAIV isolated in South Korea during 2014-2016 from wild waterfowl to poultry farms in multiple provinces. The virus, initially introduced into the western part of South Korea, which contains large populations of domestic ducks, was subsequently disseminated into other regions throughout the country. Furthermore, domestic ducks likely played a pivotal role in the persistent circulation of the virus, with minor poultry also serving as a source population. In addition, sequence analysis and http://www.gisaid.org) and accession no. EPI_ISL_157609-EPI_ISL_410213.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: GISAID EpiFlu database  .Y-GB: writing. Y-JL: supervision and funding acquisition. E-KL: project administration. D-HL: writing—review and editing. Y-JS and Y-RP: NGS sequencing. DC: host transition analysis. J-HK: posterior tree analysis. G-BH: virus isolation. All authors have read and agreed to the published version of the manuscript.This work was supported by the Animal and Plant Quarantine Agency (APQA) and Ministry of Agriculture, Food and Rural Affairs of South Korea (MAFRA), via grant B-1543418-2021-23-01.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The neurotrophin brain-derived neurotrophic factor (BDNF), which acts as a transducer, is responsible for improving cerebral stroke, neuropathic pain, and depression. Exercise can alter extracellular nucleotide levels and purinergic receptors in central nervous system (CNS) structures. This inevitably activates or inhibits the expression of BDNF via purinergic receptors, particularly the P2X receptor (P2XR), to alleviate pathological progression. In addition, the significant involvement of sensitive P2X4R in mediating increased BDNF and p38-MAPK for intracerebral hemorrhage and pain hypersensitivity has been reported. Moreover, archetypal P2X7R blockade induces mouse antidepressant-like behavior and analgesia by BDNF release. This review summarizes BDNF-mediated neural effects via purinergic receptors, speculates that P2X4R and P2X7R could be priming molecules in exercise-mediated changes in BDNF, and provides strategies for the protective mechanism of exercise in neurogenic disease. The neurotrophin brain-derived neurotrophic factor (BDNF) is a key mediator of neuroplastic changes induced by exercise. Physical activity has demonstrated positive effects, such as increased adult neurogenesis, in preventing and ameliorating a wide range of brain diseases. Disquietingly, it is possible to affirm that 46.6% of Parkinson’s disease (PD) patients in the Netherlands were less physically active since the COVID-19 lockdown, and reduced physical activity correlated with exacerbating PD symptoms  . MeanwhiMore evidence is still needed to clearly define the exercise-related BDNF mechanisms. On the other hand, the evidence supporting a role for exercise in purines has remained ambiguous. ATP was not only thought to be solely the universal energy currency but also in the nervous system is released into the extracellular space by neurons, astrocytes, and microglia through Panx1 channels to mediate inflammation and glial cell proliferation, which is engaged in the development of diseases such as stroke, epilepsy, and chronic pain . PurinerPhysical exercise is a well-known and established method for the prevention and treatment of many diseases  recognized by the American College of Sports Medicine (ACSM) –29. ImpoA group of researchers investigated the role of physical exercise in preventing and treating diseases and its relationship with purinergic signaling. Studies were carried out to investigate the modulation of the purinergic system  by physical exercise. Submitting rats to 20 min of a daily moderate treadmill for 2 weeks can significantly reduce adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis in the hippocampal synaptosomes and blood serum , suggestBDNF has been described to increase neuronal excitability and synaptic plasticity , 18, whe2+/calmodulin-dependent kinase (CaM kinase) [2+ excitability [It is accepted that P1 and P2Rs induce changes in BDNF expression in relevant CNS structures. A2AR activation is a determinant of the inhibitory actions of BDNF on GABA release and Glu release , the exp kinase) . Howevertability .Typically, neuropathic pain is characterized by hyperexcitability of the primary afferent sensory neurons accompanied by the release of transmitters or mediators such as BDNF  and glutGrowing evidence suggests that exercise alleviates neuropathic pain in rats. Six weeks of voluntary rotation before the onset of chronic compression injury prevented the full development of ectopic pain, normalized the expression of excitatory interleukin (IL)-1β, and decreased the expression of the P2X4R-BDNF axis in the dorsal spinal cord of the ipsilateral spinal cord . High-in2+ influx, phosphorylation of p38, and synthesis and release of BDNF, causing disinhibition of nociceptive projection neurons [+ production has traditionally been explained by the exercise-induced increased production of lactic acid [P2X4R expression in microglia and the subsequent release of BDNF are both required for hyperalgesia. Specifically, P2X4R activation by ATP at the cell surface leads to Ca neurons . When P2 neurons . Basical neurons , 10 and  neurons . Moreove neurons . Further neurons . Therefotic acid .Brain expression of P2X7R has been described in all intrinsic cells of the CNS, and functional P2X7R and downstream signaling pathways are extremely debated. Previous observations have described that longer C-terminal domains of P2X7R are responsible for the activation of downstream signaling pathways, including the ERK pathway and activation of caspase-3, resulting in the initiation of apoptosis , 54. Gen−/− mice compared with P2X7R+/+ controls indicate tonic inhibitory regulation of BDNF production through P2X7R [Traditionally, the expression of P2X7 is negatively correlated with BDNF. The P2X7R antagonist A-804598  was used to investigate BDNF signaling in the rat frontal cortex and ventral and dorsal hippocampus. The results showed that antagonizing P2X7R may activate TrkB and mediate an increase in BDNF-AKT-p70 S6 kinase levels in the ventral hippocampus, which produces a related antidepressant effect . Elevategh P2X7R . In linegh P2X7R . Unfortugh P2X7R , 62. Anogh P2X7R . More imgh P2X7R . It has gh P2X7R . TherefoInterestingly, preconditioning exercise significantly reduced infarct volume and apoptosis, in which P2X7R and BDNF in the ischemic brain were significantly upregulated. Following intracerebral hemorrhage, ATP administration had the ability to relieve cerebral hemorrhage–induced injury and improve cerebral neurological function by upregulating the mBDNF/proBDNF ratio and p38-MAPK. However, whether P2X7R has a clear regulatory effect on BDNF has not been clearly demonstrated in the area of stroke improvement .Owing to the relatively few discussions of the purinergic constituents in neuroprotection during or after exercise, it is pressing to explore their physiology. As discussed in this review, some P1/P2Rs have been recognized as significant mediators of BDNF expression, which participate in multiple pathologies, including stroke, neuropathic pain, and even depression. Both P2X4R and P2X7R influence BDNF activation, leading to accumulation of p38-MAPK and upregulation of Iba1 and pERK1/2. A1, A2A, A2B, P2X4, P2X7, and P2Y11Rs have been shown to participate in regulating BDNF expression. However, interactions between the downstream signaling mechanisms of P2X7R may also occur. Eventually, P2X4R and P2X7R may complement neural effects due to the release of BDNF by exercise.