A 2024 human HPAI H5N1 case in British Columbia showed mixed viral populations containing HA-190D (28%) and HA-226H (35%) variants in tracheal aspirate sequencing. In this study, solid-phase binding assay and molecular docking were used to evaluate the contribution of HA-E190D/Q226H mutations to the viral receptor profiles. Our results showed that HA-E190D marginally reduced sialic acid α2,3 receptors' affinity, while HA-Q226H impaired both sialic acid α2,3 and α2,6 receptors' binding. These results demonstrate that neither mutation strengthens viral binding to human-type receptors, indicating that such substitutions are unlikely to heighten the public health threat posed by the virus for now.

H9N2 avian influenza viruses inherently carry cross-species transmission potential, making continuous surveillance critical for pandemic prevention. This study focused on monitoring the 2024 H9N2 epidemic in Jiangsu Province’s external environment, analyzing its molecular evolution and receptor binding properties, assessing cross-species transmission and pandemic risks, and investigating serological antibody levels across different human populations. Environmental samples were collected from live poultry markets, farms, slaughterhouses, and bird habitats across Jiangsu, screened via quantitative PCR (qPCR), with positive samples used for virus isolation and whole-genome sequencing. Receptor binding properties were tested by hemagglutination assay, and H9N2 antibody levels were measured in 370 occupationally exposed individuals and 240 non-exposed individuals using hemagglutination inhibition (HI) assays. Among the 5779 collected samples, 6.89% tested H9N2-positive, and 12 strains belonging to the Eurasian lineage Y280-like clade G57 genotype were successfully isolated. All strains carried the HA-Q226L mutation, with 11 showing preferential binding to human α-2,6 receptors and one strain possessing dual receptor binding capability. Internal genes harbored mammalian adaptation mutations, and M2 proteins contained mutations conferring complete resistance to amantadine-class antiviral drugs. Serological tests revealed antibody positive rates of 4.05% in exposed populations and 2.5% in non-exposed populations, with no statistically significant difference between groups. These findings confirm that Jiangsu’s circulating H9N2 viruses have acquired human receptor preference and mammalian adaptation, posing silent infection and pandemic risks. Enhanced surveillance and the development of candidate vaccine stockpiles are strongly recommended.

H5N1, a highly pathogenic avian influenza virus, presents pandemic risks due to its ability to adapt and spread among mammalian species. Vaccination may control its spread, but the effectiveness of existing H5N1 vaccines against circulating strains, especially clade 2.3.4.4b, remains uncertain. In this study, we assess neutralizing antibody responses to global circulating H5N1 strains, using sera from individuals vaccinated with an inactivated H5N1 vaccine (NCT00535665). Neutralization is measured against 17 pseudoviruses, representing circulating and vaccine H5 strains. Our results indicate that broad protective effects are observed only when high antibody titers are achieved by vaccination. Correlation analysis estimates that a pseudovirus-based neutralization titer of at least 1:980 is required to achieve a cross-protection rate above 60%. The findings suggest that the current H5N1 vaccine can elicit cross-neutralization of circulating H5N1 strains, if high antibody titers are achieved. Until updated H5N1 vaccines are developed, this vaccine may serve as a bridging measure.

Multiple subtypes of avian influenza virus (AIV), including H5N1, H5N6, and H5N8 viruses, are currently co-circulating in wild birds and poultry and causing sporadic human infections. Vaccine development is essential for pandemic preparedness. In this study, we constructed a candidate vaccine virus (CVV) using reverse genetics (RG) based on the sequence of the first human-infected H5N8 subtype AIV, A/Astrakhan/3212/2020 (H5N8). We evaluated the immunogenicity of the rH5N8/PR8 vaccine strain in combination with Alum, ISA51, and MF59 adjuvants, and we optimized immunization strategies including dosage, administration route, and immunization interval in BALB/c mice. Our results demonstrated that a 10 μg dose of inactivated rH5N8/PR8 with MF59 adjuvant, administered intramuscularly twice at 7-day intervals, induced the strongest immune response and effectively protected mice against challenge with wild-type H5N8 AIVs. Since pandemic influenza vaccines typically require tailored vaccination doses and routes specific to their characteristics, this study provides valuable insights for the development of similar vaccine strains with pandemic potential.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both alpha 2,3 and alpha 2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn. [GRAPHICS]

Pigs are vital genetic mixing vessels for human and avian influenza viruses because their tracheal epitheliums possess both sialic acid α-2,6-Gal and α-2,3-Gal receptors. Cross-species transmission of influenza A viruses from swine to humans occurs occasionally. The first case of human infection with the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) genotype G4 was detected in Jiangsu Province, China, in February 2023, and backtracking epidemiological investigations did not reveal a clear source of the infection. The hemagglutination (HA) and neuraminidase (NA) amino acid variant sites, antiviral drug susceptibility, and antigenic variation of the isolated A/Jiangsu/27271/2023 (JS/27271/23) virus were analyzed, and we evaluated the protective effect of sera collected from occupationally exposed populations in 2024 against the virus. Compared with the vaccine strain, the nucleotide sequence similarities of JS/27271/23 HA and NA were 96.5 % and 95.2 %, respectively. JS/27271/23 was sensitive to polymerase inhibitors (favipiravir and baloxavir), and the antigenicity of its HA protein was 8-fold different from that of the vaccine strain. The percentage of occupationally exposed population with antibody titers of ≥ 40 against A/Hunan/42443/2015 (HN/42443/15) and A/Jiangsu/1/2011 (JS/1/11) were 7.25 % and 2.25 %, respectively, and the geometric mean titers (GMT) were 6.24 and 5.34, respectively. Out of 400 serum samples examined, none had antibody titers of ≥ 40 against JS/27271/23. This suggests that low serum levels of antibodies to EAH1N1 SIVs in occupationally exposed populations may not provide adequate protection because of significant differences in amino acid sites and antigenicity between this virus and the current vaccine strain of EAH1N1 SIVs. There is no evidence of human-to-human transmission of EAH1N1 SIVs. Therefore, surveillance for EAH1N1 SIVs and the development of new vaccine strains are required.

Since 2022, three human cases of a novel H3N8 avian influenza virus infection have been reported in three provinces in China. Specific vaccines are important means of preparing for the potential influenza pandemic. Thus, H3N8 viruses [A/Henan/cnic410/2022 (HN410) and A/Changsha/1000/2022(CS1000)] were isolated from the infected patients as prototype viruses to develop candidate vaccine viruses (CVVs) using the reverse genetics (RG) technology. Five reassortant viruses with different HA and NA combinations were constructed based on the two viruses to get a high-yield and safe CVV. The results showed that all viruses had similar antigenicity but different growth characteristics. Reassortant viruses carrying NA from CS1000 exhibited better growth ability and NA enzyme activity than the ones carrying HN410 NA. Furthermore, the NA gene of CS1000 had one more potential N-glycosylation site at position 46 compared with HN410. The substitution of position 46 showed that adding or removing N-glycosylation sites to different reassortant viruses had different effects on growth ability. A reassortant virus carrying HN410 HA and CS1000 NA with high growth ability was selected as a CVV, which met the requirements for a CVV. These data suggest that different surface gene combinations and the presence or absence of potential N-glycosylation sites on position 46 in the NA gene affect the growth characteristics of H3N8 CVVs.

Background The first and only case of human infection with the avian influenza A (H7N4) virus in China emerged in 2018. The H7N4 virus was distinct from previous H7N9 viruses and raised public concerns. Therefore, developing a suitable H7N4 candidate vaccine virus (CVV) remains crucial for potential pandemic preparedness. Methods We constructed a reassortant virus with a (6 + 2) genome composition, then introduced the polymerase basic protein 1 (PB1) from a wild-type virus to develop a (5 + 3) reassortant virus through reverse genetics. We performed whole-genome sequencing to confirm the genome stability, assessed the growth ability in MDCK cells, and analyzed virus antigenicity using hemagglutination inhibition assays. Subsequently, the effect of homologous PB1 on polymerase activity, viral protein yield, and pathogenicity was assessed. Results The (5 + 3) virus harbouring the homologous PB1 gene exhibited significantly improved growth characteristics, higher viral protein yield, and polymerase activity than the (6 + 2) virus. After successive passage in embryonated eggs, glutamic acid (E) substituted glycine(G) at position 218 (H3 numbering) in the hemagglutinin (HA) gene of both (5 + 3) and (6 + 2) viruses. The substitution improved the growth of the (6 + 2) virus but exhibited no significant effect or alteration on the antigenicity of the (5 + 3) virus. Moreover, the (5 + 3) virus exhibited low pathogenicity in chickens and ferrets. Conclusion Homologous PB1 of the H7N4 virus improves the growth ability while sustaining low pathogenicity. Collectively, the gene composition of the (5 + 3) reassortant virus is a suitable H7N4 CVV for potential pandemic preparedness.

The recent rise in the frequency of influenza A(H5N6) infections in China has raised serious concerns about whether the risk for human infection has increased. We surveyed epidemiologic, clinical, and genetic data of human infections with A(H5N6) viruses. Severe disease occurred in 93.8% of cases, and the fatality rate was 55.4%. Median patient age was 51 years. Most H5N6 hemagglutinin (HA) genes in human isolates in 2021 originated from subclade 2.3.4.4b; we estimated the time to most recent common ancestor as June 16, 2020. A total of 13 genotypes with HA genes from multiple subclades in clade 2.3.4.4 were identified in human isolates. Of note, 4 new genotypes detected in 2021 were the major causes of increased H5N6 virus infections. Mammalian-adapted mutations were found in HA and internal genes. Although we found no evidence of human-to-human transmission, continuous evolution of H5N6 viruses may increase the risk for human infections.

Seasonal influenza A (H3N2) virus has been a concern since its first introduction in humans in 1968. Accumulating antigenic changes in viral hemagglutinin (HA), particularly recent cocirculations of multiple HA genetic clades, allow H3N2 virus evade into humans annually. From 2010, the binding of neuraminidase (NA) to sialic acid made the traditional assay for HA inhibition antibodies (Abs) unsuitable for antigenicity characterization. Here, we investigated the serum anti-NA response in a cohort with a seroconversion of microneutralizing (MN) Abs targeting the circulating strain, A/Singapore/INFIMH-16-0019/2016 (H3N2, 3C.2a1)-like, a virus during 2018/2019 flu seasons. We discovered that MN Ab titers show no difference between children and adults. Nevertheless, higher titers of Abs with NA activity inhibition (NI) activity of 129 and seroconversion rate of 68.42% are presented in children aged 7-17 years (n = 19) and 73.47 and 41.17% in adults aged 21-59 years (n = 17), respectively. The MN Abs generated in children display direct correlations with HA- and NA-binding Abs or NI Abs. The NI activity exhibited cross-reactivity to N2 of H3N2 viruses of 2007 and 2013, commonly with 329-N-glycosylation and E344 in N2, a characteristic of earlier 3C.2a H3N2 virus in 2014. The percentage of such viruses pronouncedly decreased and was even replaced by those dominant H3N2 viruses with E344K and 329 non-glycosylation, which have a significantly low activity to the tested antisera. Our findings suggest that NI assay is a testable assay applied in H3N2 infection in children, and the antigenic drift of current N2 should be considered for vaccine selection.

Influenza vaccines represent the most effective preventive strategy to control influenza virus infections; however, adaptive mutations frequently occur in the hemagglutinin (HA) glycoprotein during the preparation of candidate vaccine virus and production of vaccine in embryonated eggs. In our previous study, we constructed candidate vaccine virus (HA-R) to match the highly pathogenic avian influenza H7N9 viruses A/Guangdong/17SF003/2016 as part of a pandemic preparedness program. However, mixed amino acids (R, G, and I) were presented at position 220 (H3 numbering) in HA during passage in embryonated eggs. The residue at position 220 is located close to the receptor-binding site and the biological characteristics of this site remain to be elucidated. Therefore, in this study, using reverse genetics, we constructed two viruses carrying the single substitution in position 220 of HA (HA-G and HA-I) and evaluated the biological effects of substitution (R with G/I) on receptor binding, neuraminidase (NA) activity, growth characteristics, genetic stability, and antigenicity. The results revealed both mutant viruses exhibited lower HA binding affinities to two receptor types (sialic acid in alpha2,3- and alpha2,6-linkage to galactose, P < 0.001) and significant better growth characteristics compared to HA-R in two cells. Moreover, under similar NA enzymatic activity, the two mutant viruses eluted more easily from agglutinated erythrocytes than HA-R. Collectively, these results implied the balance of HA and NA in mutant viruses was a stronger determinant of viral growth than the individual amino acid in the HA position 220 in HA-R without strong binding between HA and sialylated receptors. Importantly, both the substitutions conferred altered antigenicity to the mutant viruses. In conclusion, amino acid substitutions at position 220 can substantially influence viral biological properties.

Objective:To understand the agglutination characteristics of different subtypes of avian influenza viruses, we selected erythrocytes from different sources to find suitable erythrocytes for influenza environmental sample detection.Methods:Different subtypes of avian influenza viruses, which were isolated from environmental sample between 2009 and 2016 in China, were selected to do hemagglutination assay using 5 animal erythrocytes (chicken, turkey, guinea pig, horse, and sheep). Flow cytometry was used to detect expression level and type of sialic acid receptors of different erythrocytes, and the characteristics of the receptor binding domain (RBD) of the viral hemagglutinin protein were analyzed by amino acid sequence.Results:In this study, a total of 28 strains of avian influenza virus including 14 subtypes were detected. The result showed that all viruses could agglutinate with turkey and guinea pig erythrocytes and the rest three erythrocytes were unable to produce agglutination with some viruses; among them, one H9N2 virus (A/environment/Anhui/43762/2015) did not agglutinate with chicken erythrocytes, one H1N1 virus (A/environment/Shandong/76972/2014) and two H9N2 viruses (A/environment/Chongqing/79449/2014 and A/environment/Anhui/43762/2015) did not agglutinate with horse erythrocytes, two viruses of H9N2 (A/environment/Chongqing/79449/2014 and A/environment/Anhui/43762/2015) and two viruses of H13N8 (A/environment/Qinghai Lake/166/2012 and A/environment/Qinghai Lake/13/2012) did not agglutinate with sheep erythrocytes. The result of flow cytometry showed that two sialic acid receptors, α-2, 3 and α-2, 6, were detected on the surface of erythrocytes of turkey, chicken and guinea pig, but the expression ratios of the two receptors were different. Only the expression of α-2, 3 sialic acid receptors was detected in horse and sheep erythrocytes. Sequence analysis suggested that amino acid substitution in key regions of viral hemagglutinin protein RBD may be an important factor affecting the binding properties of different erythrocytes.Conclusions:Our result suggested that turkey and guinea pig erythrocytes are the most sensitive in the hemagglutination test. Receptor expression and type of erythrocytes from different sources can significantly affect the agglutination reaction of different subtypes of avian influenza virus, and the amino acid changes in key regions of RBD can also affect the result of agglutination reaction.

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC 50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log 10 TCID 50 /ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.

Objective In China, 24 cases of human infection with highly pathogenic avian influenza (HPAI) H5N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5N6 candidate vaccine viruses (CVVs). Methods In accordance with the World Health Organization (WHO) recommendations, we constructed two reassortant viruses using reverse genetics (RG) technology to match the two different epidemic H5N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo. Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs. Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics, which may aid in our preparedness for a potential H5N6 pandemic.

Objective Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection in vivo. Methods We performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs of Ifitm3-/- and wild-type mice. Results Ifitm3-/- mice developed more severe inflammation and apoptotic responses compared to wild-type mice. Moreover, the NK cell activation was higher in the lungs of Ifitm3-/- mice during acute influenza infection. Conclusions Based on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in Ifitm3-/- mice.

Since the first confirmed case of H7N9 infection was reported in China, there have been five epidemic waves of human H7N9 infections between 2013 and 2017. The fifth wave differed from the previous four waves in that highly pathogenic avian influenza (HPAI) H7N9 viruses with multiple basic amino acids at the cleavage site were detected in humans, poultry and environmental samples. The HPAI H7N9 viruses were genetically and antigenically distinct from previous H7N9 viruses. Therefore, a new candidate vaccine virus(CVV) derived from a HPAI A/Guangdong/17SF003/2016-like virus was proposed by the World Health Organization(WHO). According to the WHO recommendations, we constructed a new CVV using reverse genetic technology, with a (6+2) gene constitution. The (6+2) reassortant virus possessed hemagglutinin(HA) with multiple basic amino acids removed and the neuraminidase from A/Guangdong/SF003/2016 in a high-yield A/Puerto Rico/8/34 virus backbone. Sequence analysis confirmed that no mutations had occurred in the HA of V1E1(the initial CVV rescued in Vero cells and followed by passage in eggs), but a mixture of arginine (R)/glycine (G)/isoleucine (I) was detected at position 220 (H3 numbering) in the HA of V1E2 to V1E5 with different percentages. Furthermore, V1E5 showed improved growth characteristics and immunogenicity compared with V1E1, and retained low pathogenicity in chickens and chicken embryos, but the mutation changed its antigenicity. Our study indicates that antigenic changes should be closely monitored during the development of H7N9 CVV in eggs. Additionally, although V1E5 changes the antigenicity, the antisera had some reactivity to previous H7N9 CVVs.

Objective To isolate the cross-reactive antibodies against hemagglutinin of influenza virus and identify its biological function.Methods The antibodies gene reservoir of cross-reactive and H5N1 pseudotype particles neutralizing B cell circulating in peripheral blood of a human H5N1 case was recovered by in vitro B cell culture,screening,RT-PCR and expression vector cloning techniques.The Ab gene pairing was screened by transient transfection of human kidney 293T cells and detected using ELISA and neutralization test.The heterosubtypic antibodies were prepared and characterized.Results We discovered the VH1-2-based heterosubtypic antibodies from two B cell lineages could neutralize GX-H5N1 pseudotype particles and have broader binding with Group 1 (including H1,H5,H6 and H9) and H7 subtype.Conclusions Cross-reactive antibodies can be induced by H5N1 infection.

With no or low virulence in poultry, avian influenza A(H7N9) virus has caused severe infections in humans. In the current fifth epidemic wave, a highly pathogenic avian influenza (HPAI) H7N9 virus emerged. The insertion of four amino acids (KRTA) at the haemagglutinin (HA) cleavage site enabled trypsin-independent infectivity of this virus. Although maintaining dual receptor-binding preference, its HA antigenicity was distinct from low-pathogenic avian influenza A(H7N9). The neuraminidase substitution R292K conferred a multi-drug resistance phenotype.

Although exhibiting no or low virulence in poultry, avian influenza virus H7N9 has caused around 1400 confirmed human infections in China with a case-fatality rate of 30% since 2013. A highly pathogenic H7N9 virus (HP-H7), with the HA antigenicity distinct from the previous, were recently detected in patients and poultry. Therefore, convenient rapid diagnosis with reliability will allow early antiviral use and management for H7N9 infection. Here, a sandwich ELISA targeting the conserved viral antigen, neuraminidase (NA) was developed. The immunoassay employed mouse monoclonal antibody (mAb) 3C1 to specifically capture the N9 and 3E9 for the detection. Its limit of detection is 6.25ng/ml for N9 protein of A/Anhui/1/2013(H7N9, AH1/2013) and 0.125HAU/50μL for live virus, AH1/2013 and A/Environment/Jiangxi/28/2009 (H11N9), respectively. When applied to test the five clinic throat swabs from H7N9 patients confirmed by nuclear acid testing (NAT) using quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), two samples showed positive result in sandwich ELISA while all were negative using commercial Flu A and H7 subtype rapid antigen tests (RAT). The ELISA using anti-N9 mAbs provided a valuable approach to detect H7N9 virus and quantify the N9 protein.

Bioactive materials with osteostimulation properties have the potential to promote bone regeneration. We have found that silicate-based biomaterials have the osteostimulation ability for regeneration of large bone defects; however, the corresponding mechanism is unclear. In this study, we set out to elucidate the potential mechanism of silicate-based biomaterials with osteostimulation ability. A model silicate bioceramic, nagelschmidtite (NAGEL, Ca7P2Si2O16), was applied to study their ionic products on the effect of the Bone morphogenic protein (BMP) signaling pathway for osteoblast MC3T3-E1 as NAGEL has been previously shown to have excellent in vitro and in vivo bone-forming activity. BMP signaling, especially BMP2, is involved in bone formation during mammalian development and exhibits versatile regulatory functions in the body. It is found that NAGEL bioceramics significantly enhance the migration and osteoblastic differentiation of MC3T3-E1. mRNA and protein expression of BMP2 is enhanced by NAGEL bioceramics in a dose-dependent manner. Moreover, NAGEL bioceramics activate the Smad-dependent BMP signaling pathway and induce the activation of the BMP downstream cascade (OCN, OPN and Runx2). The accumulation of phosphorylated-Smad1/5 is induced by NAGEL bioceramics in the MC3T3-E1 cell nucleus. It is further found that NAGEL bioceramic-mediated migration, osteoblastic differentiation and the activation of the BMP downstream cascade are significantly downregulated by inhibition of BMP2 activity. Our results suggest that silicate-based NAGEL bioceramics possess excellent in vitro osteostimulation properties and the possible mechanism of silicate-based biomaterials with distinct osteostimulation may be directly related to the activation of the BMP2 signaling pathway of osteoblasts by release of Si-containing bioactive ionic products.