Spatio-temporal spread and evolution of Lassa virus in West Africa.

BACKGROUND: Lassa fever is a hemorrhagic disease caused by Lassa virus (LASV), which has been classified by the World Health Organization as one of the top infectious diseases requiring prioritized research. Previous studies have provided insights into the classification and geographic characteristics of LASV lineages. However, the factor of the distribution and evolution characteristics and phylodynamics of the virus was still limited. METHODS: To enhance comprehensive understanding of LASV, we employed phylogenetic analysis, reassortment and recombination detection, and variation evaluation utilizing publicly available viral genome sequences. RESULTS: The results showed the estimated the root of time of the most recent common ancestor (TMRCA) for large (L) segment was approximately 634 (95% HPD: [385879]), whereas the TMRCA for small (S) segment was around 1224 (95% HPD: [10301401]). LASV primarily spread from east to west in West Africa through two routes, and in route 2, the virus independently spread to surrounding countries through Liberia, resulting in a wider spread of LASV. From 1969 to 2018, the effective population size experienced two significant increased, indicating the enhanced genetic diversity of LASV. We also found the evolution rate of L segment was faster than S segment, further results showed zinc-binding protein had the fastest evolution rate. Reassortment events were detected in multiple lineages including sub-lineage IIg, while recombination events were observed within lineage V. Significant amino acid changes in the glycoprotein precursor of LASV were identified, demonstrating sequence diversity among lineages in LASV. CONCLUSION: This study comprehensively elucidated the transmission and evolution of LASV in West Africa, providing detailed insights into reassortment events, recombination events, and amino acid variations.

c-Abl kinase-mediated phosphorylation of γ-tubulin promotes γ-tubulin ring complexes assembly and microtubule nucleation.

Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function.

Genetic characteristics of classical astroviruses in Shenzhen, China, 2016-2019.

Human astrovirus (HAstV) is a single-stranded, positive-sense RNA virus and is the leading cause of viral gastroenteritis. However, despite its prevalence, astroviruses still remain one of the least studied enteroviruses. In this study, we sequenced 11 classical astrovirus strains from clinical samples collected in Shenzhen, China from 2016 to 2019, analyzed their genetic characteristics, and deposited them into GenBank. We conducted phylogenetic analysis using IQ-TREE software, with references to astrovirus sequences worldwide. The phylogeographic analysis was performed using the Bayesian Evolutionary Analysis Sampling Trees program, through Bayesian Markov Chain Monte Carlo sampling. We also conducted recombination analysis with the Recombination Detection Program. The newly sequenced strains were categorized as HAstV genotype 1, which is the predominant genotype in Shenzhen. Phylogeographic reconstruction indicated that HAstV-1 may have migrated from the United States to China, followed by frequent transmission between China and Japan. The recombination analysis revealed recombination events within and across genotypes, and identified a recombination-prone region that produced relatively uniform recombination breakpoints and fragment lengths. The genetic analysis of HAstV strains in Shenzhen addresses the current lack of astrovirus data in the region of Shenzhen and provides key insights to the evolution and transmission of astroviruses worldwide. These findings highlight the importance of improving surveillance of astroviruses.

Characterization and Diversity of Klebsiella pneumoniae Prophages.

Klebsiella pneumoniae is a common human commensal and opportunistic pathogen. In recent years, the clinical isolation and resistance rates of K. pneumoniae have shown a yearly increase, leading to a special interest in mobile genetic elements. Prophages are a representative class of mobile genetic elements that can carry host-friendly genes, transfer horizontally between strains, and coevolve with the host's genome. In this study, we identified 15,946 prophages from the genomes of 1437 fully assembled K. pneumoniae deposited in the NCBI database, with 9755 prophages on chromosomes and 6191 prophages on plasmids. We found prophages to be notably diverse and widely disseminated in the K. pneumoniae genomes. The K. pneumoniae prophages encoded multiple putative virulence factors and antibiotic resistance genes. The comparison of strain types with prophage types suggests that the two may be related. The differences in GC content between the same type of prophages and the genomic region in which they were located indicates the alien properties of the prophages. The overall distribution of GC content suggests that prophages integrated on chromosomes and plasmids may have different evolutionary characteristics. These results suggest a high prevalence of prophages in the K. pneumoniae genome and highlight the effect of prophages on strain characterization.

A Standardized Framework for Better Understanding of Phenotypic Differences within Bacterial Phyla Based on Protein Domain.

We propose a standardized framework to classify target species based on their protein domains, which can be utilized in different contexts, like eukaryotes and prokaryotes. In this study, by applying the framework to the bacterial kingdom as an implementation example and comparing the results with the current taxonomy standards at the phylum level, we came to the conclusion that the sequence of domains rather than the content of domains in a protein and the presence of one domain rather than the number of occurrences of one domain play more important roles in deciding bacterial phenotypes as well as matching the current taxonomy. In addition, the comparison also helps us to better focus on the species that conflict with the current phylum category, as well as to further investigate their phenotypic or genotypic differences. IMPORTANCE A 3-step framework was designed which can be applied to clustering species based on their protein domains, and different candidate models are proposed in each step for better adaptation of various scenarios. We show its implementation for the bacterial kingdom as an example, which helps us to find the most appropriate model combination that will best reflect the relationship between domains and phenotypes in this context. In addition, identifying species that are distant in the results but should be closely related phylogenetically can help us to focus on the mismatch for better understanding of their key phenotypic or genotypic differences.

Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells.

Upregulation of the neuropeptide neurotensin (NTS) in a subgroup of lung cancers has been linked to poor prognosis. However, the regulatory pathway centered on NTS in lung cancer remains unclear. Here we identified the NTS-specific enhancer in lung adenocarcinoma cells. The AF4/FMR2 (AFF) family protein AFF1 occupies the NTS enhancer and inhibits NTS transcription. Clustering analysis of lung adenocarcinoma gene expression data demonstrated that NTS expression is highly positively correlated with the expression of the oncogenic factor CPS1. Detailed analyses demonstrated that the IL6 pathway antagonizes NTS in regulating CPS1. Thus, our analyses revealed a novel NTS-centered regulatory axis, consisting of AFF1 as a master transcription suppressor and IL6 as an antagonist in lung adenocarcinoma cells.

Comparative Genomic Analysis of Vibrio cincinnatiensis Provides Insights into Genetic Diversity, Evolutionary Dynamics, and Pathogenic Traits of the Species.

Vibrio cincinnatiensis is a poorly understood pathogenic Vibrio species, and the underlying mechanisms of its genetic diversity, genomic plasticity, evolutionary dynamics, and pathogenicity have not yet been comprehensively investigated. Here, a comparative genomic analysis of V. cincinnatiensis was constructed. The open pan-genome with a flexible gene repertoire exhibited genetic diversity. The genomic plasticity and stability were characterized by the determinations of diverse mobile genetic elements (MGEs) and barriers to horizontal gene transfer (HGT), respectively. Evolutionary divergences were exhibited by the difference in functional enrichment and selective pressure between the different components of the pan-genome. The evolution on the Chr I and Chr II core genomes was mainly driven by purifying selection. Predicted essential genes in V. cincinnatiensis were mainly found in the core gene families on Chr I and were subject to stronger evolutionary constraints. We identified diverse virulence-related elements, including the gene clusters involved in encoding flagella, secretion systems, several pili, and scattered virulence genes. Our results indicated the pathogenic potential of V. cincinnatiensis and highlighted that HGT events from other Vibrio species promoted pathogenicity. This pan-genome study provides comprehensive insights into this poorly understood species from the genomic perspective.

Identification of immunogenic outer membrane proteins and evaluation of their protective efficacy against Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia (S. maltophilia) is an emerging global multiple-drug-resistant organism. It becomes increasingly challenging to treat S. maltophilia infection effectively. Novel therapeutic and preventive approaches targeting S. maltophilia infection are still lacking. This study aims to isolate outer membrane proteins (Omps) from S. maltophilia and use immunoproteomic technology to identify potential vaccine candidates of Omps against S. maltophilia infections. METHODS: Omps from S. maltophilia culture were separated by two-dimensional electrophoresis and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry and nano liquid chromatography coupled fourier transform ion cyclotron resonance tandem mass spectrometry. Recombinant Omps were prepared and used to immunize mice, and the potency of mouse anti-Omp serum was tested in opsonophagocytic killing assay (OPKA). The effects of immunization with recombinant Omp on blood and tissue bacterial loads in a mouse model of S. maltophilia-induced infection were analyzed. RESULTS: Outer membrane protein A (OmpA) and Smlt4123 were identified by mass spectrometry. Mouse anti-Smlt4123 serum significantly reduced the bacterial counts in healthy individuals' blood in OPKA (P < 0.05) but mouse anti-OmpA serum did not. Enzyme-linked immunosorbent assay revealed that the antibody subtype of mouse anti-Smlt4123 antibody was IgG1. Eight hours after an intraperitoneal challenge with S. maltophilia, the bacterial loads in mouse blood were significantly lower in the mice receiving immunization with recombinant Smlt4123 than in the control mice receiving no immunization (P < 0.05), whereas the bacterial loads in other organs, such as the liver, spleen, lung, and kidney were similar in the two groups. CONCLUSIONS: The results revealed that the immunoproteomic approach was an efficient way to screen the immunogenic protein of Stenotrophomonas maltophilia. Moreover, the recombinant Smlt4123 had potential to protect mice from bacteremia caused by S. maltophilia in the early stages.

Gene flow, recombination, and positive selection in Stenotrophomonas maltophilia: mechanisms underlying the diversity of the widespread opportunistic pathogen.

Stenotrophomonas maltophilia is a global multidrug-resistant human opportunistic pathogen in clinical environments. Stenotrophomonas maltophilia is also ubiquitous in aqueous environments, soil, and plants. Various molecular typing methods have revealed that S. maltophilia exhibits high levels of phenotypic and genotypic diversity. However, information regarding the genomic diversity within S. maltophilia and the corresponding genetic mechanisms resulting in said diversity remain scarce. The genome sequences of 17 S. maltophilia strains were selected to investigate the mechanisms contributing to genetic diversity at the genome level. The core and large pan-genomes of the species were first estimated, resulting in a large, open pan-genome. A species phylogeny was also reconstructed based on 344 orthologous genes with one copy per genome, and the contribution of four evolutionary mechanisms to the species genome diversity was quantified: 15%-35% of the genes showed evidence for recombination, 0%-25% of the genes in one genome were likely gained, 0%-44% of the genes in some genomes were likely lost, and less than 0.3% of the genes in a genome were under positive selection pressures. We observed that, among the four main mechanisms, homologous recombination plays a key role in maintaining diversity in S. maltophilia. In this study, we provide an overview of evolution in S. maltophilia to provide a better understanding of its evolutionary dynamics and its relationship with genome diversity.

Phylogeography of Avian influenza A H9N2 in China.

BACKGROUND: During the past two decades, avian influenza A H9N2 viruses have spread geographically and ecologically in China. Other than its current role in causing outbreaks in poultry and sporadic human infections by direct transmission, H9N2 virus could also serve as an progenitor for novel human avian influenza viruses including H5N1, H7N9 and H10N8. Hence, H9N2 virus is becoming a notable threat to public health. However, despite multiple lineages and genotypes that were detected by previous studies, the migration dynamics of the H9N2 virus in China is unclear. Increasing such knowledge would help us better prevent and control H9N2 as well as other future potentially threatening viruses from spreading across China. The objectives of this study were to determine the source, migration patterns, and the demography history of avian influenza A H9N2 virus that circulated in China. RESULTS: Using Bayesian phylogeography framework, we showed that the H9N2 virus in mainland China may have originated from the Hong Kong Special Administrative Region (SAR). Southern China, most likely the Guangdong province acts as the primary epicentre for multiple H9N2 strains spreading across the whole country, and eastern China, most likely the Jiangsu province, acts as an important secondary source to seed outbreaks. Our demography inference suggests that during the long-term migration process, H9N2 evolved into multiple diverse lineages and then experienced a selective sweep, which reduced its genetic diversity. Importantly, such a selective sweep may pose a greater threat to public health because novel strains confer higher fitness advantages than strains being replaced and could generate new viruses through reassortment. CONCLUSION: Our analyses indicate that migratory birds, poultry trade and transportation have all contributed to the spreading of the H9N2 virus in China. The ongoing migration and evolution of H9N2, which poses a constant threat to the human population, highlights the need for a more comprehensive surveillance of wild birds and for the enhancement of biosafety for China's poultry industry.

Herpes B virus gD interaction with its human receptor--an in silico analysis approach.

BACKGROUND: The glycoprotein D (gD) is essential for Herpes B virus (BV) entry into mammalian cells. Nectin-1, an HSV-1 gD receptor, is found to be the receptor which mediated BV induced cell-cell fusion, while HVEM does not mediate fusion by BV glycoprotein. However, the specific sequence and structural requirements of the BV gD for the recognition of and binding to Nectin-1 are unknown. Moreover, the 3D structures of BV gD and the BV gD-receptor complex have not been determined. In this study, we propose a reliable model of the interaction of the BV gD with receptor using bioinformatics tools. RESULTS: The three-dimensional structures of two BV gD-receptor complexes were constructed using homology modelling and docking strategy. Based on the models of these complexes, the BV gD receptor interaction patterns were calculated. The results showed that the interface between the BV gD and nectin-1 molecule is not geometrically complementary. The computed molecular interactions indicated that two terminal extensions were the main region of BV gD that binds to nectin-1 and that hydrophobic contacts between the two molecules play key roles in their recognition and binding. The constructed BV gD-HVEM complex model showed that this complex had a lower shape complementarity value and a smaller interface area compared with the HSV-1 gD-HVEM complex, and the number of intermolecular interactions between BV gD-HVEM were fewer than that of HSV-1 gD-HVEM complex. These results could explain why HVEM does not function as a receptor for BV gD. CONCLUSION: In this study, we present structural model for the BV gD in a complex with its receptor. Some features predicted by this model can explain previously reported experimental data. This complex model may lead to a better understanding of the function of BV gD and its interaction with receptor and will improve our understanding of the activation of the BV fusion and entry process.

Analysis on the interaction domain of VirG and apyrase by pull-down assay.

VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2). Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) were transformed into E. coli BL21 (DE3) and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG.

Predicting environmental risks of pharmaceutical residues by wastewater surveillance: An analysis based on pharmaceutical sales and their excretion data.

Pharmaceutical residues are increasingly becoming a significant source of environmental water pollution and ecological risk. This study, leveraging official national pharmaceutical sales statistics, predicts the environmental concentrations of 33 typical pharmaceuticals in the Tianjin area. The results show that 52 % of the drugs have a PEC/MEC (Predicted Environmental Concentration/Measured Environmental Concentration) ratio within the acceptable range of 0.5-2, including atenolol (1.21), carbamazepine (1.22), and sulfamethoxazole (0.91). Among the selected drugs, tetracycline, ciprofloxacin, and acetaminophen had the highest predicted concentrations. The EPI (Estimation Programs Interface) biodegradation model, a tool from the US Environmental Protection Agency, is used to predict the removal efficiency of compounds in wastewater treatment plants. The results indicate that the EPI predictions are acceptable for macrolide antibiotics and ß-blockers, with removal rates of roxithromycin, spiramycin, acetaminophen, and carbamazepine being 14.1 %, 61.2 %, 75.1 %, and 44.5 %, respectively. However, the model proved to be less effective for fluoroquinolone antibiotics. The ECOSAR (Ecological Structure-Activity Relationships) model was used to supplement the assessment of the potential impacts of drugs on aquatic ecosystems, further refining the analysis of pharmaceutical environmental risks. By combining the concentration and detection frequency of pharmaceutical wastewater, this study identified 9 drugs with significant toxicological risks and marked another 24 drugs as substances of potential concern. Additionally, this study provides data support for addressing pharmaceutical residues of priority concern in subsequent research.

Detecting genetic gain and loss events in terms of protein domain: Method and implementation.

Continuous gain and loss of genes are the primary driving forces of bacterial evolution and environmental adaptation. Studying bacterial evolution in terms of protein domain, which is the fundamental function and evolutionary unit of proteins, can provide a more comprehensive understanding of bacterial differentiation and phenotypic adaptation processes. Therefore, we proposed a phylogenetic tree-based method for detecting genetic gain and loss events in terms of protein domains. Specifically, the method focuses on a single domain to trace its evolution process or on multiple domains to investigate their co-evolution principles. This novel method was validated using 122 Shigella isolates. We found that the loss of a significant number of domains was likely the main driving force behind the evolution of Shigella, which could reduce energy expenditure and preserve only the most essential functions. Additionally, we observed that simultaneously gained and lost domains were often functionally related, which can facilitate and accelerate phenotypic evolutionary adaptation to the environment. All results obtained using our method agree with those of previous studies, which validates our proposed method.

Prediction of Potential Suitable Areas and Priority Protection for Cupressus gigantea on the Tibetan Plateau.

Cupressus gigantea (C. gigantea) is an endemic endangered species on the Tibetan Plateau; its potential suitable areas and priority protection in the context of global climate change remain poorly predicted. This study utilized Biomod2 and Marxan to assess the potential suitable areas and priority protection for C. gigantea. Our study revealed that the suitable areas of C. gigantea were concentrated in the southeastern Tibetan Plateau, with the center in Lang County. Temperature was identified as a crucial environmental factor influencing the distribution of C. gigantea. Over the coming decades, the suitable range of C. gigantea expanded modestly, while its overall distribution remained relatively stable. Moreover, the center of the highly suitable areas tended to migrate towards Milin County in the northeast. Presently, significant areas for improvement are needed to establish protected areas for C. gigantea. The most feasible priority protected areas were located between the Lang and Milin counties in Tibet, which have more concentrated and undisturbed habitats. These results provide scientific guidance for the conservation and planning of C. gigantea, contributing to the stability and sustainability of ecosystems.

Comparative genomic analysis provides insights into the genetic diversity and pathogenicity of the genus Brucella.

Some Brucella spp. are important pathogens. According to the latest prokaryotic taxonomy, the Brucella genus consists of facultative intracellular parasitic Brucella species and extracellular opportunistic or environmental Brucella species. Intracellular Brucella species include classical and nonclassical types, with different species generally exhibiting host preferences. Some classical intracellular Brucella species can cause zoonotic brucellosis, including B. melitensis, B. abortus, B. suis, and B. canis. Extracellular Brucella species comprise opportunistic or environmental species which belonged formerly to the genus Ochrobactrum and thus nowadays renamed as for example Brucella intermedia or Brucella anthropi, which are the most frequent opportunistic human pathogens within the recently expanded genus Brucella. The cause of the diverse phenotypic characteristics of different Brucella species is still unclear. To further investigate the genetic evolutionary characteristics of the Brucella genus and elucidate the relationship between its genomic composition and prediction of phenotypic traits, we collected the genomic data of Brucella from the NCBI Genome database and conducted a comparative genomics study. We found that classical and nonclassical intracellular Brucella species and extracellular Brucella species exhibited differences in phylogenetic relationships, horizontal gene transfer and distribution patterns of mobile genetic elements, virulence factor genes, and antibiotic resistance genes, showing the close relationship between the genetic variations and prediction of phenotypic traits of different Brucella species. Furthermore, we found significant differences in horizontal gene transfer and the distribution patterns of mobile genetic elements, virulence factor genes, and antibiotic resistance genes between the two chromosomes of Brucella, indicating that the two chromosomes had distinct dynamics and plasticity and played different roles in the survival and evolution of Brucella. These findings provide new directions for exploring the genetic evolutionary characteristics of the Brucella genus and could offer new clues to elucidate the factors influencing the phenotypic diversity of the Brucella genus.

Monkeypox: Can we count on the current smallpox immunization?

Two vaccines ACAM 2000 and JYNNEOS have obtained approval from the Food and Drug Administration as preventive measures against monkeypox, contributing significantly to the management of the monkeypox epidemic. Nonetheless, research has demonstrated that smallpox vaccination offers approximately 88.8% protection against monkeypox, while immunization with these vaccines generates relatively low levels of neutralizing antibodies. In this work, we performed a comprehensive comparison of antigens between the 2022-2023 monkeypox strains and the smallpox vaccine strains. Our analysis has revealed considerable amino acid changes in all 27 antigens, including core and envelope proteins. Amino acid substitutions within B cell epitopes were observed in 26 of these antigens, with at least half of the antigen substitutions occurring within B cell epitopes in 20 out of the 26 antigens analyzed. These findings may raise potential concerns regarding the efficacy of these vaccines.

ABL1-mediated phosphorylation promotes FOXM1-related tumorigenicity by Increasing FOXM1 stability.

The transcription factor FOXM1, which plays critical roles in cell cycle progression and tumorigenesis, is highly expressed in rapidly proliferating cells and various tumor tissues, and high FOXM1 expression is related to a poor prognosis. However, the mechanism responsible for FOXM1 dysregulation is not fully understood. Here, we show that ABL1, a nonreceptor tyrosine kinase, contributes to the high expression of FOXM1 and FOXM1-dependent tumor development. Mechanistically, ABL1 directly binds FOXM1 and mediates FOXM1 phosphorylation at multiple tyrosine (Y) residues. Among these phospho-Y sites, pY575 is indispensable for FOXM1 stability as phosphorylation at this site protects FOXM1 from ubiquitin-proteasomal degradation. The interaction of FOXM1 with CDH1, a coactivator of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which is responsible for FOXM1 degradation, is significantly inhibited by Y575 phosphorylation. The phospho-deficient FOXM1(Y575F) mutant exhibited increased ubiquitination, a shortened half-life, and consequently a substantially decreased abundance. Compared to wild-type cells, a homozygous Cr-Y575F cell line expressing endogenous FOXM1(Y575F) that was generated by CRISPR/Cas9 showed obviously delayed mitosis progression, impeded colony formation and inhibited xenotransplanted tumor growth. Overall, our study demonstrates that ABL1 kinase is involved in high FOXM1 expression, providing clear evidence that ABL1 may act as a therapeutic target for the treatment of tumors with high FOXM1 expression.

Leucine-rich repeat 11 of Toll-like receptor 9 can tightly bind to CpG-containing oligodeoxynucleotides, and the positively charged residues are critical for the high affinity.

TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-κB activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.