ObjectiveThis study leverages structural data from antigen-antibody complexes of the influenza A virus neuraminidase (NA) protein to investigate the spatial recognition relationship between the antigenic epitopes and antibody paratopes. MethodsStructural data on NA protein antigen-antibody complexes were comprehensively collected from the SAbDab database, and processed to obtain the amino acid sequences and spatial distribution information on antigenic epitopes and corresponding antibody paratopes. Statistical analysis was conducted on the antibody sequences, frequency of use of genes, amino acid preferences, and the lengths of complementarity determining regions (CDR). Epitope hotspots for antibody binding were analyzed, and the spatial structural similarity of antibody paratopes was calculated and subjected to clustering, which allowed for a comprehensively exploration of the spatial recognition relationship between antigenic epitopes and antibodies. The specificity of antibodies targeting different antigenic epitope clusters was further validated through bio-layer interferometry (BLI) experiments. ResultsThe collected data revealed that the antigen-antibody complex structure data of influenza A virus NA protein in SAbDab database were mainly from H3N2, H7N9 and H1N1 subtypes. The hotspot regions of antigen epitopes were primarily located around the catalytic active site. The antibodies used for structural analysis were primarily derived from human and murine sources. Among murine antibodies, the most frequently used V-J gene combination was IGHV1-12*01/IGHJ2*01, while for human antibodies, the most common combination was IGHV1-69*01/IGHJ6*01. There were significant differences in the lengths and usage preferences of heavy chain CDR amino acids between antibodies that bind within the catalytic active site and those that bind to regions outside the catalytic active site. The results revealed that structurally similar antibodies could recognize the same epitopes, indicating a specific spatial recognition between antibody and antigen epitopes. Structural overlap in the binding regions was observed for antibodies with similar paratope structures, and the competitive binding of these antibodies to the epitope was confirmed through BLI experiments. ConclusionThe antigen epitopes of NA protein mainly ditributed around the catalytic active site and its surrounding loops. Spatial complementarity and electrostatic interactions play crucial roles in the recognition and binding of antibodies to antigenic epitopes in the catalytic region. There existed a spatial recognition relationship between antigens and antibodies that was independent of the uniqueness of antibody sequences, which means that antibodies with different sequences could potentially form similar local spatial structures and recognize the same epitopes.

Objective:To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods:The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δ wza were detected by measuring the optical density OD 600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δ wza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δ wza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δ wza mutant strain. Results:The PCR results revealed that the Δ wza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δ wza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δ wza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δ wza mutant strain (45.963±2.795) μg/ml compared with wild-type strain W14 (138.800±5.201) μg/ml. There was a statistical difference between the two groups ( t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion:Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Objective:To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hv Kp) serotype K1 and validate its function. Methods:Phage that infected serotype K1-type hv Kp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results:A lytic phage (phiA2) that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates. Conclusion:Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp, which has potential application value in treating bacterial infection.

Objective:To analyze the growth characteristics of different genotypes of Japanese encephalitis virus (JEV) in different cell lines, and to provide scientific basis for the selection of cell lines in the study of JEV.Methods:BHK-21, Vero, C6/36, PK-15, DF-1, N2a, SH-sy5y and MDCK cell lines were selected. The proliferation ability of genotype 1 (NX1889 strain), genotype 3 (P3 strain) and genotype 5 (XZ0934 strain) JEV in these cell lines was evaluated by plaque assay and RT-qPCR.Results:Significant cytopathogenic effects (CPE) were observed in BHK-21, Vero, C6/36, DF-1, N2a and PK-15 cell lines across all three JEV genotypes. However, no significant differences in CPE characteristics were observed within the same cell line. SH-sy5y and MDCK cell lines did not show significant CPE, but virus proliferation was detected in SH-sy5y cell line, while MDCK cell line were found to be insensitive to JEV. No significant difference was observed in the proliferation curves of G1, G3 and G5 JEV in BHK-21, Vero and SH-sy5y cell lines. In C6/36 and PK-15 cell lines, the titer of G1 JEV was higher than that of G3 and G5. In DF-1 cell line, G5 demonstrated a higher titer than the other two genotypes, whereas in N2a cell line, G5 showed a lower titer than the other two.Conclusions:There are differences in the proliferation of three different genotypes of JEV in different cell lines, which can provide reference for the study of JEV in different directions.

Objective:To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods:The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δ wza were detected by measuring the optical density OD 600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δ wza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δ wza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δ wza mutant strain. Results:The PCR results revealed that the Δ wza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δ wza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δ wza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δ wza mutant strain (45.963±2.795) μg/ml compared with wild-type strain W14 (138.800±5.201) μg/ml. There was a statistical difference between the two groups ( t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion:Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Objective:To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hv Kp) serotype K1 and validate its function. Methods:Phage that infected serotype K1-type hv Kp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results:A lytic phage (phiA2) that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates. Conclusion:Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp, which has potential application value in treating bacterial infection.

Objective:To understand the monkeypox knowledge awareness, risk perception and vaccination intention in men who have sex with men (MSM) in five cities in northeast China.Methods:A cross-sectional study was conducted by using electronic questionnaire in MSM selected by convenience sampling in five cities in northeast China (Shenyang, Panjin, Changchun, Harbin and Jiamusi) from June 28 to July 8, 2023 by local centers for disease control and prevention and MSM communities. The sample size was estimated to be 220. Information about their demographics, monkeypox-related knowledge awareness, perceived concern about epidemic risk perception, and monkeypox vaccination intention were collected. Logistic regression model was used to analyze related factors for MSM's monkeypox vaccination intention.Results:In 355 MSM, 63.9% (227/355) had monkeypox vaccination intentions, and 55.5% (197/355) had high awareness of monkeypox related knowledge with a mean knowledge awareness score of 3.7±1.5. MSM with education level of high-school and above (a OR=1.93, 95% CI:1.01-3.69), higher knowledge awareness score (a OR=1.19, 95% CI:1.02-1.40) and higher risk perception of monkeypox infection (a OR=1.82, 95% CI:1.15-2.88), were more willing to receive monkeypox vaccination. The main reasons for willingness to receive monkeypox vaccine were preventing monkeypox (86.3%, 196/227) and worrying about appearance being affected (62.1%, 141/227). The main reasons for unwillingness for the vaccination included concerns about vaccine safety (53.1%, 68/128), clinical progression of AIDS being affected (46.1%, 59/128) and efficacy of antiretroviral therapy being affected (44.5%, 57/128). Conclusions:The levels of knowledge awareness and vaccine intentions still need to be improved among MSM in five cities of northeast China. It is necessary to improve the awareness of monkeypox and intention of monkeypox vaccination, promote protected sex behavior and self-assessment of infection risk, reduce vaccine hesitancy and increase monkeypox vaccination intention in MSM in 5 cities in northeast China.

Circular RNA(circRNA)represents a unique class of closed-loop structured non-coding RNAs involved in various biological processes such as cell proliferation,differentiation,and apopto-sis.They play a significant role in the development of numerous diseases,and also serve as poten-tial biomarkers and therapeutic targets.To explore the impact of circRNA on viral replication,this study performed an omics measurement and analysis of circRNA differential expression in MC38 cells infected with HY12 enterovirus.It was found that,following HY12 virus infection,the ex-pressionlevels of 570 circRNAs were upregulated,while 381 circRNAs were downregulated.A-mong the upregulated circRNAs,the significantly upregulated circRNA mmu_circ_0001083 was selected for further investigation into its association with HY12 infection and its impact on viral replication.The results indicated that after HY12 virus infection,the expression of host circRNA mmu_circ_0001083 significantly increased,and its expression level was dependent on the virus dos-age and time.Compared to normal MC38 cells infected with the HY12 virus,cells with knocked down expression of circRNA mmu_circ_0001083 showed reduced expression of the 2C protein and significantly lower viral titers.Conversely,after HY12 virus infection in MC38 cells with overexpressed circRNA mmu_circ_0001083,there was an increase in the expression of the 2C pro-tein and a significant rise in viral titers.These results suggest that the upregulation of host cir-cRNA mmu_circ_0001083 is significantly positively correlated with the replication of HY12 virus,meaning mmu_circ_0001083 plays a positive regulatory role in the replication of HY12.This find-ing lays a foundation for future in-depth studies on the regulatory mechanisms of circRNA on viral replication.

A recombinant enzyme-mediated nucleic acid amplification(RAA)technology combined with colloidal gold test strips was developed for the rapid detection of bovine enterovirus(BEV).Using the highly conserved BEV 5'UTR as the target sequence,the primers were designed and screened.Downstream primer labeled with biotin at the 5'end and the probe labeled with 6-FAM at the 5'end were used to establish the RT-RAA method.The test strips were assembled by using mouse-derived anti-6-FAM monoclonal antibody as the gold standard antibody,with a streptavidin encapsulated in the detection line and sheep anti-mouse IgG encapsulated in the quality control line.A RT-RAA-LFD method was established by combing RAA technique with the prepared later-al flow device test strips for the detection of bovine enterovirus nucleic acids.The specificity,sensi-tivity,repeatability,and clinical application of the method are also evaluated.The results showed that the optimal primer concentration of this method was 5 μmol/L,and the amplification of BEV nucleic acids was accomplished by reacting at 35 ℃ for 8 min with the lowest detection limit of 101 copies/μL.No cross-reactivity with bovine viral diarrhea virus,bovine parvovirus,and foot-and-mouth disease virus was observed.The efficacy for the prepared test strips was at least for 90 d kept at 4 ℃.Detection of 74 clinical samples yielded a similar result compared with RT-PCR method.The above results demonstrated that the BEV RT-RAA-LFD method established in this study has high sensitivity,specificity,and more convenient to use,which is suitable for clinical de-tection on-site and provides a new technical tool for the diagnosis and epidemiological investigation of BEV infection.