DEAR EDITOR,Dengue fever（DF）, caused by dengue virus（DENV） infection,is a highly prevalent mosquito-borne infectious disease. The development of antiviral drugs and vaccines relies on the clinical utility of dengue infection models, particularly those involving antibody-dependent enhancement（ADE）. In this study, we co-incubated DENV-2 anti-prM with DENV-3 for 2 h,

Dengue virus (DV) has occasionally emerged at epidemic levels in Yunnan, China. Vaccine development is limited by antibody-dependent enhancement and a lack of good animal models. Thus, the study investigated cross infection based on maternal immunity in BALB/c mice and assessed the risk of cross infection by DV2-D13113 and DV3-YNWS2 epidemic virus strains. DV replicated within the organs of the BALB/c infant mice, even causing death. Particularly, DV3-infected infant mice were at higher risk of severe disease if their mothers were infected with DV2. Although BALB/c adults and pups survived DV2/DV3 infection and produced anti-DV antibodies after 5-8 days, extensive subcutaneous vascular leakage was observed after secondary DV infection. Furthermore, vascular permeability in the lung and kidney significantly increased in offspring born to heterotypic virus-infected mothers. Thus, vascular leakage indicates severe DV infection. The results indicate that maternal immunity increases the severity of subsequent heterotypic infection. Additionally, secondary cross infection by D13113 and YNWS2 represents a risk of serious disease. This study has implications for studies of DV cross infection and vaccine development.

Lipid-membrane-targeting strategies hold great promise to develop broad-spectrum antivirals. However, it remains a big challenge to identify novel membrane-based targets of viruses and virus-infected cells for development of precision targeted approaches. Here, it is discovered that viroporins, viral-encoded ion channels, which have been reported to mediate release of hydrogen ions, trigger membrane acidification of virus-infected cells. Through development of a fine-scale library of gradient pH-sensitive (GPS) polymeric nanoprobes, the cellular membrane pH transitions are measured from pH 6.8-7.1 (uninfection) to pH 6.5-6.8 (virus-infection). In response to the subtle pH alterations, the GPS polymer with sharp response at pH 6.8 (GPS(6.8)) selectively binds to virus-infected cell membranes or the viral envelope, and even completely disrupts the viral envelope. Accordingly, GPS(6.8) treatment exerts suppressive effects on a wide variety of viruses including SARS-CoV-2 through triggering viral-envelope lysis rather than affecting immune pathway or viability of host cells. Murine viral-infection models exhibit that supplementation of GPS(6.8) decreases viral titers and ameliorates inflammatory damage. Thus, the gradient pH-sensitive nanotechnology offers a promising strategy for accurate detection of biological pH environments and robust interference with viruses.

Dengue fever, as a mosquito-borne viral disease widely spread in tropical and subtropical regions, remarkably threatens public health, while the mechanism involved in host-DENV interaction has not been fully elucidated. Firstly, we analyzed the expression levels of long non-coding RNAs (lncRNAs) in THP-1 cells after DENV-3 infection and Antibody- Dependent Enhancement of viral infection (ADE-VI) by RNA-Seq. Secondly, through the RT-qPCR to confirm those differentially expressed (DE) lncRNAs. Then, we also analyzed the competitive endogenous RNA (CeRNA) regulatory network of DE lncRNAs. Finally, we predicted the encode ability of DE lncRNAs. It was found that on the X and Y chromosomes, the expression levels of lncRNAs in THP-1 cells after ADE-VI were significantly different from those in the negative control and the DENV-3 infection groups. There were 71 DE lncRNAs after DENV-3 infection, including 42 up-regulated and 29 down-regulated lncRNAs. A total of 70 DE lncRNAs after ADE-VI were detected, including 38 up-regulated and 32 down- regulated lncRNAs. After ADE-VI and DENV-3 infection, there were 35 DE lncRNAs, including 11 up-regulated and 24 down-regulated lncRNAs. The analysis of the CeRNA regulatory network of DE lncRNAs revealed that, TRIM29, STC2, and IGFBP5 were correlated with the ADE-VI. Additionally, it was found that lncRNAs not only participated in the CeRNA regulatory network, but also maybe encoded small peptides. Our findings provided clues for further investigation into the lncRNAs associated antiviral mechanism of ADE-VI and DENV-3 infection.

目的 分析亚洲区西尼罗河病毒(West Nile virus,WNV)的流行情况,探讨其系统发育和分子进化特征.方法 从NCBI数据库中分别获取亚洲区流行的WNV 全基因组序列和E 蛋白序列,进行比对和同源性分析后构建系统进化树,分析来自不同株型病毒的结构蛋白E 的核苷酸和氨基酸的突变率,进一步找出氨基酸的替代位点.结果 在中国、马来西亚、印度、以色列、巴基斯坦、伊朗和土耳其等地有血清学感染或回顾性检测阳性报道.将获取的WNV全基因组序列进行系统进化树分析,发现WNV 以色列株(HM152773、AF481864)在亲缘关系上与谱系la株最为接近,而以色列株(AY688948)在亲缘关系上与谱系2株较为接近,印度株(KU978770)与谱系1c接近;将获取的WNV E 蛋白序列进行系统进化分析,发现WNV 中国株(JX442278,JX42280-JX442282)在亲缘关系上与谱系la株最为接近.氨基酸序列和核酸序列分析表明,不同株型的E蛋白与谱系2相比,表现为碱基和氨基酸的突变率高,与谱系la相比,印度株(GQ851605、JX041632、GQ851604和KU978770)的第154和第156位氨基酸分别由天冬酰胺突变为天冬氨酸和丝氨酸(N154D,N154S),丝氨酸突变为脯氨酸和丙氨酸(S156P,S156A),而中国株(AY490240)、巴基斯坦株(JX070655)和以色列株(HM051416)的第156 位氨基酸分别由丝氨酸突变为脯氨酸、苯丙氨酸和脯氨酸(S156P、S156F、S156P).结论 通过谱系分析、系统进化树构建、包膜蛋白E氨基酸序列及编码核苷酸序列的比较分析发现,有些位点的改变可能与病毒的毒力、致病性、神经侵袭力、流行等有关,为研究亚洲区不同国家不同株型间WNV 的生物学和流行致病性差异提供了参考.

Glucokinase-maturity onset diabetes of the young (GCK-MODY) represents a rare genetic disorder due to mutation in the glucokinase (GCK) gene. The low incidence of vascular complications in GCK-MODY makes it a natural paradigm for interrogating molecular mechanisms promoting vascular health under prolonged hyperglycemia. Clinical rate of misdiagnosis has remained high, and a reliable serum lipid biomarker that precedes genetic screening can facilitate correct diagnosis and treatment. Herein, we comprehensively quantitated 565 serum lipids from 25 classes in 105 subjects (42 nondiabetic controls, 30 GC K-MODY patients, 33 drug-naïve, and newly-onset T2D patients). At false-discovery rate (FDR) < 0.05, several phosphatidylcholines (PCs) and plasmalogen PCs were specifically increased in GCK-MODY, while triacylglycerols (TAGs) and diacylglycerols (DAGs) were reduced. Correlation matrices between lipids uncovered coregulation between plasmalogen PCs (PCps) and glycerolipid precursors was distinctly enhanced in GCK-MODY compared to T2D. Strengthened positive correlations between serum PCps and circulating HDLs was specifically observed in hyperglycemic subjects (i.e. T2D and GCK-MODY) compared to normglycemic controls, suggesting that HDL-PCps may elicit distinct physiological effects under hyperglycemia. Amongst GCK-MODY patients, individuals harboring variants of GCK mutations with elevated PCps also exhibited higher HDLs. Isolated HDLs displayed localized increases (p < 0.05) in very-long-chain PUFA-PCs and PCps in GCK-MODY. Protein analyses revealed elevated levels of HDL-resident ATGL (P = 0.003) and CEPT1 (P < 0.0001), which mediate critical steps of PCps production along the TAG-DAG-PC axis, in GCK-MODY relative to T2D. A panel of four lipids differentiated GCK-MODY from T2D with AUC of 0.950 (95% CI 0.903–9.997). This study provides the first evidence that enhanced recruitment of CEPT1 and ATGL onto HDLs essentially underlie the atheroprotective profiles associated with GCK-MODY. Resultant increases in the production of HDL-PCps and PUFA-PCs provides an active, circulating form of protection towards the vasculature of GCK-MODY, thereby lowering the incidence of vascular complications despite chronic exposure to hyperglycemia since birth.

ObjectivesTo compare the safety, immunogenicity, and immune persistence of hepatitis A (HA) vaccines between HBs-Ag-positive and -negative participants.Method9000 participants were enrolled in the phase IV study of live attenuated HA (HA-L) or inactivated HA (HA-I) vaccines. The HBs-Ag-positive subjects were detected and became an independent observation group. Adverse reactions (ARs), geometric mean concentrations (GMCs) and seroconversion rates (SRs) of the vaccines were analyzed at five time points until three years after vaccination. Results: 120 HBs-Ag-positive subjects were screened out, only 1 participant had grade 1 experienced ARs after HA-L injection. Except the time point of two years, the SRs of HBs-Ag-positive group were 100% for both vaccines. The GMCs were not statistically different between HBs-Ag-positive and -negative groups after the HA-L vaccination. The logarithmically transformed GMCs for HBs-Ag-positive and -negative groups were 3.21 mIU/mL (95% CI, 2.03-4.39 mIU/mL) and 2.95 mIU/mL (95% CI, 2.88-3.02 mIU/mL) 28 days after the HA-L vaccination, respectively.ConclusionsBoth HA-L and HA-I vaccines were safe for HBs-Ag-positive participants and may provide an excellent long-term protection against HAV in this study. The results indicated that people positive or negative for HBs-Ag can receive both HA-L and HA-I vaccines (ClinicalTrials.gov number, NCT02601040).

Dengue virus infection mainly causes dengue hemorrhagic fever (DHF) and/or dengue shock syndrome (DSS). However, ADE (antibody-dependent enhancement) is one of the main pathogenic factors, and its pathogenic mechanism has not been fully elucidated. Recently, with the development of high-throughput sequencing, an increased number of RNAs have been confirmed to play a vital regulatory role in the process of virus infection. However, there is a lack of research on dengue virus infection and ADE. In this study, we used RNA-Seq to detect differentially expressed RNAs (DE RNAs) profiles in mock-infected, DENV-3-infected, and ADE-infected THP-1 cells. Firstly, we found 69 circRNAs, 259 miRNAs, and 18 mRNAs were differentially expressed in THP-1 vs DENV-3. In THP-1 vs ADE, 94 circRNAs, 263 miRNAs, and 111 mRNAs were differentially expressed. In DENV-3 vs ADE, 68 circRNAs, 105 miRNAs, and 94 mRNAs were differentially expressed. Functional enrichment analysis of these DE RNAs mainly focused on immune system, viral infectious diseases, cytokine-cytokine receptor interactions, and NOD/RIG-I-like receptor signaling pathways. In DENV-3 vs ADE, notably, the expression of HBB was up-regulated, which was a Fcγ Receptor-mediated phagocytosis protein. Additionally, we predicted the encoding ability of DE circRNAs, and it was found that a small peptide was encoded by novel_circ_001562 and that its amino acid sequence was consistent with that of DDX60L, which is a class of interferon-stimulated genes. Finally, we constructed the ceRNA regulatory network pathway. Therefore, our study provides a new strategy for further investigation on DENV-host interactions.

Objective:B-cell-activating factor (BAFF) has been determined to be involved in HIV-1 infection and is correlated with disease progression, while its homologous molecule, a proliferation-inducing ligand (APRIL), is less frequently reported, and its role remains unclear. We aimed to characterize the APRIL levels in subjects with different HIV-1 infection statuses and determine the relationships with disease progression and immune activation. Methods:The plasma levels of APRIL were compared among 17 long-term non-progressors (LTNPs), 17 typical progressors (TPs), 10 ART-treated patients, and 10 healthy donors (HDs). Seventeen LTNPs and a subset of TPs (n= 6) who initiated ART were assessed longitudinally. The correlations between the APRIL levels and markers of disease progression, B-cell count and specific antibody response, and markers of immune activation and functional cells were analyzed. Results:The circulating APRIL levels were significantly elevated in the LTNPs relative to the TPs, ART-treated patients, and HDs. The longitudinal investigation revealed that the APRIL levels were decreased during follow-up in the LTNPs. ART did not significantly influence the APRIL levels. The levels of plasma APRIL were negatively correlated with the plasma HIV-1 viral load and cellular HIV-1 DNA levels and positively correlated with the CD4(+)T-cell count and CD4/CD8 ratio. An inverse correlation was observed between the APRIL and BAFF levels. Furthermore, the APRIL levels were negatively correlated with the frequency of activated CD8(+)T cells and levels of interferon gamma-induced protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1). Finally, positive correlations were observed among the APRIL levels, the frequency of CD8(+)CD28(+)T cells, and natural killer (NK) cell count. Conclusion:The APRIL levels were elevated in the LTNPs and negatively correlated with disease progression and immune activation, suggesting likely protective activity in HIV-1 infection.

The reactivation of HIV latency cell will be necessary to curing HIV infection. Although many latency-reversal agents (LRAs) have proven effective to reactivate the latency cell, there is a lack of any systematic analysis of the molecular targets of these LRAs and related pathways in the context of transcriptome. In this study, we performed an integrated analysis of the target profile of bryostatin and transcriptome of the reactivated CD4 + T cells after exposing to bryostatin. The result showed a distinct gene expression profile between latency cells and bryostatin reactivated cells. We found bryostatin can target multiple types of protein other than only protein kinase C. Functional network analysis of the target profile and differential expressed genes suggested that bryostatin may activate a few novel pathways such as pyrimidine metabolism, purine metabolism and p53 signaling pathway, besides commonly known pathways DNA replication, cell cycle and so on. The results suggest that bryostatin may reactivate the HIV-latent cells through up-regulation of pyrimidine and purine metabolism or through starting the cell-cycle arrest and apoptosis induced by up-regulation of p53 signaling pathway. Our study provides some novel insights into the role of bryostatin and its affected pathways in controlling HIV latency and reactivation.

AIMS:The study aimed to screen the HNF1A and HNF4A mutation in a large Chinese cohort of high clinical suspicion of maturity-onset diabetes of the young (MODY) patients and characterize the clinical features of those patients. The performance of hsCRP as a biomarker to differentiate MODY3 from early onset T2DM was also evaluated.METHODS:A total of 74 patients with a strong clinical suspicion of MODY from 59 families and 33 newly diagnosed early-onset T2DM were included. HNF1A and HNF4A mutations were analyzed by Sanger sequencing. ROC curves were used to identify the optimal cutoff of hsCRP.RESULTS:One novel (c.864_865insG) and six recurrent HNF1A mutations (R203H, R263H, P379T, L422P, P519L and c.873delC) in 17 patients from 8 families (13.6%), as well as one novel HNF4A (R331H) mutation were identified. Nonspecific clinical presentations were observed in MODYX compared to MODY3 patients. MODY3 subjects exhibited with younger, lower BMI, TG, fasting and postprandial C-peptide, higher HDL than T2DM. Particularly, we confirmed serum hsCRP was lower in MODY3 than T2DM. ROC curve showed a good discrimination with an AUC of 0.852 and identified a cutoff hsCRP of 0.79 (75% sensitivity and 83% specificity). Good glycemic control was observed in all identified patients after switching to glimepiride therapy.CONCLUSIONS:The prevalence of HNF1A mutation was relatively lower in Mainland China and HNF4A mutation was rare. Serum hsCRP concentrations performed well in discriminating MODY3 from T2DM. Molecular diagnosis of MODY3/1 did transform management in clinical practice and facilitated the glycemic control.

Objective To optimize the condition for ultracentrifugation for verification of inactivation effect of inactivated enterovirus 71 (EV71) vaccine,and provide a basis for treatment of samples of inactivation verification test of the vaccine.Methods The harvested liquid EV71 was ultracentrifuged at various rotation rates (45 000,40 000,35 000,30 000 and 25 000 r/min) for various (120,90,60 and 30) minutes,of which the titers before and after ultracentrifugation were determined,and recovery rates were calculated to compare the recovery effects under various conditions.Results The precipitation effects of EV71 at the same rotation rate were positively related to the time for ultracentrifugation (R2 increased from 0.77 to 0.99),while those at the same time point after ultracentrifugation were positively related to the rotation rate (R2 increased from 0.88 to 0.99).When the rotation rate was more than 40 000 r/min,the recovery rates of EV71 after ultracentrifugation for various minutes were more than 90％.When the time for ultracentrifugation was 120 min,and the rotation rate was 45 000,the precipitation effect of EV71 was satisfactory,while the recovery rate reached a maximum of 98％.The recovery rate of EV71 after ultracentrifugation at 45 000 r/min for 60 min was 97％,which showed no significant difference with that after ultracentrifugation for 120 min.However,the recovery rate after ultracentrifugation for 30 min showed significant difference with those after ultracentrifugation for 120,90 and 60 min (P ＜0.05).Conclusion The precipitation effect of EV71 was positively related to rotation rate and time for ultracentrifugation.The effect was improved,while the recovery rate increased,with the increasing rotation rate and time.

Objective To elucidate the clinical characteristics and genotype-phenotype relationships of Chinese patients with neonatal diabetes mellitus (NDM) caused by ATP-sensitive potassium channel (KATP ) gene mutations (KATP-NDM). Methods Total of 23 suspected patients with NDM were recruited from Peking Union Medical College Hospital. Amplification and direct sequencing of all exons and exon-intron boundaries of the KCNJ11 and ABCC8 gene were done for molecular genetic diagnosis. Patients with a definite diagnosis of KATP-NDM were switched from insulin injection to oral glibenclamide. Results Thirteen patients with KATP-NDM were identified, including 9 cases of KCNJ11-NDM and 4 cases of ABCC8-NDM. Four novel mutations of KATP were identified: 1 in KCNJ11 (I182M) and 3 in ABCC8 (Q211R, I585F, R653W). Among 9 patients with KCNJ11-NDM, 2 were transient NDM (TNDM) and 7 were permanent NDM (PNDM), and 3 of which had intermediate developmental delay, neonatal diabetes (iDEND syndrome). Four patients with ABCC8-NDM were all PNDM, 2 of them had iDEND syndrome. Switching from insulin to glibenclamide monotherapy was successful in 9 patients (69.2%). Conclusions Different clinical severities and treatment responses to sulfonylureas exist even among patients with the same mutation or with different amino acids at the same mutation site. Early diagnosis of KATP-NDM and initiation of sulfonylureas are of great importance in the prevention of neurological abnormalities in patients with iDEND syndrome.

Background: Familial renal glucosuria (FRG) is an inherited renal tubular disorder characterized by persistent isolated glucosuria with normal blood glucose. SLC5A2 gene mutation was the causative of FRG.Methods: Molecular genetic analysis of SLC5A2 gene by Sanger sequencing was conducted in two unrelated non consanguineous Chinese families with isolated glucosuria. Extensive laboratory test and physical examination were performed. In silico algorithms were used to explore the potential effect of novel mutation on SGLT2 function. We also summarized the reported SLC5A2 mutations in the Chinese patients with FRG.Results: A novel missense mutation (c.877A > T, p.Ser293Cys) in exon 3 was detected in proband 1 with weight loss accompanying by glucosuria and in her father with normal phenotype. In family 2, a previously reported compound heterozygous mutation (c.229G > C, p.Gly77Arg; c.1540C > T, p.Pro514Ser) was identified, and her healthy parents were heterozygous mutation carriers. The p.S293C mutation was predicted to be pathogenic. No hot spot mutation was found in reported Chinese patients with FRG.Conclusions: The novel pathogenic SLC5A2 mutation p.S293C was responsible for the onset of FRG. Our study further confirmed the co-dominant inheritance trait with variable penetrance and expanded the clinical and genetic spectrum of FRG.

目的 原核表达树鼩(tree shrew,ts)白细胞介素-6(interleukin-6,IL-6)基因,并分析tsIL-6蛋白分子特征.方法 以人、猴、啮齿类动物等IL-6 mRNA的共有序列作为查询序列,从树鼩cDNA数据库中进行本地blast,获得预测tsIL-6核苷酸序列,将其翻译成氨基酸序列,用MEGA 6软件进行核酸序列和氨基酸序列比对,绘制进化树.参考预测IL-6基因序列设计引物,PCR扩增IL-6基因序列,克隆至原核表达载体pET-28a(+),构建重组表达质粒pET-28a(+)-IL-6;转化感受态E.coli BL21(ED3),IPTG诱导表达,重组蛋白经14 KD透析膜梯度透析纯化;利用蛋白质在线数据库expasy进行蛋白质结构同源建模,并用SignalP sever预测tsIL-6的信号肽序列,经模板与预测模型的结构拟合来预测树鼩IL-6的gp130结合位点.结果 tsIL-6核苷酸和氨基酸序列与高等灵长类动物有较高的同源性;重组表达质粒pET-28a(+)-IL-6经双酶切鉴定证明构建正确;重组表达蛋白相对分子质量约为24 000,纯化后可见单一目的条带,浓度为0.2mg/ml;hIL-6和tsIL-6蛋白的空间结构较类似,tsIL-6的gp130结合位点Ⅱa位于A和C螺旋间,位点Ⅲa位于A和B螺旋的柔性链区及分子近C-端.结论 tsIL-6的核苷酸和氨基酸序列与高等灵长类动物具有较高同源性,成功构建的重组原核表达质粒pET-28a(+)-IL-6,为后续IL-6相关的病理研究奠定了基础.