Combining molecular transmission network analysis and spatial epidemiology to reveal HIV-1 transmission pattern among the older people in Nanjing, China.

BACKGROUND: In China, the problem of HIV infection among the older people has become increasingly prominent. This study aimed to analyze the pattern and influencing factors of HIV transmission based on a genomic and spatial epidemiological analysis among this population. METHODS: A total of 432 older people who were aged ≥ 50 years, newly diagnosed with HIV-1 between January 2018 and December 2021 and without a history of ART were enrolled. HIV-1 pol gene sequence was obtained by viral RNA extraction and nested PCR. The molecular transmission network was constructed using HIV-TRACE and the spatial distribution analyses were performed in ArcGIS. The multivariate logistic regression analysis was performed to analyze the factors associated with clustering. RESULTS: A total of 382 sequences were successfully sequenced, of which CRF07_BC (52.3%), CRF01_AE (32.5%), and CRF08_BC (6.8%) were the main HIV-1 strains. A total of 176 sequences entered the molecular network, with a clustering rate of 46.1%. Impressively, the clustering rate among older people infected through commercial heterosexual contact was as high as 61.7% and three female sex workers (FSWs) were observed in the network. The individuals who were aged ≥ 60 years and transmitted the virus by commercial heterosexual contact had a higher clustering rate, while those who were retirees or engaged other occupations and with higher education degree were less likely to cluster. There was a positive spatial correlation of clustering rate (Global Moran I = 0.206, P < 0.001) at the town level and the highly aggregated regions were mainly distributed in rural area. We determined three large clusters which mainly spread in the intra-region of certain towns in rural areas. Notably, 54.5% of cases in large clusters were transmitted through commercial heterosexual contact. CONCLUSIONS: Our joint analysis of molecular and spatial epidemiology effectively revealed the spatial aggregation of HIV transmission and highlighted that towns of high aggregation were mainly located in rural area. Also, we found vital role of commercial heterosexual contact in HIV transmission among older people. Therefore, health resources should be directed towards highly aggregated rural areas and prevention strategy should take critical persons as entry points.

Molecular transmission network analysis of newly diagnosed HIV-1 infections in Nanjing from 2019 to 2021.

OBJECTIVE: The objective of this study was to conduct a comprehensive analysis of the molecular transmission networks and transmitted drug resistance (TDR) patterns among individuals newly diagnosed with HIV-1 in Nanjing. METHODS: Plasma samples were collected from newly diagnosed HIV patients in Nanjing between 2019 and 2021. The HIV pol gene was amplified, and the resulting sequences were utilized for determining TDR, identifying viral subtypes, and constructing molecular transmission network. Logistic regression analyses were employed to investigate the epidemiological characteristics associated with molecular transmission clusters. RESULTS: A total of 1161 HIV pol sequences were successfully extracted from newly diagnosed individuals, each accompanied by reliable epidemiologic information. The analysis revealed the presence of multiple HIV-1 subtypes, with CRF 07_BC (40.57%) and CRF01_AE (38.42%) being the most prevalent. Additionally, six other subtypes and unique recombinant forms (URFs) were identified. The prevalence of TDR among the newly diagnosed cases was 7.84% during the study period. Employing a genetic distance threshold of 1.50%, the construction of the molecular transmission network resulted in the identification of 137 clusters, encompassing 613 nodes, which accounted for approximately 52.80% of the cases. Multivariate analysis indicated that individuals within these clusters were more likely to be aged ≥ 60, unemployed, baseline CD4 cell count ≥ 200 cells/mm3, and infected with the CRF119_0107 (P < 0.05). Furthermore, the analysis of larger clusters revealed that individuals aged ≥ 60, peasants, those without TDR, and individuals infected with the CRF119_0107 were more likely to be part of these clusters. CONCLUSIONS: This study revealed the high risk of local HIV transmission and high TDR prevalence in Nanjing, especially the rapid spread of CRF119_0107. It is crucial to implement targeted interventions for the molecular transmission clusters identified in this study to effectively control the HIV epidemic.

Low level exposure to BDE-47 facilitates the development of prostate cancer through TOP2A/LDHA/lactylation positive feedback circuit.

2,2',4,4'-tetra brominated diphenyl ether (BDE-47) is one of the most widely distributed congeners of polybrominated diphenyl ethers. While the relationships between BDE-47 exposure and other hormone-dependent cancers (such as breast cancer) are well established, no previous study has examined whether BDE-47 exposure is related to the development of prostate cancer (PCa). Through bulk and single-cell RNA sequencing (scRNA-seq) analyses, as well as in vitro and in vivo experiments, this study aims to investigate the effect of BDE-47 exposure on PCa progression. Herein, we found that low dose BDE-47 promoted the growth of PCa cells (PC3 and LNCaP) in a dose-dependent manner in vitro and in vivo. Based on Comparative Toxicogenomics Database (CTD) and The Cancer Genome Atlas (TCGA), we obtained 34 BDE-47-related and PCa-related genes through screening and overlapping. These genes were significantly enriched in fatty acid metabolism-related gene ontology (GO) terms, which were also enriched for genes targeting BDE-47 obtained from the UniProt. Through scRNA-seq data, certain cell type-specific expression was observed for CYP2E1, PIK3R1, FGF2, and TOP2A in PCa tissues from men. Molecular docking simulation showed that BDE-47 was tightly bound to the protein residues of AOX1, PIK3R1, FGF2, CAV2, CYP2E1 and TOP2A. Further screening in terms of patient overall survival, receiver operating characteristics curve (ROC) curve and Gleason score grading system narrowed the candidate genes down to TOP2A. Mechanistically, the growth-promoting effect of BDE-47 on PCa cells could be reversed by TOP2A inhibitor. RNA-seq followed by experimental verification demonstrated that TOP2A promoted PCa progression through upregulating LDHA and glycolysis. Furthermore, lactate upregulated TOP2A transcription through lactylation of H3K18la in PCa cells, which could be rescued by LDHA knockdown. Taken together, low dose BDE-47 triggered PCa progression through TOP2A/LDHA/lactylation positive feedback circuit, thus revealing epigenetic shifting and metabolic reprogramming underpinning BDE-47-induced carcinogenesis of the prostate.

Genes and Pathways Underpinning Klinefelter Syndrome at Bulk and Single-Cell Levels.

Klinefelter syndrome (KS) is the most frequent genetic anomaly in infertile men. Given its unclear mechanism, we aim to investigate critical genes and pathways in the pathogenesis of KS based on three bulk and one single-cell transcriptome data sets from Gene Expression Omnibus. We merged two data sets (GSE42331 and GSE47584) with human KS whole blood samples. When comparing the control and KS samples, five hub genes, including defensin alpha 4 (DEFA4), bactericidal permeability increasing protein (BPI), myeloperoxidase (MPO), intelectin 1 (ITLN1), and Xg Glycoprotein (XG), were identified. Besides, infiltrated degree of certain immune cells such as CD56bright NK cell were positively associated with the expression of ITLN1 and XG. Kyoto Encyclopedia of Genes and Genomes analysis identified upregulated phosphatidylinositol 3-kinase (PI3K)/AKT pathway in KS. Gene set enrichment analysis followed by gene set variation analysis confirmed the upregulation of G2M checkpoint and heme metabolism in KS. Thereafter, the GSE200680 data set was used for external validation of the expression variation of hub genes from healthy to KS testicular samples, and each hub gene yielded excellent discriminatory capability for KS without exception. At the single-cell level, the GSE136353 data set was utilized to evaluate intercellular communication between different cell types in KS patient, and strong correlations were detected between macrophages/ dendritic cells/ NK cells and the other cell types. Collectively, we provided hub genes, pathways, immune cell infiltration degree, and cell-cell communication in KS, warranting novel insights into the pathogenesis of this disease.

Transmitted drug resistance and transmission clusters among ART-naïve HIV-1-infected individuals from 2019 to 2021 in Nanjing, China.

Background: Transmitted drug resistance (TDR) is an increasingly prevalent problem worldwide, which will significantly compromise the effectiveness of HIV treatments. However, in Nanjing, China, there is still a dearth of research on the prevalence and transmission of TDR among ART-naïve HIV-1-infected individuals. This study aimed to understand the prevalence and transmission of TDR in Nanjing. Methods: A total of 1,393 participants who were newly diagnosed with HIV-1 and had not received ART between January 2019 and December 2021 were enrolled in this study. HIV-1 pol gene sequence was obtained by viral RNA extraction and nested PCR amplification. Genotypes, TDR and transmission cluster analyses were conducted using phylogenetic tree, Stanford HIV database algorithm and HIV-TRACE, respectively. Univariate and multivariate logistic regression analyses were performed to identify the factors associated with TDR. Results: A total of 1,161 sequences were successfully sequenced, of which CRF07_BC (40.6%), CRF01_AE (38.4%) and CRF105_0107 (6.3%) were the main HIV-1 genotypes. The overall prevalence of TDR was 7.8%, with 2.0% to PIs, 1.0% to NRTIs, and 4.8% to NNRTIs. No sequence showed double-class resistance. Multivariate logistic regression analysis revealed that compared with CRF01_AE, subtype B (OR = 2.869, 95%CI: 1.093-7.420) and female (OR = 2.359, 95%CI: 1.182-4.707) were risk factors for TDR. Q58E was the most prevalent detected protease inhibitor (PI) -associated mutation, and V179E was the most frequently detected non-nucleoside reverse transcriptase inhibitor (NNRTI) -associated mutation. A total of 613 (52.8%) sequences were segregated into 137 clusters, ranging from 2 to 74 sequences. Among 44 individuals with TDR (48.4%) within 21 clusters, K103N/KN was the most frequent TDR-associated mutation (31.8%), followed by Q58E/QE (20.5%) and G190A (15.9%). Individuals with the same TDR-associated mutations were usually cross-linked in transmission clusters. Moreover, we identified 9 clusters in which there was a transmission relationship between drug-resistant individuals, and 4 clusters in which drug-resistant cases increased during the study period. Conclusion: The overall prevalence of TDR in Nanjing was at a moderate level during the past 3 years. However, nearly half of TDR individuals were included in the transmission clusters, and some drug-resistant individuals have transmitted in the clusters. Therefore, HIV drug-resistance prevention, monitoring and response efforts should be sustained and expanded to reduce the prevalence and transmission of TDR in Nanjing.

COVID-19 of differing severity: from bulk to single-cell expression data analysis.

Coronavirus disease 2019 (COVID-19) is raging worldwide and causes an immense disease burden. Despite this, the biomarkers and targeting drugs of COVID-19 of differing severity remain largely unknown. Based on the GSE164805 dataset, we identified modules most critical for mild COVID-19 (mCOVID-19) and severe COVID-19 (sCOVID-19) through WGCNA, respectively. We subsequently constructed a protein-protein interaction network, and detected 16 hub genes for mCOVID-19 and 10 hub genes for sCOVID-19, followed by the prediction of upstream transcription factors (TFs) and kinases. The enrichment analysis then showed downregulation of TNFA signaling via NFKB for mCOVID-19, as well as downregulation of MYC targets V1 for sCOVID-19. Infiltration degrees of many immune cells, such as macrophages, were also sharply different between mCOVID-19 and sCOVID-19 samples. Predicted protein targeting drugs with the highest scores nearly all belong to naturally derived or synthetic glucocorticoids. For the two single-cell RNA-seq datasets, we explored the expression distribution of hub genes for mCOVID-19/sCOVID-19 in each cell type. The expression levels of PRKCA, MCM5, TYMS, RBBP4, BCL6, FLOT1, KDM6B, and TLR2 were found to be cell-type-specific. Furthermore, the expression levels of 10 hub genes for mCOVID-19 were significantly upregulated in PBMCs between eight healthy controls and eight mCOVID-19 patients at our institution. Collectively, we detected critical modules, pathways, TFs, kinases, immune cells, targeting drugs, hub genes, and their expression distributions in different cell types that may involve the pathogenesis of COVID-19 of differing severity, which may propel earlier diagnosis and more effective treatment of this intractable disease in the future.

Trends in HIV Prevalence and HIV-Related Risk Behaviors Among Male Students Who Have Sex With Men From 2016 to 2020 in Nanjing, China: Consecutive Cross-Sectional Surveys.

Background: The growing HIV epidemic among student men who have sex with men (MSM) necessitates immediate attention from public health. In China, male students who have sex with men (SMSM) were also at an increasing risk of HIV transmission. The aim of this study was to investigate the trends in HIV prevalence, HIV-related risk behaviors, and HIV testing, as well as analyze the factors associated with HIV infection among SMSM in Nanjing. Methods: Data were collected through face-to-face questionnaire interviews and laboratory testing in Nanjing. The participants were recruited among SMSM by snowball sampling and internet-recruited convenience sampling annually from 2016 to 2020. The self-report data primarily included demographics, HIV knowledge, HIV-related behaviors, and HIV testing, while the laboratory test results of HIV and syphilis were collected. Linear-by-linear chi-square test was used to analyze the trends of HIV/syphilis prevalence and its risk behaviors. The binary logistic regression model was used to explore the factors associated with HIV infection. Results: During the 5 years from 2016 to 2020, a total of 775 SMSM were recruited in our survey (220, 112, 171, 142, and 120, respectively). The HIV prevalence was 5.2, 6.3, 5.3, 7.0, and 8.3%, without a significant increasing trend (P = 0.277). Syphilis prevalence fluctuated from 5.7% in 2016 to 4.2% in 2020, without a significant decreasing trend (P = 0.318). The proportion of consistent condom use in anal intercourse (48.5 to 56.2%, P < 0.05), and HIV testing in the past 12 months (51.0 to 59.2%, P < 0.05) were increasing. A remarkable growing trend has been reported in the percentage of MSM with more than one male sex partner (46.2 to 59.2%, P < 0.05). Multivariate analysis showed that HIV testing in the past 12 months was a protective factor against HIV infection. MSM who had unprotected anal intercourse (UAI) in the past 6 months, recreational drug use, and currently syphilis infection were risk factors for HIV infection. Conclusions: We observed stable HIV/ syphilis prevalence, increasing consistent condom use, increasing HIV testing rate, and increasing multiple male sex partners dramatically among SMSM in China. The original comprehensive intervention measures should be continuously strengthened for the subgroup. To satisfy the current HIV prevention requirements, new biological interventions should be introduced and carried out as major components of combination prevention programs.

Identification of recombinant coxsackievirus A6 variants in hand, foot and mouth disease in Nanjing, China, 2013.

PURPOSE: Enteroviruses (EV) 71 and coxsackievirus A (CVA) 16 are the most prevalent EV serotypes responsible for hand, foot and mouth disease (HFMD). Nevertheless, CVA6 was found to be the leading cause of HFMD in the Nanjing area, of China in 2013. This study aims to provide insights into the occurrence of the emergent recombinant CVA6 through examination of the evolutionary history and the involved recombination events. METHODOLOGY: The viral protein1 (VP1) and non-structural (NS) 2C and 3D of 28 Nanjing CVA6 strains were aligned, among which the full-length sequences of eight strains were further characterized. RESULTS: We revealed the co-existence of two recombinant forms (RFs), RF-A and RF-J, in the local area. RF-J is a novel RF group, comprising a proportion of local and Shanghai CVA6 strains from 2013. The appearance of RF-J CVA6 strains was most likely the result of two recombination events, with the co-circulating CVA4 and CVA8 providing the regions beyond positions 4001~4045 and 4866~4873, respectively. Evolutionary history analysis showed that the VP1 sequences of RF-J derived from RF-A, which was also probably the ancestor of several other RF groups. The 3D region of RF-J was closely related to CVA8. The point in time of emergence of the most recent common ancestor (tMRCA) of RF-J in China was estimated to be around 2011 in both terms of VP1 and 3D region. CONCLUSION: The emerging recombinant CVA6 variants belong to a novel RF-J group which was most likely formed by at least two recombination events. Continued monitoring on the geographical distribution of various CVA6 RFs is essential.

Genetic characteristics of coxsackievirus A16 associated with hand, foot, and mouth disease in Nanjing, China.

INTRODUCTION: Coxsackievirus A16 (CVA16) is a main pathogen in hand, foot, and mouth disease (HFMD) worldwide. This study intended to clarify the genetic characteristics of CVA16 associated with HFMD in a defined area in Nanjing, China. METHODOLOGY: A total of 175 CVA16 strains isolated from throat swabs between 2011 and 2013 were obtained through sentinel hospitals in Nanjing. Multiplex polymerase chain reaction (PCR) was used to amplify the VP1 sequence of local CVA16 strains, and their genetic relationship with 138 CVA16 strains isolated in China and other countries of the world was compared. RESULTS: Phylogenetic analysis based on complete VP1 sequences revealed that subgenotype B1a and B1b were predominantly circulating in Nanjing and B1b strains were spread more widely. The evolution of CVA16 strains is very conservative, with a mean distance of less than 9%. Moreover, six reported conservative regions in VP1 protein were examined, and three of them exhibited high conservation in all CVA16 genotypes except the G-10 prototype and may serve for further vaccine research. CONCLUSIONS: The CVA16 strains circulating in Nanjing, China, in 2011 to 2013 belonged to different genotypes and evolved in a conservative way. To provide further evidence for epidemiological linkage and evolutionary recombination events in CVA16, persistent surveillance of HFMD-associated pathogens is required.

Molecular Diversity of Sapovirus Infection in Outpatients Living in Nanjing, China (2011-2013).

Aim. To gain insight into the molecular diversity of sapovirus in outpatients with acute gastroenteritis in Nanjing, China. Methods. The specimens from outpatients clinically diagnosed as acute gastroenteritis were detected by real-time PCR; RT-PCR was then performed to amplify part of VP1 sequences. The PCR products were cloned into pGEM-T Easy vector and bidirectionally sequenced. All sequences were edited and analyzed. A phylogenetic tree was drawn with the MEGA 5.0 software. Results. Between 2011 and 2013, 16 sapovirus positive cases were confirmed by real-time PCR. The infected cases increased from two in 2011 and six in 2012 to eight in 2013. The majority was children and the elderly (15, 93.75%) and single infections (15, 93.75%). Of the 16 real-time positive specimens, 14 specimens had PCR products and the analysis data of the 14 nucleic sequences showed that there was one GI genogroup with four genotypes, two GI.2 in 2011, three GI.2, and one GI.1 in 2012 and one GI.2, three GI.1, two GI.3, and two GI.5 in 2013. Conclusion. Our data confirmed continuous existing of GI genogroup and GI.2 genotype from 2011 to 2013 in Nanjing and the successive appearance of different genotypes from outpatients with gastroenteritis.

Genetic characterization of emergent GII.17 norovirus variants from 2013 to 2015 in Nanjing, China.

During the 2014/15 winter season, a newly emergent GII.P17-GII.17 variant overwhelmed currently dominant GII.4 viruses, causing outbreaks of acute gastroenteritis in China and Japan. In Nanjing area, this novel GII.17 variant was first identified in a sporadic case of acute gastroenteritis in July 2013, 18 months ahead of reports from other parts of China. In this study, epidemiological features and genotyping of noroviruses from 2013 to 2015 were depicted. Twenty-eight local GII.17 sequences of capsid N-terminus originating from July 2013 to August 2015 were aligned, among which complete genome of seven strains obtained from two outbreaks and five sporadic cases was extensively characterized. The differences of local GII.17 variants led to at least two clusters, with strains from 2013/14 season and those from 2014/15 season grouped differently. Multiple nucleotide and amino acid variations between different clusters of GII.17 were elucidated, including residue substitutions and insertion occurring in or near antigenic and receptor-binding sites of viral protein 1. In addition, sequence hypervariability from residue 279 through 406 of viral protein 2 was identified. The modifications may reveal a distinctive adaptive process which could in part explain the rapid spread of emerging GII.17 variants. Continued monitoring on novel GII.17 is essential.

[Phylogenetic Analyses and Characterization of the VP7 Genes of Serotype G9 Rotaviruses Circulating in Nanjing (China), 2012-2013].

Group-A rotaviruses are recognized as the most common cause of acute diarrhea. Phylogenetic analyses of the VP7 genes of rotaviruses circulating in Nanjing (China) could aid in the development of rotavirus vaccines. A total of 908 stool specimens were collected from patients suffering from acute diarrhea in Nanjing between October 2012 and December 2013, and were tested further for rotaviruses. Fifty rotavirus isolates selected randomly were typed by reverse transcription-polymerase chain reaction using serotype-specific primers for G genotyping. VP7 genes of 19 G9 strains were sequenced for further genetic characterization. Among the 908 stool specimens examined during the surveillance period, 103 (11.34%) were rotavirus-positive. G9 was the most predominant genotype (78.0%), followed by G2, G1 and G3. Sequence and phylogenetic analyses of the VP7 genes of serotype G9 rotaviruses revealed these strains to comprise two lineages (G9-VI, G9-III) and to be dominated by the G9-VI lineage (which belonged to a unique subcluster of Japanese and Chinese G9 strains). Amino-acid sequences of the four antigenic regions (A, B, C or F) were variant among a portion of strains, which may have contributed to the prevalence of G9 rotaviruses in this area.