应用免疫学和病毒学指标辅助判断HIV-1新发感染的可行性分析

目的 分析免疫学和病毒学指标作为预测1型艾滋病病毒(HIV-1)新发感染标志的可行性.方法 对2017-2018年浙江省、北京市、云南省三个现场的HIV抗体初筛、确证阳性样本1 382份,进行新发感染、CD4+T淋巴细胞(简称CD4细胞)计数和病毒载量检测,采用Logistic回归模型比较新发感染和既往感染两组样本之间...     

套式PCR对HIV—l env基因检测的敏感度分析

采用套式PCR对含HIV—1膜蛋白基因的B亚型质粒和HIV—1感染者外周血单核细胞(PBMC)进行env基因部分片断的扩增,套式PCR可使常规PCR的敏感性提高100至1000倍,常规PCR只扩增出12份PBMC中的2份样品,套式PCR则扩增出全部样品中HIV—1膜蛋白基因片断.     

针对MSM人群的HIV-1早期感染检测策略研究

目的建立针对男男性行为人群（MSM）艾滋病病毒（HIV）感染的高敏感检测策略,减少窗口期漏检。方法收集来自黑龙江、辽宁、内蒙古、宁夏和青海省（自治区）的MSM人群专项调查样本,分别用中国现行的HIV抗体常规检测策略、新检测策略一、新检测策略二和新检测策略三进行检测,对常规策略与3种新策略的检测结果进行对比、分析。结果11023份调查样本中,常规策略[三代酶联免疫吸附试验（ELISA）＋蛋白免疫印迹试验（WB）]检出的抗体阳性、阴性和不确定标本分别为282份、10716份和25份;新策略一（四代ELISA＋WB）检出的抗体阳性、阴性和不确定标本分别为282份、10676份和65份;新策略二（三代ELISA＋WB＋集合核酸）检出的核酸阳性,抗体阳性、阴性和不确定标本分别为28份、282份、10688份和25份;新策略三（四代ELISA＋WB＋集合核酸）检出的核酸阳性,抗体阳性、阴性、不确定标本分别为13份、282份、10663份和65份。常规策略、新策略一至三的检测敏感性分别为90．96％、90．96％、100％和95．16％,新策略二和新策略三的检测敏感性显著高于常规策略（P〈0．05）,且新策略对早期及急性HIV感染者的检测能力优于常规策略。结论针对MSM人群的HIV新检测策略,可以减少早期及急性HIV感染者的漏检,应根据实验室检测能力和样本量尽快在该人群中推广使用。     

Challenges in the Design of a T Cell Vaccine in the Context of HIV-1 Diversity

The extraordinary variability of HIV-1 poses a major obstacle to vaccine development. The effectiveness of a vaccine is likely to vary dramatically in different populations infected with different HIV-1 subtypes, unless innovative vaccine immunogens are developed to protect against the range of HIV-1 diversity. Immunogen design for stimulating neutralizing antibody responses focuses on “breadth” – the targeting of a handful of highly conserved neutralizing determinants on the HIV-1 Envelope protein that can recognize the majority of viruses across all HIV-1 subtypes. An effective vaccine will likely require the generation of both broadly cross-neutralizing antibodies and non-neutralizing antibodies, as well as broadly cross-reactive T cells. Several approaches have been taken to design such broadly-reactive and cross-protective T cell immunogens. Artificial sequences have been designed that reduce the genetic distance between a vaccine strain and contemporary circulating viruses; “mosaic” immunogens extend this concept to contain multiple potential T cell epitope (PTE) variants; and further efforts attempt to focus T cell immunity on highly conserved regions of the HIV-1 genome. Thus far, a number of pre-clinical and early clinical studies have been performed assessing these new immunogens. In this review, the potential use of these new immunogens is explored.     

Sequence Length of HIV-1 Subtype B Increases over Time: Analysis of a Cohort of Patients with Hemophilia over 30 Years

We aimed to investigate whether the sequence length of HIV-1 increases over time. We performed a longitudinal analysis of full-length coding region sequences (FLs) during an HIV-1 outbreak among patients with hemophilia and local controls infected with the Korean subclade B of HIV-1 (KSB). Genes were amplified by overlapping RT-PCR or nested PCR and subjected to direct sequencing. Overall, 141 FLs were sequentially determined over 30 years in 62 KSB-infected patients. Phylogenetic analysis indicated that within KSB, two FLs from plasma donors O and P comprised two clusters, together with 8 and 12 patients with hemophilia, respectively. Signature pattern analysis of the KSB of HIV-1 revealed 91 signature nucleotide residues (1.1%). In total, 48 and 43 signature nucleotides originated from clusters O and P, respectively. Six positions contained 100% specific nucleotide(s) in clusters O and P. In-depth FL analysis for over 30 years indicated that the KSB FL significantly increased over time before combination antiretroviral therapy (cART) and decreased with cART. This increase occurred due to the significant increase in env and nef genes, originating in the variable regions of both genes. The increase in sequence length of HIV-1 over time suggests an evolutionary direction.     

A Novel High Throughput,Parallel Infection Assay for Determining the Replication Capacities of 346 Primary HIV-1 Isolates of the Zurich Primary HIV-1 Infection Study in Primary Cells

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSF_WT and HIV-1 JR-CSF_{K65R_M184V}. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells.     

Prediction of SARS-CoV-2 Variant Lineages Using the S1-Encoding Region Sequence Obtained by PacBio Single-Molecule Real-Time Sequencing

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (n = 11), N501Y (n = 6), or E484K (n = 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.     

Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.     

Evaluation of the use of dried spots and of different storage conditions of plasma for HIV-1 RNA quantification

OBJECTIVES: The aim of the study was to evaluate the use of dried plasma spots to determine HIV-1 RNA viral loads. METHODS: The viral loads of 30 liquid plasma samples were compared with those of corresponding dried plasma spots on filter paper (DPS-FP) and in tubes (DPS-T), both of which were left for 7 days at 22 degrees C. Also, 10 liquid plasma samples with detectable viral load were stored at 4, 22 or 37 degrees C for 7 days and five further liquid plasma samples were air-dried for up to 54 h to assess the effects of temperature and the drying step on HIV-1 viral load. RESULTS: The viral loads of the 30 liquid plasma samples correlated significantly with those of the paired dried spots DPS-FP and DPS-T, but with median losses of 0.64 and 0.69 log(10) HIV-1 RNA copies/mL, respectively, and a limit of detection of 3 log(10) copies/mL. The 10 liquid plasma samples stored for 1 week at 37 degrees C showed a weaker correlation and had a significantly reduced median viral load (-0.92 log(10); P=0.005) when compared with the viral load of the matched plasma stored at - 80 degrees C. Most of the loss happened during the drying step. CONCLUSIONS: Reliable measurement of HIV-1 RNA viral load requires good plasma storage conditions. HIV RNA stability was affected by desiccation and 1 week of storage at 37 degrees C. However, our findings suggest that liquid plasma can be kept at 4 or 22 degrees C for a week with no effect on viral load.     

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-β Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis

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