A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

A strongly selected mutation in the HIV-1 genome is independent of T cell responses and neutralizing antibodies.

BACKGROUND: Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. RESULTS: Strongly selected mutations were identified by analyzing 5'-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 and fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. CONCLUSIONS: The rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.

[Comparison of Classifcation and Regulation of in Vitro Diagnostic Products for Clinical Pathology in China and Foreign Country].

The classification and the regulatory requirement among U.S., E.U., Japan and China were summarized and compared for the immunohistochemistry and in situ hybridization products. The results indicate that:the regulatory classifications of the related products are higher in Japan and China, than U.S.and E.U.; the classification and regulatory requirement are adjusted more flexibly in the centralized system. The difference was discussed and accordingly some suggestions and implications were given for the China's regulation.

Job security and work performance in Chinese employees: The mediating role of organisational identification.

This research focuses on investigating whether organisational identification mediates the effects of job security on in-role behaviour and extra-role behaviour and how these mediation mechanisms differ according to gender. Through analysing 212 supervisor-subordinate dyads from a Chinese air transportation group, the research indicated that organisational identification partially mediated the effect of job security on in-role behaviour and fully mediated the effect of job security on extra-role behaviour. A multi-group analysis also showed that there were significant differences between male and female employees in these relationships. In addition, moderated mediation analyses showed that gender moderated the indirect effects of job security on in-role behaviour and extra-role behaviour through organisational identification. Limitations and implications of these findings are discussed.

Both Rbx1 and Rbx2 exhibit a functional role in the HIV-1 Vif-Cullin5 E3 ligase complex in vitro.

Rbx1 and Rbx2 are essential components of Cullin-RING E3 Ligases. Vif is generally believed to preferentially recruit the Cul5-Rbx2 module to induce proteasomal degradation of antiretroviral enzyme APOBEC3G, although some investigators have found that the Cul5-Rbx1 module is recruited. Here, to investigate the function of the two Rbx proteins in the Vif-Cul5 complex, we analyzed the performance of Cul5-Rbx1/Cul5-Rbx2 module in the activity of Vif E3 ligase and evaluated the interactions between Rbx1/Rbx2 and Cul5. We found that either Rbx1 or Rbx2 could promote ubiquitination of APOBE3G (A3G) in vitro. We also found that both Rbx1 and Rbx2 could bind Cul5 in cells and Rbx2 could dose-dependently inhibit the interaction of Rbx1 with Cul5. Furthermore, only the decrease of endogenous Rbx2 but not Rbx1 could impair the Vif-induced A3G degradation in cells. These findings indicate that Rbx1 and Rbx2 can both activate Cul5-Vif E3 ligase in vitro, but they may undergo a more delicate selection mechanism in vivo.

Design of hyperthermophilic lipase chimeras by key motif-directed recombination.

Recombination of diverse natural evolved domains within a superfamily offers greater opportunity for enzyme function leaps. How to recombine protein modules from distant parents with less disruption in cross-interfaces is a challenging issue. Here, we identified the existence of a key motif, the sequence VVSVN(D)YR, within a structural motif ψ loop in the α/ß-hydrolase fold superfamily, by using a MEME server and the PROMOTIF program. To obtain thermostable lipase-like enzymes, two chimeras were engineered at the key motif regions through recombination of domains from a mesophilic lipase and a hyperthermophilic esterase/peptidase with amino acid identity less than 21 %. The chimeras retained the desirable substrate preference of their mesophilic parent and exhibited more than 100-fold increased thermostability at 50 °C. Through site-directed mutation, we further improved activity of the chimera by 4.6-fold. The recombination strategy presented here enables the creation of novel catalysts.

Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation.

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Characteristics of Vaginal Microbiota of Women of Reproductive Age with Infections.

The vaginal microbiota can be classified into five major community state types (CSTs) based on the bacterial content. However, the link between different CST subtypes and vaginal infection remains unclear. Here, we analyzed 2017 vaginal microbiota samples from women of a reproductive age with vaginal infections that were published in the last decade. We found that L. iners was the most dominant in 34.8% of the vaginal samples, followed by L. crispatus (21.2%). CST I was common in healthy individuals, whereas CST III and IV were associated with dysbiosis and infection. CST III-B, IV-A, IV-B, and IV-C0 were prevalent in patients with bacterial vaginosis (BV). Based on the relative abundance of bacteria at the (sub)genus level, a random forest classifier was developed to predict vaginal infections with an area under the curve of 0.83. We further identified four modules of co-occurring bacterial taxa: L. crispatus, Gardnerella, Prevotella, and Bacteroides. The functional prediction revealed that nucleotide biosynthesis pathways were upregulated in patients with human papilloma virus, and carbohydrate degradation pathways were downregulated in patients with BV. Overall, our study identified the bacterial signatures of healthy and infected vaginal microbiota, providing unique insights into the clinical diagnosis and health status prediction of women of a reproductive age.

Rapid inference of antibiotic resistance and susceptibility for Klebsiella pneumoniae by clinical shotgun metagenomic sequencing.

OBJECTIVES: The study aimed to develop a genotypic antimicrobial resistance testing method for Klebsiella pneumoniae using metagenomic sequencing data. METHODS: We utilized Lasso regression on assembled genomes to identify genetic resistance determinants for six antibiotics (Gentamicin, Tobramycin, Imipenem, Meropenem, Ceftazidime, Trimethoprim/Sulfamethoxazole). The genetic features were weighted, grouped into clusters to establish classifier models. Origin species of detected antibiotic resistant gene (ARG) was determined by novel strategy integrating "possible species", "gene copy number calculation" and "species-specific kmers". The performance of the method was evaluated on retrospective case studies. RESULTS: Our study employed machine learning on 3928 K. pneumoniae isolates, yielding stable models with AUCs > 0.9 for various antibiotics. GenseqAMR, a read-based software, exhibited high accuracy (AUC 0.926-0.956) for short-read datasets. The integration of a species-specific kmer strategy significantly improved ARG-species attribution to an average accuracy of 96.67%. In a retrospective study of 191 K. pneumoniae-positive clinical specimens (0.68%-93.39% genome coverage), GenseqAMR predicted 84.23% of AST results on average. It demonstrated 88.76%-96.26% accuracy for resistance prediction, offering genotypic AST results with a shorter turnaround time (mean ± SD: 18.34 ± 0.87 hours) than traditional culture-based AST (60.15 ± 21.58 hours). Furthermore, a retrospective clinical case study involving 63 cases showed that GenseqAMR could lead to changes in clinical treatment for 24 (38.10%) cases, with 95.83% (23/24) of these changes deemed beneficial. CONCLUSIONS: In conclusion, GenseqAMR is a promising tool for quick and accurate AMR prediction in Klebsiella pneumoniae, with the potential to improve patient outcomes through timely adjustments in antibiotic treatment.

Interactions between HIV-1 Vif and human ElonginB-ElonginC are important for CBF-ß binding to Vif.

BACKGROUND: The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-ß-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-ß-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-ß is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-ß-Cul5 E3 ligase complex. RESULTS: The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-ß to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-ß interaction. (2) Introduction of both the Vif SLQ → AAA mutant (VifΔSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL → AAA mutant (VifΔPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-ß binding. (3) EloB-EloC but not CBF-ß could greatly enhance the folding of full-length Vif in Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifΔSLQ/VifΔPPL through the region between residues 9 and 14. CONCLUSION: Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-ß to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization.

Small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between Vif and ElonginC.

The HIV-1 viral infectivity factor (Vif) protein is essential for viral replication. Vif recruits cellular ElonginB/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. In the absence of Vif, A3G is packaged into budding HIV-1 virions and introduces multiple mutations in the newly synthesized minus-strand viral DNA to restrict virus replication. Thus, the A3G-Vif-E3 complex represents an attractive target for development of novel anti-HIV drugs. In this study, we identified a potent small molecular compound (VEC-5) by virtual screening and validated its anti-Vif activity through biochemical analysis. We show that VEC-5 inhibits virus replication only in A3G-positive cells. Treatment with VEC-5 increased cellular A3G levels when Vif was coexpressed and enhanced A3G incorporation into HIV-1 virions to reduce viral infectivity. Coimmunoprecipitation and computational analysis further attributed the anti-Vif activity of VEC-5 to the inhibition of Vif from direct binding to the ElonginC protein. These findings support the notion that suppressing Vif function can liberate A3G to carry out its antiviral activity and demonstrate that regulation of the Vif-ElonginC interaction is a novel target for small-molecule inhibitors of HIV-1.

Purification of eukaryotic tetherin/Vpu proteins and detection of their interaction by ELISA.

Tetherin/BST-2/CD317 inhibits HIV-1 release from infected cells, while HIV-1 Vpu efficiently antagonizes tetherin based on intermolecular interactions between the transmembrane domains of each protein. In this study, we successfully partially purified His-tagged tetherin with a glycophosphatidylinositol deletion (delGPI) and His-tagged full-length Vpu from transiently transfected 293T cells using affinity chromatography. The in vitro interaction between these purified proteins was observed by a pull-down assay and ELISA. Detection of the Vpu/tetherin interaction by ELISA is a novel approach that would be advantageous for inhibitor screening in vitro. Successful co-purification of the tetherin/Vpu complex also provides a basis for further structural studies.

Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses.

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

The impact of sample processing on the rapid antigen detection test for SARS-CoV-2: Virus inactivation, VTM selection, and sample preservation.

Many factors have been identified as having the ability to affect the sensitivity of rapid antigen detection (RAD) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to identify the impact of sample processing on the sensitivity of the RAD tests. We explored the effect of different inactivation methods, viral transport media (VTM) solutions, and sample preservation on the sensitivity of four RAD kits based on two SARS-CoV-2 strains. Compared with non-inactivation, heat inactivation significantly impacted the sensitivity of most RAD kits; however, ß-propiolactone inactivation only had a minor effect. Some of the VTM solutions (VTM2, MANTACC) had a significant influence on the sensitivity of the RAD kits, especially for low viral-loads samples. The detection value of RAD kits was slightly decreased, while most of them were still in the detection range with the extension of preservation time and the increase of freeze-thaw cycles. Our results showed that selecting the appropriate inactivation methods and VTM solutions is necessary during reagent development, performance evaluation, and clinical application.

Multicenter assessment of shotgun metagenomics for pathogen detection.

BACKGROUND: Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. METHODS: A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. FINDINGS: Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. INTERPRETATION: Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. FUNDING: This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

[Challenges and considerations on quality control and evaluation of pathogen metagenomic next-generation sequencing].

Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.

Accumulated mutations by 6 months of infection collectively render transmitted/founder HIV-1 significantly less fit.

OBJECTIVE: Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early infection affect viral fitness has not been well studied. METHODS: Paired infectious molecular clones (IMCs) for transmitted/founder (T/F) and 6-month (6-mo) viruses post infection were generated from 10 infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo viruses to their cognate T/F viruses. RESULTS: All ten 6-mo viruses were less fit than their cognate T/F viruses. Moreover, the fitness losses of the 6-mo viruses correlated with the decrease in viral loads from the peak of viremia. CONCLUSION: These results show that the mutations accumulated during half a year post infection collectively reduce viral fitness and thereby contribute to lowering viral loads.

[Expert consensus on microbiome sequencing and analysis].

In the past ten years, the research and application of microbiome has continued to increase. The microbiome has gradually become the research focus in the fields of life science, environmental science, and medicine. Meanwhile, many countries and organizations around the world are launching their own microbiome projects and conducting a multi-faceted layout, striving to gain a strategic position in this promising field. In addition, whether it is scientific research or industrial applications, there has been a climax of research and a wave of investment and financing, accordingly, products and services related to the microbiome are constantly emerging. However, due to the rapid development of microbiome sequencing and analysis related technologies and methods, the research and application from various countries have not yet unified on the standards of technology, programs, and data. Domestic industry participants also have insufficient understanding of the microbiome. New methods, technologies, and theories have not yet been fully accepted and used. In addition, some of the existing standards and guidelines are too general with poor practicality. This not only causes obstacles in the integration of scientific research data and waste of resources, but also gives related companies unfair competition opportunity. More importantly, China still lacks national standards related to the microbiome, and the national microbiome project is still in the process of preparation. In this context, the experts and practitioners of the microbiome worked together and developed the consensus of experts. It can not only guide domestic scientific research and industrial institutions to regulate the production, learning and research of the microbiome, the application can also provide reference technical basis for the relevant national functional departments, protect the scale and standardized corporate company's interests, strengthen industry self-discipline, avoid unregulated enterprises from disrupting the market, and ultimately promote the benign development of microbiome-related industries.

[Isolation and identification of a thermophilic bacterium from hot springs in Changbai Mountains].

OBJECTIVE: We studied physiological and biochemical properties of a thermophilic bacterium isolated from hot springs in Changbai Mountain. METHODS: The strain CBS-5 (=JCM 15484) from hot springs in Changbai Mountains was isolated by plating and screening on Olive-rich medium. It was characterized by the physiological and biochemical analysis, 16S rDNA sequencing and determination of (G+C)mol% contents. RESULTS: The cells were Gram-positive, non-motile rods, spore-forming and generally occurred singly or in pairs. The growth temperature ranges from 48 degrees C to 70 degrees C, optimum at 65 degrees C; growth pH ranges from 6.5 to 8.5, optimum at 7.7. The strain used glucose, sucrose, maltose, lactose and rhanmose as carbon and energy sources. The G+C content of DNA was 41.9 mol%. The major cellular fatty acids were iso-C15:0, C16:0 and iso-C17:0, respectively representing about 24.20%, 20.45% and 17.42% of total fatty acids. The 16S rDNA sequence analysis showed that the strain belonged to the Anoxybacillus genus, for the sequence similarity was 95.1%-98.5% to other known species of Anoxybacillus. CONCLUSION: Strain CBS-5 was a member of Anoxybacillus.

Multiplex Cell-Free DNA Reference Materials for Quality Control of Next-Generation Sequencing-Based In Vitro Diagnostic Tests of Colorectal Cancer Tolerance.

Background: Liquid biopsies based on next-generation sequencing (NGS) assays are confronted with more opportunities and challenges. Widespread clinical implementation of NGS-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials (RMs) which could provide full control of the process from nucleic acid extraction to test report generation. Quality control based on cell-free DNA (cfDNA) RMs is especially important for liquid biopsies. Methods: Here, we used genomic DNA from thirteen cell lines to establish four negative cfDNA RMs (N1-N4) and four multiplex cfDNA RMs (L1-L4) at serial allelic frequencies ranging from approximately 2% to 0.1%. All the cfDNA RMs were quantified and validated via both droplet digital polymerase chain reaction (ddPCR) and NGS. These RMs were distributed to eight domestic manufacturers to collaboratively evaluate the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton, and BGISEQ). Results: Each multiplex RM has eleven colorectal cancer-related mutations, including six KRAS mutations (G12S, G12C, G12D, G12A, G12V, and G13D), three NRAS mutations (G12D, Q61R, and Q61K), one PIK3CA mutation (H1047R), and one BRAF mutation (V600E). Each mutation in the cfDNA RMs was quantified and validated via both ddPCR and NGS, showing the good relevance of mutant allelic frequency. These RMs were distributed to eight domestic manufacturers for collaborative evaluation. All eight manufacturers provided similar results by domestic NGS-based cancer IVDs, except for manufacturer #5. The coefficient of variation (CV) was increased with decreasing mutant allelic frequency, and poor repetition occurred when the allelic frequency was lower than 0.5%. Conclusions: These results indicated that these cfDNA RMs would be pivotal for NGS-based cancer IVDs, especially for liquid biopsies of colorectal cancer-related mutations and would guide the further development of RMs covering more onco-related mutations.