Improving trans-cleavage activity of CRISPR-Cas13a using engineered crRNA with a uridinylate-rich 5'-overhang.

The engieering of Cas13a crRNA to enhance its binding affinity with the Cas enzyme or target is a promising method of improving the collateral cleavage efficiency of CRISPR-Cas13a systems, thereby amplifying the sensitivity of nucleic acid detection. An examination of the top-performing engineered crRNA (24 nt 5'7U LbuCas13a crRNA, where the 5'-end was extended using 7-mer uridinylates) and optimized conditions revealed an increased rate of LbuCas13a-mediated collateral cleavage activity that was up to seven-fold higher than that of the original crRNA. Particularly, the 7-mer uridinylates extension to crRNA was determined to be spacer-independent for enhancing the LbuCas13a-mediacted collateral cleavage activity, and also benefited the LwaCas13a system. The improved trans-cleavage activity was explained by the interactions between crRNA and LbuCas13a at the molecular level, i.e. the 5'-overhangs were anchored in the cleft formed between the Helical-1 and HEPN2 domains with the consequence of more stable complex, and experimentally verified. Consequently, the improved CRISPR-Cas13a system detected the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a sensitivity of 2.36 fM that was 160-times higher than that of the original system. Using isothermal amplification via reverse transcription-recombinase polymerase amplification (RT-RPA), the system was capable to detect SARS-CoV-2 with attomolar sensitivity and accurately identified the SARS-CoV-2 Omicron variant (20/21 agreement) in clinical samples within 40 min.

Human rotavirus strains circulating among children in the capital of China (2018-2022)_ predominance of G9P[8] and emergence ofG8P[8].

Objective: This study aimed to update the genetic diversity of Rotavirus (RV) infections in children under five years old in Beijing, China. Methods: A 5-year active hospital-based surveillance for sporadic acute gastroenteritis (AGE) from January 2018 to December 2022 in the capital of China was performed. A total of 748 fecal samples from AGE patients were collected for followed by RV antigen detection by ELSIA, RNA detection by reverse transcription PCR, G/P genotyping and phylogenetic analyzing. Results: RV antigen was detected in 11.0% of the collected samples, with 54 samples confirmed to be RV RNA positive. G9 and G8 genotypes were identified in 43 (79.6%) and 7 (13.0%) samples, respectively, all of which were allocated to P[8]. The predominant G/P combination was G9P[8] (79.6%), following by G8P[8] (13.0%), G4P[8] (5.6%) and G3P[8] (1.9%). A significant change in G/P-type distribution was observed, with the G9P[8] being predominant from 2018 to 2021, followed by the emergence of an uncommon G8P[8] genotype, which was first reported in 2021 and became predominant in 2022. Blast analysis showed that one G1 isolate had a high similarity of 99.66% on nucleotide acid with RotaTeq vaccine strain with only one amino acid difference L150V. Additionally, one P[8] isolate was clustered into a branch together with RotaTeq vaccine strain G6P[8]. Conclusions: The study reveals that G8P[8] has become the predominant genotype in pediatric outpatients in China for the first time, indicating a significant change in the composition of RV genetic diversity. The importance of RVA genotyping in surveillance is emphasized, as it provides the basis for new vaccine application and future vaccine efficacy evaluation.

Genotypes Diversity of Acute Gastroenteritis Outbreaks Caused by Human Sapovirus - Beijing Municipality, China, 2015-2021.

Introduction: Human sapovirus (HuSaV) is an enteric virus responsible for sporadic cases and outbreaks of acute gastroenteritis (AGE) globally. A seven-year active surveillance study was conducted to investigate the molecular epidemiology of HuSaVs associated with AGE outbreaks in Chaoyang District of Beijing Municipality, China from January 2015 to December 2021. Methods: Fecal and anal swab samples were obtained from patients experiencing AGE outbreaks. HuSaVs were identified through reverse transcription polymerase chain reaction (RT-PCR), and partial viral protein 1 (VP1) sequences (approximately 434 base pairs) were utilized for genotyping, single nucleotide polymorphism (SNP) analysis, and phylogenetic examination. Results: HuSaVs were identified in 71 AGE outbreaks, demonstrating a detection rate of 10.5%, second only to norovirus. The primary demographic affected by HuSaV were children under the age of 5 in kindergarten settings. Infection rates tended to peak during two distinct periods: May to June and September to December. Upon genotyping, seven distinct genotypes emerged. GII.3 was the most prevalent, accounting for 54.9% of cases, followed by GI.1 (12.7%), GI.2 (9.9%), GII.5 (7.0%), GI.5 (2.8%), GI.6 (1.4%), GII.1 (1.4%), and untyped cases (9.9%). A phylogenetic analysis of GII.3 identified three distinct groups, with 15 notable SNPs observed. Conclusions: This study offers a comprehensive analysis of the persistent prevalence of HuSaV outbreaks in Chaoyang District, Beijing Municipality, China. Over time, the diversity of HuSaV subtypes has shifted, and it is now recognized as the second leading viral agent responsible for AGE outbreaks. This highlights the importance of ongoing surveillance in the future.

Persistence of SARS-CoV-2 RNA in wastewater after the end of the COVID-19 epidemics.

Although the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely detected in wastewater in many countries to track the COVID-19 pandemic development, it is still a lack of clear understanding of the persistence of SARS-CoV-2 in raw sewage, especially after the end of the COVID-19 pandemic event. To fill this knowledge gap, this study conducted a field trial on the SARS-CoV-2 presence in various wastewater facilities after the end of the COVID-19 epidemics in Beijing. The result showed that the wastewater treatment facility is a large SARS-CoV-2 repository. The viral RNA was still present in hospital sewage for 15 days and was continually detected in municipal WWTPs for more than 19 days after the end of the local COVID-19 epidemics. The T90 values of the SARS-CoV-2 RNA in raw wastewater were 17.17-8.42 days in the wastewater at 4 â„ƒ and 26 â„ƒ, respectively, meaning that the decay rates of low titer viruses in raw sewage were much faster. The results confirmed that the SARS-CoV-2 RNA could persist in wastewater for more than two weeks, especially at lower temperatures. The sewage systems would be a virus repository and prolong the presence of the residual SARS-CoV-2 RNA. The study could enhance further understanding of the presence of SARS-CoV-2 RNA in raw wastewater.

The Use of Delphi Method and Analytical Hierarchy Process in the Establishment of Assessment Tools in Premature Ejaculation: The Scoring System for Premature Ejaculation Treatment Outcomes.

There is a lack of objective tools to comprehensively evaluate premature ejaculation (PE) treatment results clinically. We aimed to describe the development of a novel scoring system for PE treatment results as an example of using the Delphi method and an analytical hierarchy process for complex decision-making in the field of sexual medicine. A Delphi question survey was adopted to collect expert opinions from 47 Chinese specialists in andrology/urology on the assessment of PE treatment outcomes based on four primary properties, that is, the improvement in intravaginal ejaculation latency time, a couple's mental status, the ability to control ejaculation, and sexual intercourse satisfaction. Different weights on those primary properties were assigned to create a mathematical hierarchy matrix and then perform an analysis. The scores were assigned according to the calculated weights. The ratio among the combined weights of the four primary properties was 1:3:2:3. The total numerical score was 36. Scores above 27, between 26 and 18, and below 17 indicated significant improvement, moderate improvement, and no improvement in PE, respectively, with selected treatments. The scoring system with 36 points can be used by physicians, patients, and their sexual partners to comprehensively and objectively assess quantitative PE treatment results.

Emerging recombination of the C2 sub-genotype of HFMD-associated CV-A4 is persistently and extensively circulating in China.

Sporadic outbreaks caused by coxsackievirus A4 (CV-A4) have been reported worldwide. To further elucidate the detailed genetic characteristics and evolutionary recombination events of CV-A4, virus samples from nationwide hand, foot and mouth disease (HFMD) surveillance, encompassing 27 out of the 31 provinces in China, were investigated. Comprehensive and systematic phylogenetic analyses were performed by using 29 complete genomes, 142 complete CV-A4 VP1 sequences. Four genotypes (A, B, C and D) and five sub-genotypes (C1-C5) were re-identified based on the complete VP1 sequences. C2 is the predominant sub-genotype of CV-A4 associated with HFMD and has evolved into 3 clusters. Cluster 1 is a major cluster that has been persistently and extensively circulating in China since 2006 and has been associated with all severe cases. All the sequences showed high homology with the CV-A4 prototype in the P1 region, while higher identities with CV-A5, CV-14 and CV-16 in the P2 and P3 regions. Recombination analysis revealed that C2 had two specific genetic recombination patterns with other EV-A prototypes in the 5'-UTR and 3D region compared with C5. These recombination patterns might be associated with the increased transmissibility of C2 viruses, which were obtained due to their persistent and extensive circulation in populations.

The Negative Impact of Higher Body Mass Index on Sperm Quality and Erectile Function: A Cross-Sectional Study Among Chinese Males of Infertile Couples.

The objective of the current study was to explore the role of body mass index (BMI) in sperm quality and erectile function in Chinese males of infertile couples. A total of 28 fertility centers in different regions of China were enrolled. Patient data were collected from June 1, 2017, through October 31, 2017. Semen analyses and demographic data were collected and the five-item International Index of Erectile Function (IIEF-5) questionnaire was used to evaluate the erectile function in participants with different BMIs. In total, 3,174 Chinese men of infertile couples with an average age of 33.11 ± 6.08 years were enrolled. The occurrence of obesity, overweight, normal weight, and underweight were 5.4%, 36.6%, 56.8%, and 1.2%, respectively. In addition to hypertension and diabetes, primary infertility, a longer course of infertility, and chronic prostatitis were risk factors for obesity. Compared with men of normal weight, adjusted odds ratios (ORs) for men with obesity, overweight, and underweight for semen volume <2 ml were 2.53 (95% CI [1.61, 3.97]), 1.33 (95% CI [1.09, 1.62]), and 0.84 (95% CI [0.29, 2.43]); for sperm progressive motility (A + B) (%) <32, the ORs were 1.60 (95% CI [1.16, 2.22]), 1.30 (95% CI [1.12, 1.51]), and 1.03 (95% CI [0.54, 1.98]); and for IIEF-5 ≤ 21, the ORs were 1.52 (95% CI [1.10, 2.10]), 1.11 (95% CI [0.96, 1.30]), and 0.62 (95% CI [0.31, 1.26]), respectively. Obesity was associated with lower semen volume, lower sperm motility, and erectile dysfunction in Chinese males of infertile couples.

The emerging sub-genotype C2 of CoxsackievirusA10 Associated with Hand, Foot and Mouth Disease extensively circulating in mainland of China.

Coxsackievirus A10 (CV-A10) associated with Hand, foot, and mouth disease (HFMD) cases emerged increasingly in recent years. In this study, the samples from nation-wide HFMD surveillance, including 27 out of 31 provinces in China were investigated, and the continuous and extensive virological surveillance, covered 13 years, were conducted to provide a comprehensive molecular characterization analysis of CV-A10. 855 CV-A10 viruses (33 severe cases included), were isolated from HFMD children patients during 2009 to 2016 in China. 164 representative sequences from these viruses, together with 117 CV-A10 sequences downloaded from GenBank based on entire VP1 were recruited in this study. Two new genotypes (F and G) and two sub-genotypes (C1 and C2) were identified. Among 264 Chinese sequences, 9 of them were genotype B, 8 of them were C1, and the other (247) were C2, the predominant sub-genotype in China since 2012. Chinese C2 viruses showed obvious temporal characteristics and can be divided into 3 clusters (cluster 1~3). Cluster 3 viruses was circulating extensively during 2014 and 2016 with more severe cases. It is very necessary and important to continuously conduct the extensive virological surveillance for CV-A10, and further evolutionary studies will provide more evidence on its evolution and virulence.