One-base-mismatch CRISPR-based transistors for single nucleotide resolution assay.

An effective strategy for accurately detecting single nucleotide variants (SNVs) is of great significance for genetic research and diagnostics. However, strict amplification conditions, complex experimental instruments, and specialized personnel are required to obtain a satisfactory tradeoff between sensitivity and selectivity for SNV discrimination. In this study, we present a CRISPR-based transistor biosensor for the rapid and highly selective detection of SNVs in viral RNA. By introducing a synthetic mismatch in the crRNA, the CRISPR-Cas13a protein can be engineered to capture the target SNV RNA directly on the surface of the graphene channel. This process induces a fast electrical signal response in the transistor, obviating the need for amplification or reporter molecules. The biosensor exhibits a detection limit for target RNA as low as 5 copies in 100 µL, which is comparable to that of real-time quantitative polymerase chain reaction (PCR). Its operational range spans from 10 to 5 × 105 copy mL-1 in artificial saliva solution. This capability enables the biosensor to discriminate between wild-type and SNV RNA within 15 min. By introducing 10 µL of swab samples during clinical testing, the biosensor provides specific detection of respiratory viruses in 19 oropharyngeal specimens, including influenza A, influenza B, and variants of SARS-CoV-2. This study emphasizes the CRISPR-transistor technique as a highly accurate and sensitive approach for field-deployable nucleic acid screening or diagnostics.

Epidemiological and Genomic analysis of Vibrio parahaemolyticus isolated from imported travelers at the port of Shanghai, China (2017-2019).

BACKGROUND: Vibrio parahaemolyticus is the predominant etiological agent of seafood-associated foodborne illnesses on a global scale. It is essential to elucidate the mechanisms by which this pathogen disseminates. Given the existing research predominantly concentrates on localized outbreaks, there is a pressing necessity for a comprehensive investigation to capture strains of V. parahaemolyticus cross borders. RESULTS: This study examined the frequency and genetic attributes of imported V. parahaemolyticus strains among travelers entering Shanghai Port, China, between 2017 and 2019.Through the collection of 21 strains from diverse countries and regions, Southeast Asia was pinpointed as a significant source for the emergence of V. parahaemolyticus. Phylogenetic analysis revealed clear delineation between strains originating from human and environmental sources, emphasizing that underlying genome data of foodborne pathogens is essential for environmental monitoring, food safety and early diagnosis of diseases. Furthermore, our study identified the presence of virulence genes (tdh and tlh) and approximately 120 antibiotic resistance-related genes in the majority of isolates, highlighting their crucial involvement in the pathogenesis of V. parahaemolyticus. CONCLUSIONS: This research enhanced our comprehension of the worldwide transmission of V. parahaemolyticus and its antimicrobial resistance patterns. The findings have important implications for public health interventions and antimicrobial stewardship strategies, underscoring the necessity for epidemiological surveillance of pathogen at international travel hubs.

Ultra-Fast Single-Nucleotide-Variation Detection Enabled by Argonaute-Mediated Transistor Platform.

"Test-and-go" single-nucleotide variation (SNV) detection within several minutes remains challenging, especially in low-abundance samples, since existing methods face a trade-off between sensitivity and testing speed. Sensitive detection usually relies on complex and time-consuming nucleic acid amplification or sequencing. Here, a graphene field-effect transistor (GFET) platform mediated by Argonaute protein that enables rapid, sensitive, and specific SNV detection is developed. The Argonaute protein provides a nanoscale binding channel to preorganize the DNA probe, accelerating target binding and rapidly recognizing SNVs with single-nucleotide resolution in unamplified tumor-associated microRNA, circulating tumor DNA, virus RNA, and reverse transcribed cDNA when a mismatch occurs in the seed region. An integrated microchip simultaneously detects multiple SNVs in agreement with sequencing results within 5 min, achieving the fastest SNV detection in a "test-and-go" manner without the requirement of nucleic acid extraction, reverse transcription, and amplification.

Application of whole-genome tiling array at Shanghai port, China: An alternative method for SARS-CoV-2 surveillance.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the critical role of genomic surveillance in tracking rapidly spreading viruses and their evolving lineages. The emergence of the SARS-CoV-2 tiling array, a comprehensive tool capable of capturing the entire viral genome, has presented a promising avenue for variants. This study introduces the SARS-CoV-2 tiling array as a novel method for port inspection. Using next-generation sequencing as a benchmark, 35 positive samples underwent sequencing through both methodologies, including the Alpha variant (B.1.1.7), Delta variants (AY.120, AY.122, AY.23.1), and Omicron variants (BA.1, BA.2, BA.2.75, BA.4, BA.5, BE.1, BF.7, BN.1, BQ.1, XBB.1) within the sample set. The whole-genome tiling array demonstrated successful identification of various sublineages of SARS-CoV-2. The average sequencing coverage rates were 99.22% (96.82%-99.92%) for the whole-genome tiling array and 98.56% (92.81%-99.59%) for Illumina sequencing, respectively. The match rates of these two methods ranged from 92.81%-99.59%, with an average rate of 98.56%. Among the benefits of the whole-genome tiling array are its cost-effectiveness and equipment simplification, making it particularly suitable for identifying SARS-CoV-2 variants in the front-line inspection department. The aforementioned findings provide valuable insights into the surveillance of COVID-19 and present a pragmatic solution for improving quarantine measures at entry points.

The efficacy of acupuncture combined with other therapies in post stroke cognitive impairment: A network meta-analysis.

BACKGROUND: The network meta-analysis was used to evaluate the efficacy of acupuncture combined with other therapies in the treatment of post stroke cognitive impairment (PSCI). METHODS: The China National Knowledge Infrastructure, Wanfang DATA, Vip Chinese Periodic Service Platform, PUBMED, Cochrane Library, Web of Science, and Embase were searched for randomized controlled trials (RCTs) published before March 18, 2023. Two researchers independently reviewed articles and extracted data, and then qualified papers were included in the study. STATA 14.0 was used for network meta-analysis. RESULTS: A total of 29 articles including 2241 patients were included in this study. The treatment of the intervention group includes acupuncture combined with traditional Chinese medicine prescriptions (TCMP), acupuncture combined with hyperbaric oxygen (HBO), acupuncture combined with repetitive transcranial magnetic stimulation (rTMS), acupuncture combined with cognitive rehabilitation (CR), acupuncture combined with donepezil. The intervention of the control group includes acupuncture, HBO, rTMS, CR, TCMP, and donepezil. In terms of improving the score of Minimum Mental State Examination (MMSE), acupuncture combined with TCMP was most likely to be the best treatment (P < .05). In terms of improving the score of Montreal Cognitive Assessment (MoCA), acupuncture combined with TCMP was most likely to be the best treatment (P < .05). In terms of improving the total effective rate of clinical treatment, acupuncture combined with rTMS was most likely to be the best treatment (P < .05). CONCLUSION: Acupuncture combined with TCMP may be the best treatment method among all of the above treatments for PSCI.

Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution.

Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

A highly sensitive bead-based flow cytometric competitive binding assay to detect SARS-CoV-2 neutralizing antibody activity.

Accurate detection of SARS-CoV-2 neutralizing antibody (nAb) is critical for assessing the immunity levels after virus infection or vaccination. As fast, cost-effective alternatives to viral infection-based assays, competitive binding (CB) assays were developed to quantitate nAb by monitoring the ability of sera to inhibit the binding of viral spike (S) protein to the angiotensin converting enzyme 2 (ACE2) receptor. Herein, we established a bead-based flow cytometric CB assay and tested the detection performance of six combination models, i.e. immobilized ACE2 and soluble Fc-tagged S1 subunit of S protein (iACE2/S1-Fc), immobilized ACE2 and soluble Fc-tagged receptor binding domain (RBD) of S protein (iACE2/RBD-Fc), immobilized S1 and soluble Fc-tagged ACE2 (iS1/ACE2-Fc), immobilized S1 and soluble His-tagged ACE2 (iS1/ACE2-His), immobilized RBD and soluble Fc-tagged ACE2 (iRBD/ACE2-Fc), and immobilized RBD and soluble His-tagged ACE2 (iRBD/ACE2-His). Using SARS-CoV-2 monoclonal antibodies and sera of convalescent COVID-19 patients and vaccinated subjects, the combination models iACE2/RBD-Fc, iACE2/S1-Fc and iS1/ACE2-His were identified to be able to specifically detect SARS-CoV-2 nAb, among which iACE2/RBD-Fc model showed the highest sensitivity, superior to a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) ELISA kit. Further studies demonstrated that the sensitivity and specificity of CB assays were affected by the tag of ACE2, type of spike and method of measuring binding rate between ACE2 and spike. Moreover, the iACE2/RBD-Fc model showed good performance in detecting kinetic development of nAb against both the prototype SARS-CoV-2 strain and an omicron variant of SARS-CoV-2 in people immunized by an inactivated SARS-CoV-2 vaccine, and the results of iACE2/RBD-Fc model are correlated well with those of live virus-based and pseudovirus-based neutralization tests, demonstrating the potential to be developed into a highly sensitive, specific, versatile and high-throughput method for detecting SARS-CoV-2 nAb in clinical practice.

Internet of medical things-enabled CRISPR diagnostics for rapid detection of SARS-CoV-2 variants of concern.

Previous studies have highlighted CRISPR-based nucleic acid detection as rapid and sensitive diagnostic methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we reported an optimized CRISPR-Cas12a diagnostic platform for the safe and rapid detection of SARS-CoV-2 variants of concern (VOCs). This platform, which was referred to as CALIBURN-v2, could complete the diagnosis on extracted RNA samples within 25 min in a closed-lid reaction mode and had 100-fold increase in detection sensitivity in comparison with previous platforms. Most importantly, by integrating a portable device and smartphone user interface, CALIBURN-v2 allowed for cloud server-based data collection and management, thus transforming the point-of-care testing (POCT) platform to internet of medical things (IoMT) applications. It was found that IoMT-enabled CALIBURN-v2 could achieve 95.56% (172 out of 180) sensitivity for SARS-CoV-2 wild type and 94.38% (84 out of 89) overall sensitivity for SARS-CoV-2 variants including Delta and Omicron strains. Therefore, our study provides a feasible approach for IoMT-enabled CRISPR diagnostics for the detection of SARS-CoV-2 VOCs.

Research on safety and compliance of imported microbial inoculants using high-throughput sequencing.

Microbial inoculants are widely used in wastewater treatment, soil remediation, and biological control. Safety and compliance for active constituents are considered to be the most important measures of imported microbial inoculants. Microbial inoculants composition was commonly identified by phenotypic culture, which is time-consuming and labor intense with occasionally false negative results provided, and can only be tested for specific species. High-throughput sequencing (HTS), known for its non-targeted detection of unknown species composition in samples, is suitable for composition consistency identification and biosafety analysis of imported microbial inoculants. In this study, the application of HTS for microflora distribution and resistance gene was verified in microbial inoculants for environmental protection and then applicated in imported microbial inoculants. Both Illumina- and Nanopore-based HTS methods identified the same dominant bacterial species successfully in the imported microbial inoculants. The main component of bacterial species was Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Enterococcus faecium, and further confirmed with traditional methods. The antibiotic resistance genes Bacillus subtilis mprF, bcrA, blt, lmrB, rphB, tet(L), tmrB, vmlR, ykkC, and ykkD were detected in all samples. Our results indicated that HTS processes the application potential to identify the active ingredients of microbial inoculants. Therefore, rapid and accurate identification of the microbial compositions in microbial formulation products is of high importance for port biosafety supervision.

Integrated System for On-Site Rapid and Safe Screening of COVID-19.

Since the outbreak of coronavirus disease 2019 (COVID-19), the epidemic has been spreading around the world for more than 2 years. Rapid, safe, and on-site detection methods of COVID-19 are in urgent demand for the control of the epidemic. Here, we established an integrated system, which incorporates a machine-learning-based Fourier transform infrared spectroscopy technique for rapid COVID-19 screening and air-plasma-based disinfection modules to prevent potential secondary infections. A partial least-squares discrimination analysis and a convolutional neural network model were built using the collected infrared spectral dataset containing 857 training serum samples. Furthermore, the sensitivity, specificity, and prediction accuracy could all reach over 94% from the results of the field test regarding 968 blind testing samples. Additionally, the disinfection modules achieved an inactivation efficiency of 99.9% for surface and airborne tested bacteria. The proposed system is conducive and promising for point-of-care and on-site COVID-19 screening in the mass population.

Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants.

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.

Increased genetic variation of A(H3N2) virus from influenza surveillance at the end of the 2016/2017 season for Shanghai port, China.

Influenza A(H3N2) virus exhibited complex seasonal patterns to evade pre-existing antibodies, resulting in changes in the antigenicity of the viron surface protein hemagglutinin (HA). To monitor the currently imported influenza viruses as well as to assess the capacity of health emergencies at the Shanghai port, we collected respiratory specimens of passengers from different countries and regions including some of Europe with influenza-like illness at the Shanghai port during 2016/2017, examined amino acid substitutions, and calculated the perfect-match vaccine efficacy using the p epitope model. Phylogenetic analysis of the HA genes revealed that influenza A(H3N2) viruses belonging to eight subclades were detected, and three amino acid substitutions in the subclade 3C.2a.4 were also added. Besides, two epidemic influenza virus strains were found in the 2016/2017 winter and 2016 summer. The results of lower predicted vaccine effectiveness in summer suggest that the imported A(H3N2) strains were not a good match for the A/Hong Kong/4801/2014 vaccine strain since the summer of 2017. Therefore, the Shanghai Port might stop the risk of the international spread of influenza for the first time, and curb the entry of A(H3N2) from overseas at the earliest stage of a probable influenza pandemic.

Genomic phylogenetic analyses of four major hand, foot and mouth disease-related enteroviruses.

Enteroviruses had diverged into many types, some of which cause hand, foot and mouth disease (HFMD) in children. The predominant enterovirus types associated with HFMD are EVA71, CVA16, CVA6 and CVA10. Four enterovirus types were classified into subtypes based on VP1 sequences. However, the phylogenetics of these enteroviruses is rarely concerned at the genomic level. In this study, we performed the phylogenetic analyses of the EVA71, CVA16, CVA6 and CVA10 using available full-length genomic sequences. We found that the topologies of phylogenetic trees of full-length genomic sequences and VP1 sequences were almost consistent, except few subtypes of EVA71 and CVA10. The mean genetic divergence was 15.8-27% between subtypes and less than 12% within subtypes/sub-subtypes at genomic level. Comparison of phylogenetic topologies between genomic and VP1 sequences helped us to identify two new EVA71 inter-subtype recombinants RF01_CC4 and RF02_CC4. Furthermore, EVA71 subtypes C1 and C2 and CVA10 subtype D were found to originate through inter-subtype recombination. The genomic reference sequences of these enteroviruses are provided here for subtyping. The results provide important insights into the understanding of the evolution and epidemiology of the four enteroviruses. Keywords: enterovirus; hand; foot and mouth disease; classification; genetic distance; recombination.

Programmed Exercise Attenuates Familial Hypertrophic Cardiomyopathy in Transgenic E22K Mice via Inhibition of PKC-α/NFAT Pathway.

Familial hypertrophic cardiomyopathy (FHCM), an autosomal dominant disease, is caused by mutations in genes encoding cardiac sarcomeric proteins. E22K, a mutation in the myosin regulatory light chain sarcomere gene, is associated with the development of FHCM. However, the molecular mechanisms by which E22K mutation promotes septal hypertrophy are still elusive. The hypertrophic markers, including beta-myosin heavy chain, atrial natriuretic peptide and B-type natriuretic peptide, were upregulated, as detected by fluorescence quantitative PCR. The gene expression profiles were greatly altered in the left ventricle of E22K mutant mice. Among these genes, nuclear factor of activated T cells (NFAT) and protein kinase C-alpha (PKC-α) were upregulated, and their protein expression levels were also verified to be elevated. The fibrosis markers, such as phosphorylated Smad and transforming growth factor beta receptor, were also elevated in transgenic E22K mice. After receiving 6 weeks of procedural exercise training, the expression levels of PKC-α and NFAT were reversed in E22K mouse hearts. In addition, the expression levels of several fibrosis-related genes such as transforming growth factor beta receptor 1, Smad4, and alpha smooth muscle actin in E22K mouse hearts were also reversed. Genes that associated with cardiac remodeling such as myocyte enhancer factor 2C, extracellular matrix protein 2 and fibroblast growth factor 12 were reduced after exercising. Taken together, our results indicate that exercise can improve hypertrophy and fibrosis-related indices in transgenic E22K mice via PKC-α/NFAT pathway, which provide new insight into the prevention and treatment of familial hypertrophic cardiomyopathy.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single "pot". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.

Complete genome sequence of GII.9 norovirus.

Norovirus is recognized as one of the leading causes of acute gastroenteritis outbreaks. Genotype GII.9 was first detected in Norfolk, VA, USA, in 1997. However, the complete genome sequence of this genotype has not yet been determined. In this study, a complete genome sequence of GII.9[P7] norovirus, SCD1878_GII.9[P7], from a patient was determined using high-throughput sequencing and rapid amplification of cDNA ends (RACE) technology. The complete genome sequence of SCD1878_GII.9[P7] is 7544 nucleotides (nt) in length with a 3' poly(A) tail and contains three open reading frames. Sequence comparisons indicated that SCD1878_GII.9[P7] shares 92.1%-92.3% nucleotide sequence identity with GII.P7 (AB258331 and AB039777) and 96.7%-97.4% identity with GII.9 (AY038599 and DQ379715). The results suggested that SCD1878_GII.9[P7] is a member of P genotype GII.P7 and G genotype GII.9. This viral sequence fills a gap at the whole-genome level for the GII.9 genotype.

Imported human norovirus in travelers, Shanghai port, China 2018: An epidemiological and whole genome sequencing study.

BACKGROUND: Global mobility of the population has accelerated spread of the Human Norovirus (HuNoV), with long-distance travel in enclosed spaces increasing the opportunity for viral outbreaks. However, surveillance of HuNoV transmission is still lacking, especially in cross-border transportation. METHOD: From 533 self-reported patients, 83 swab samples (15.6%) tested positive for HuNoV by RT-qPCR. Positive samples were sequenced using next-generation sequencing (NGS). Epidemiological investigation and whole genome analysis were then conducted. RESULTS: Most cases occurred in February and March, with large outbreaks involving more than 34 people. A total of 74 HuNoV sequences that could be genotyped were obtained, with near-complete genomes (>7 kb) accounting for most sequences (57/74). A total of 19 different genotypes of viral whole genome sequences were included. The first whole genome sequence of GII.9[P7] was obtained. Rarely reported genotypes including GI.3[P10], GI.3[P13], GII.7[P7], GII.8[P8], and GIX.1[GII.P15] were sequenced and assembled successfully. Four possible sources of virus outbreaks in China were traced. Beyond HuNoV, whole genome sequences of food-borne viruses including Salivirus, Kobuvirus, and Enterovirus were obtained in further assembly. CONCLUSIONS: Surveillance of the etiology and epidemiology of HuNoV global spread through travelers will improve pre-travel health advice, empirical treatment, and estimates of vaccine-preventable diseases.

Evolutionary dynamics of group A and B respiratory syncytial virus in China, 2009-2018.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and is a public health threat globally. To investigate the spatiotemporal dynamics of RSV evolution, we performed systematic phylogenetic analysis using all available sequences from the GenBank database, together with sequences from Shanghai, China. Both RSV-A and RSV-B appear to have originated in North America, with an inferred origin time of 1954.0 (1938.7-1967.6) and 1969.7 (1962.6-1975.5), respectively. BA-like strains of RSV-B, with a 60-nt insertion, and the ON1 strain of RSV-A, with a 72-nt insertion, emerged in 1997.6 (1996.2-1998.6) and 2010.1 (2009.1-2010.3), respectively. Since their origin, both genotypes have gradually replaced the former circulating genotypes to become the dominant strain. The population dynamic of RSV-A showed a seasonal epidemic pattern with obvious expansion in the periods of 2006-2007, 2010-2011, 2011-2012, and 2013-2014. Thirty fixed amino acid substitutions were identified during the divergence of NA4 from GA1 genotypes of RSV-A, and 13 were found during the divergence of SAB4 from GB1 of RSV-B. Importantly, ongoing evolution has occurred since the emergence of ON1, including four amino acid substitutions (I208L, E232G, T253K, and P314L). RSV-A genotypes GA5, NA4, NA1, and ON1 and RSV-B genotypes CB1, SAB4, BA-C, BA10, BA7, and BA9 were co-circulating in China from 2005 to 2015. In particular, RSV-A genotype ON1 was first detected in China in 2011, and it completely replaced GA2 to become the predominant strain after 2016. These data provide important insights into the evolution and epidemiology of RSV.

Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis.

Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.