Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism.

Sphingosine-1-phosphate receptor 1 (S1PR1) is a master regulator of lymphocyte egress from the lymph node and an established drug target for multiple sclerosis (MS). Mechanistically, therapeutic S1PR1 modulators activate the receptor yet induce sustained internalization through a potent association with ß-arrestin. However, a structural basis of biased agonism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of Gi-bound S1PR1 in complex with S1P, fingolimod-phosphate (FTY720-P) and siponimod (BAF312). In combination with functional assays and molecular dynamics (MD) studies, we reveal that the ß-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxxY motifs. Specifically, the intermediate flipping of W2696.48 and the retained interaction between F2656.44 and N3077.49 are the key features of the ß-arrestin bias. We further identify ligand-receptor interactions accounting for the S1PR subtype specificity of BAF312. These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.

Generation of porcine circovirus type 4 virus-like particles and their use to detect serum antibodies.

Porcine circovirus type 4 (PCV4), first identified in 2019 as a newly emerging pathogen, has been found in several provinces of China, as well as in Korea and Thailand. Since PCV4 is not included in immunization programs, epidemiological investigations should be conducted for detection of anti-PCV4 antibodies. Virus-like particles (VLPs) are frequently used for serological analysis of pathogen infections. However, there have been no reports on using PCV4 VLPs for serological investigation of PCV4 infection. In this study, we generated self-assembled PCV4 VLPs using an E. coli expression system, purified them using a two-step process, and used them to develop an indirect ELISA. This ELISA method was found to be highly specific, sensitive, and repeatable, making it suitable for PCV4 antibody detection in serum samples. Finally, the ELISA was used to analyze 422 serum samples collected from across several regions in China, 134 of which tested positive. Thus, the PCV4-VLP-based ELISA can effectively detect antibodies against PCV4 in serum samples, making it a useful tool for PCV4 epidemiology.

Synthesis of Novel Acetyl-11-keto-ß-boswellic Acid Derivatives as Potential Anti-GBM Agents.

Acetyl-11-keto-ß-boswellic acid (AKBA) is known to inhibit the growth of glioblastoma (GBM) cells and subcutaneous GBM. A series of acetyl-11-keto-ß-boswellic acid (AKBA) derivatives containing the oxime-ester functionality or amide side chains were synthesized, and their anti-GBM activities were evaluated. Some of these compounds exhibited significant inhibitory activity against cell proliferation in U87 and U251 GBM cell lines, with IC50 values in the micromolar concentration range. Cellular thermal shift analysis showed that A-01 and A-10 improved the thermal stability of FOXM1, indicating that these highly active compounds may directly bind to FOXM1 in cells. Docking studies of the two most active compounds, A-01 and A-10, revealed key interactions between these compounds and the active site of FOXM1, in which the amide moiety at the C-24 position was essential for improving the activity. These results suggested that A-10 is a suitable lead molecule for the development of FOXM1 inhibitors. Thus, the rational design of AKBA derivatives with amide side chains holds significant potential for discovering of a new class of triterpenoids capable of inhibiting GBM cell proliferation.

Organic Semiconductor Nanosheets for Sulfite Detecting Based on Activation of Sulfite and a Synergetic Chemiluminescence Resonance Energy Transfer Process in a Mild System of Fe2+-SO32.

Sulfur dioxide (SO2) as one kind of air pollution not only causes extreme environmental pollution but also negatively affects human health. Chemiluminescence (CL) methods applied for sulfite analysis with high selectivity based on activating sulfite with oxidants are always implemented in acid media with a high background rise. In this work, we proposed to develop a mild CL system of Fe2+-SO32- to detect sulfite under neutral conditions and provide in situ CL spectral data for deeply studying the CL mechanism of Fe2+-SO32-. Herein, we first synthesized one type of water-soluble supramolecular nanosheets, APDI NSs, which had a strong oxidation potential (+2.9 V) due to a π-conjugated system for activation of sulfite to enhance the generation of SO3̇- and other active radicals, and strong a CL signal from the APDI NSs-Fe2+-SO32- system was generated. By studying the CL mechanism under acidic and neutral conditions, a new CL reaction pathway (path-1) and a key intermediate, S2O42-, from the reaction of Fe2+ and SO32- were found. The CL signal was emitted by SO2* after oxidation of S2O42- by strong oxidants like SO4•- and further amplified by APDI NSs through the CL resonance energy transfer (CRET) process. Based on the APDI NSs-Fe2+-SO32- system under neutral conditions, a CL method for detecting SO32- was established. The detection limit was 2.7 × 10-8 M (S/N = 3), and the recovery rates in spiked water samples were in the range of 87%-101%. This study strengthens the understanding of the CL reaction process of the Fe2+-SO32- system and provides a mild sulfite sensing platform for environmental samples.

New Perylene-Based Chemiluminescent Polymer Nanoparticles for Highly Selective Detection of the Superoxide Anion In Vivo.

The superoxide anion (O2•-) is one of the primary reactive oxygen species in biological systems. Developing a determination system for O2•- in vivo has attracted much attention thanks to its complex biological function. Herein, we proposed a new perylene-based chemiluminescence (CL) probe, the SH-PDI polymer, which was capable of generating strong CL signals with O2•- in comparison with other ROS. The CL mechanism involved was proposed to be a kind of oxidation reaction induced by the breakage of the S-S and S-H bonds into sulfoxide bonds by O2•-. Subsequently, a nanoprecipitation method was introduced, using cumene-terminated poly(styrene-co-maleic anhydride) as the amphiphilic agent, to obtain water-soluble nanoparticles, SPPS NPs, which exhibited not only stronger CL intensity but also higher selectivity toward O2•- than the SH-PDI polymer. Moreover, the CL wavelength of the SPPS-O2•- system was found to be located at 580 and 710 nm, which was conducive to CL imaging. By virtue of these advantages, SPPS NPs were utilized to evaluate the O2•- level in vitro in the range of 0.25-60 µM at pH 7.0, with a detection limit of 8.2 × 10-8 M (S/N = 3). Moreover, SPPS NPs were also capable of imaging O2•- in an LPS-induced acute inflammation mice model and drug-induced acute kidney injury (AKI).

Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus-host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Δfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development.

Clinical characteristics of liver injury induced by immune checkpoint inhibitors in patients with advanced biliary tract carcinoma.

Immune-related liver injuries are closely associated with the liver's fundamental state. Patients with advanced biliary tract carcinoma (BTC) have poor liver function. We evaluated the clinical data of immune-related liver injury in patients with advanced BTC and gastric cancer (GC) during immune checkpoint inhibitor (ICI) treatment between February 2019 and July 2022 at Peking University First Hospital. Twenty-five patients with advanced BTC were identified. Fifteen patients (60%) experienced immune-related liver injury during ICI treatment. We also evaluated the clinical status of patients with GC in another group receiving immunotherapy. The results demonstrated that the incidence of immune-related liver injury was higher in patients with BTC than in GC cancer (p=0.040). Multivariate analysis suggested that the type of malignant tumor and baseline liver function status were high-risk factors for grade 2 and higher immune-related liver injuries. Two patients were diagnosed with immune-related cholangitis. Both biliary enzymes can be decreased to a certain degree by corticosteroid and ursodeoxycholic acid (UDCA) therapy but are difficult to reduce to normal levels. Liver function normalized, and symptoms improved after local treatment for cholestasis (stent implantation or PTBD). We observed a higher incidence of immune-related liver injury after ICI treatment in patients with advanced BTC. Effect of baseline liver function on the incidence of liver injury associated with immunotherapy. Interventional therapy provides rapid relief from cholestasis and is an indispensable and effective approach to the treatment of immune-related cholangitis.

The presence of human respiratory syncytial virus in the cerebrospinal fluid of a child with Anti-N-methyl-D-aspartate receptor encephalitis of unknown trigger.

BACKGROUND: Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is an important type of brain inflammation caused by autoantibody. As one of the primary agents responsible for respiratory tract infection, the human respiratory syncytial virus (hRSV) has also been reported to be capable of causing extrapulmonary diseases. Here, we first describe a case of anti-NMDAR encephalitis when hRSV was shown to be present in the cerebrospinal fluid. CASE PRESENTATION: The child was noted to have ataxia and positive anti-NMDA receptors in the cerebrospinal fluid, diagnosed as anti-NMDA receptor encephalitis in combination with cranial MRI images. After high-dose hormone pulse therapy and medication, the disease improved, and he was discharged. However, a relapse occurred almost a year later, and the cranial MRI imaging showed progressive cerebellar atrophy. An hRSV strain from group B was detected in his cerebrospinal fluid, and the whole genome sequence was recovered using transcriptome sequencing. CONCLUSIONS: To our knowledge, this is the first report of hRSV being found in the cerebrospinal fluid of a patient with anti-NMDAR encephalitis. Even though more clinical records and experimental evidence are needed for validation, this work expands the types of diseases linked to hRSV and the likely cause of anti-NMDAR encephalitis.

Chemiluminescence-assisted study on a peracetic acid-based advanced oxidation process activated with cobalt phosphide.

In recent years, the elimination of organic pollutants using advanced oxidation processes (AOPs) based on peracetic acid (PAA) has drawn increasing attention due to the high oxidative potential and low byproducts. However, to explore more efficient and stable PAA-based AOPs, there is still great room for study on the activation of PAA and degradation mechanism in the reaction process. In this study, a new PAA-based AOP activated by metal-organic framework-derived cobalt phosphide (CoP) and accompanied by chemiluminescence (CL) behaviour was explored. The CoP/PAA system could efficiently degrade 99.98% of RhB (20 mg L-1 ) within 5 min at pH 7 compared with the conventional Co3 O4 /PAA system (merely 17.29%), and the degradation process was matched well with the pseudo-first-order kinetic, and the kinetic constants was ~23.7 times higher than that of Co3 O4 (0.546 min-1 for CoP vs. 0.023 min-1 for Co3 O4 ). In the CoP/PAA/RhB process, the CL intensity was related to the concentration of 1 O2 , O2 •- and acetyl peroxyl radicals [CH3 C(O)OO• and CH3 C(O)O•]. Therefore, CL analysis, combined with quenching tests and electron paramagnetic resonance analysis, was used to study the degradation mechanism in detail, and 1 O2 was confirmed as the dominant contributor for the dye degradation.

Biochar nanosphere-functionalized carbon fibers for in-tube solid-phase microextraction of polycyclic aromatic hydrocarbons in environmental water followed by liquid chromatography and diode array detection.

In order to improve the extraction ability of carbon fibers (CFs) for microextraction of polycyclic aromatic hydrocarbons (PAHs), biochar nanospheres derived from glucose were in-situ grown onto the surface of CFs via hydrothermal synthesis. The surface morphology and elemental composition of biochar nanospheres-CFs were investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. Thereafter, the biochar nanosphere-CFs were pulled into the polyetheretherketone tube for solid-phase microextraction, and the tube was combined with high-performance liquid chromatography-diode array detector to online detect PAHs. With the help of π-stacking, hydrophobic, and hydrophilic effect of biochar nanospheres, the extraction efficiency of CFs was greatly enhanced (enrichment factor increased by 293% compared with  the original). The conditions affecting the analytical performance (sampling volume, sampling rate, methanol content, and desorption time) were investigated. Under the optimal conditions, an online analytical method for microextraction and determination of several PAHs was developed, and satisfactory results were achieved. The limits of detection were 0.003-0.010 ng mL-1 owing to high enrichment effect (2973-3600), linearity ranged from  0.010-15.0 ng mL-1, and relative standard deviations were 0.4%-1.6% (intra-day) and 2.4%-4.4% (inter-day), respectively. The method was applied to analyze environmental water samples (rain water, snow water, and river water), and spiked recoveries within 80.0%-119% were obtained.

Design, synthesis and insecticidal activities of 4-propargyloxybenzene sulfonamide derivatives substituted with amino acids.

Sixty-nine 4-propargyloxybenzene sulfonamide derivatives with different amino acids as amino substituent were synthesized and evaluated for their insecticidal activity against third-instar Mythimna separate. The bioassay results revealed that some derivatives bearing amino acid ester group performed good insecticidal activity against third-instar M.separata, such as the LC50 values of D18 and D19 were 4.28 and 2.96 mg/ml after 48 h, in particular, the LC50 of D16 was 2.38 mg/ml and the activity was improved by 14 times compared to celangulin V (34.48 mg/ml). The above results provided theoretical and experimental basis for the discovery of novel insecticidal active compounds.

Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex.

Flower-like Gold Nanoparticles for In Situ Tailoring Luminescent Molecules for Synergistic Enhanced Chemiluminescence.

In recent years, gold nanoparticles (AuNPs) have attracted much attention due to their ease of surface modification, excellent biocompatibility, and extraordinary optoelectronic and catalytic activities. Herein, based on a AuNP-catalyzed reaction, a strategy for tailoring luminescent molecules in situ is proposed to trigger an ultrastrong chemiluminescence (CL). In the strategy, flower-like AuNPs are prepared using CL molecular probes (Probe-OH for NaClO/ONOO-) via one-pot synthesis and subsequently act as a tailor for Probe-OH to generate novel CL molecules, allowing a synergistic CL enhancement about 4 times that of initial Probe-OH. Furthermore, by modification with poly(vinylpyrrolidone) (PVP) on the surface, the CL signals (only for NaClO) are amplified by 100 times based on an intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism. Given the improved sensitivity and selectivity over Probe-OH, the thus-formed CIEEL nanoplatform (PVP-Au) is successfully developed for detecting NaClO in a wide range of 2.5-100 µM, and the detection limit is 10.68 nM. This work provides unprecedented perspectives for expanding this facile and effective strategy for CL amplification based on AuNP catalysis.

Efficient Photoinduced Thermocatalytic Chemiluminescence System Based on the Z-Scheme Heterojunction Ag3PO4/Ag/Bi4Ti3O12 for H2S Sensing.

Cataluminescence as a highly efficient gas transduction principle has attracted wide attention among research in environmental monitoring and clinical diagnosis with increasing awareness of human safety. Nowadays, the development of innovation sensing systems and the construction of the sensing mechanism to improve the analytical performance of compounds remain a major challenge. Herein, we construct an advanced photoinduced thermocatalytic chemiluminescence (PI-TC-CL) gas-sensing system via the introduction of a Z-scheme heterojunction Ag3PO4/Ag/Bi4Ti3O12 to achieve higher efficient detection of H2S. The unique electron transport path of the Z-scheme heterojunction and the LSPR effect of Ag nanoparticles fascinate the generation of the photoinduced electron-hole pair on the surface of catalysts when stimulated by LED lamps and slow down the recombination of electron-hole pairs under thermal conditions. Thus, based on the cooperative effect of the Z-scheme heterojunction AgPO/Ag/BTO and PI-TC-CL system, we have successfully established an efficient H2S CTL detection system, which has a response three times higher than that on the traditional CTL system and even 45 times higher than that on BTO and ranges among the best of the state-of-the-art CTL performance in H2S detection with the linear range of 0.095-8.87 µg mL-1 and a limit of detection of 0.0065 µg mL-1. Besides, to explore the gas-sensing mechanism, the synergetic effects of photoinduction and thermal catalysis are investigated thoroughly via conductivity and electrochemical experiments. This research provides a new perspective of engineering highly efficient catalysts and ingenious sensor systems through designing the nanostructure of materials and synergism catalytic mechanism.

Cataluminescence System Coupled with Vacuum Desorption-Sampling Methodology for Real-Time Ozone Sensing during the Self-Decomposition Process on Functional Boron Nitride.

The treatment and detection of ozone have been widely studied in recent decades with respect to toxicity and contamination, while the measurement method of ozone is relatively toneless. Fortunately, a new concept of the cataluminescence (CTL) sensor provides a scheme of real-time ozone sensing in a tiny system. Here, a novel CTL sensor system was specially developed with silica-hydroxyl functional boron nitride as the sensing material for rapid and sensitive ozone detection. Coupled with the construction of a pulse vacuum static sampling system, ozone on the surface of sensing material can be desorbed rapidly and can step into the next detection circulation in a few seconds. Based on the strong emission initiated by the transient of reactive oxygen species (ROS) including singlet oxygen, a trioxide group, and an oxygen radical, the detection limit of ozone could be optimized to be as low as 51.2 ppb. Besides, the sensor system exhibited remarkable anti-interference performance in which humidity changes and common VOCs do not disturb or weakly disturb ozone sensing, and the CTL mechanism of the multistep degradation process was further discussed on the basis of multiple pieces of experimental evidence and a DFT transient calculation. A real-time degradation-sensing module was further attached to the system to realize the functions of ozone decomposition and real-time monitoring.

Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway.

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the roles of porcine APN (pAPN) during PDCoV infection of nonsusceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter nonsusceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly colocalized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with the potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters nonsusceptible cells but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate antiviral drugs.

Transient Chemiluminescence Assay for Real-Time Monitoring of the Processes of SO32--Based Advanced Oxidation Reactions.

The hydroxyl radical (·OH) is a strong oxidizing agent in situ generated in advanced oxidation processes (AOPs) and crucial for assessing the performances of AOPs toward organic contaminants' degradation. Herein, we developed a specific luminescent probe, APDI (N' N'-di(propylethylenediamine)-perylene-3,4,9,10-tetracarboxylic diimide), to selectively detect ·OH among diverse reactive oxygen species and other radicals. Based on the transient chemiluminescence (TCL) spectra, the in situ concentration profile of ·OH within 0.01 s interval time in classical Fenton reactions and four kinds of SO32--based AOPs was obtained, which provides insights into the high dynamic processes of the whole ·OH generation and consumption processes. Besides, compared with acidic conditions, reduced degradation efficiencies in Fe2+-SO32- and Fe2+-SO32--H2O2 systems were found under neutral conditions. The complete depletion of active free radicals due to SO2-̇ radicals generated from Fe2+ and SO32- should account most for decreased degradation efficiencies evidenced by a new SO2* TCL signal discovered in the TCL spectra. In addition, similar phenomena have also been found in other M(n-1)+-SO32--related AOPs. As SO32- and HSO3- often exist naturally in wastewater, more efforts are needed to improve the performance of Fe2+-H2O2 systems. This discovery has important significance for organic contaminant degradation in a natural environment.

Anti-inflammatory protein TNFα-stimulated gene-6 (TSG-6) reduces inflammatory response after brain injury in mice.

BACKGROUND: Current research suggests that the glial scar surrounding penetrating brain injuries is instrumental in preserving the surrounding uninjured tissue by limiting the inflammatory response to the injury site. We recently showed that tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), a well-established anti-inflammatory molecule, is present within the glial scar. In the present study we investigated the role of TSG-6 within the glial scar using TSG-6 null and littermate control mice subjected to penetrating brain injuries. RESULTS: Our findings show that mice lacking TSG-6 present a more severe inflammatory response after injury, which was correlated with an enlarged area of astrogliosis beyond the injury site. CONCLUSION: Our data provides evidence that TSG-6 has an anti-inflammatory role within the glial scar.

A potential new recombinant echovirus 18 strain detected in a 4-year-old child with encephalitis in China in 2019.

The species Enterovirus B includes diverse serotypes that can cause a wide spectrum of human diseases, such as aseptic encephalitis, myocarditis, and paralysis. In this study, a 4-year-old child was diagnosed with viral encephalitis, but the causative agent could not be identified using routine immunological tests. Using metagenomic RNA sequencing, a novel strain of enterovirus B, strain PC06, was identified, and its genome sequence was determined by RT-PCR and Sanger sequencing. The viral genome sequence was most similar to that of echovirus E18 strain E18-HeB15-54498/HeB/CHN/2015 (GenBank accession MG720261), with 87.73% nucleotide sequence identity, while the viral proteins shared 96.98% amino acid sequence identity with those of E18 strain Jena/AN1365/10 (GenBank accession no. KX139452). Phylogenetic analysis based on the VP1 and 3D genes revealed discrepant placement of PC06 in the two trees. In the 3D tree, PC06 formed a separate branch together with other recombinant E18 strains. Further recombination tests revealed that PC06 had possibly undergone recombination at a site between the structural and non-structural regions during its evolutionary history. Based on the analysis of VP1 phylogeny and using online genotyping tools, this potential recombinant is tentatively considered a strain of echovirus 18. This information might contribute to the diagnosis and prevention of related diseases.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.