RABV induces biphasic actin cytoskeletal rearrangement through Rac1 activity modulation.

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

Combining binding pocket mutagenesis and substrate tunnel engineering to improve an (R)-selective transaminase for the efficient synthesis of (R)-3-aminobutanol.

(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi, virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency (kcat/Km) than the wild-type enzyme. Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of (R)-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of (R)-3-aminobutanol.

Identification and evolution of non-traditional nitrilase from Spirosoma linguale DSM 74 with high hydration activity.

Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.

Engineering an (R)-selective transaminase for asymmetric synthesis of (R)-3-aminobutanol.

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (kcat/KM) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the Tm value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.

Molecular docking and molecular dynamics simulation of wheat gluten-derived antioxidant peptides acting through the Keap1-Nrf2 pathway.

BACKGROUND: In our previous study, we successfully identified five peptides from wheat gluten: Ala-Pro-Ser-Tyr (APSY), Leu-Tyr (LY), Pro-Tyr (PY), Arg-Gly-Gly-Tyr (RGGY) and Tyr-Gln (YQ). Molecular docking and molecular dynamics simulation methods were employed to investigate the interaction between these antioxidant peptides and the Kelch-like ECH-associated protein 1 (Keap1 protein), revealing the molecular mechanism of their non-competitive binding. In addition, the total antioxidant capacity of the five peptides was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. RESULTS: The affinities of APSY, LY, PY, RGGY and YQ were -8.9, -8.3, -8.5, -9.1 and - 7.9 kcal mol-1, respectively. The five peptides effectively bound to Keap1 protein through hydrogen, π-σ, π-alkyl and alkyl interactions. Significant roles were observed for the P1 pocket residue ARG-415 and the P3 pocket residue ALA-556 in the interactions of the Keap1-peptide complexes. Molecular dynamics simulations further elucidated the dynamic process of peptide binding to the Keap1 protein. All five peptides formed stable complexes with Keap1 protein, with van der Waals forces playing crucial roles in these complex systems, indicative of the peptides' strong binding ability to Keap1 protein. The van der Waals forces were -178.74, -123.11, -134.36, -132.59, and -121.44 kJ mol-1 for the Keap1-APSY, Keap1-LY, Keap1-PY, Keap1-RGGY and Keap1-YQ complexes, respectively. These peptides exhibited excellent antioxidant effects. Among them, the YQ peptide exhibited the highest total antioxidant capacity, with an activity value of 1.18 ± 0.06 mmol Trolox equivalent (TE) L-1 at a concentration of 0.10 mg mL-1. The RGGY, PY, LY and APSY peptides followed in descending order, with activity values of 0.91 ± 0.05, 0.72 ± 0.06, 0.62 ± 0.04 and 0.60 ± 0.05 mmol TE L-1, respectively. CONCLUSION: These results unveiled the molecular mechanism by which the five antioxidant peptides act on active pockets through the Keap1-Nrf2 signaling pathway, providing a theoretical basis for the development of antioxidants. © 2024 Society of Chemical Industry.

Species of associated bryophytes and their effects on the yield and quality of Dendrobium nobile.

BACKGROUND: Dendrobium nobile has unique growth environment requirements, and unstable yields and high management costs are the key factors restricting the development of its imitation wild cultivation industry. The present study explored the effects of different associated bryophyte species on the yield and quality of D. nobile to clarify the dominant bryophyte species associated with D. nobile and to provide a scientific basis for the rational cultivation and quality evaluation of D. nobile. RESULTS: The growth of D. nobile was closely related to the microenvironment of the Danxia stone, and the different associated bryophytes had different effects on D. nobile growth. There was a rich variety of bryophytes associated with D. nobile, with a total of 15 families, 24 genera and 31 species of bryophytes identified in the study area, including 13 families, 22 genera and 29 species of mosses and 2 families, 2 genera and 2 species of liverworts, and mosses predominated in the association with D. nobile. Usually, 3-9 species of bryophytes were growing in association with D. nobile, among which associations of 5-6 bryophytes species were more common, and the bryophytes associated with D. nobile were only related to the species to which they belonged. The dry matter accumulation, quality and mineral content of D. nobile differed significantly among different bryophyte species. The coefficients of variation of dry matter accumulation, dendrobine content and content of 11 mineral elements of D. nobile in the 35 sample quadrats were 25.00%, 21.08%, and 11.33-57.96%, respectively. The biomass, dendrobine content and mineral content of D. nobile were analysed by principal component analysis (PCA) and membership function. The results showed that each evaluation method initially screened Trachycystis microphylla and Leucobryum juniperoideum as the dominant associated bryophytes in the preliminary identification analysis, and the frequency of occurrence and coverage of the two bryophytes were significantly higher than those of the remaining bryophytes. It was determined that T. microphylla and L. juniperoideum were the dominant associated bryophytes. CONCLUSIONS: There is a rich variety of bryophytes associated with D. nobile. The yield and quality of D. nobile differed significantly among different bryophyte species. T. microphylla and L. juniperoideum were the dominant associated bryophytes, and were the two bryophytes associated with D. nobile through mixed growth.

Molecular Engineering of AIE Photosensitizers for Inactivation of Rabies Virus.

Rabies is a zoonotic neurological disease caused by the rabies virus (RABV) that is fatal to humans and animals. While several post-infection treatment have been suggested, developing more efficient and innovative antiviral methods are necessary due to the limitations of current therapeutic approaches. To address this challenge, a strategy combining photodynamic therapy and immunotherapy, using a photosensitizer (TPA-Py-PhMe) with high type I and type II reactive oxygen species (ROS) generation ability is proposed. This approach can inactivate the RABV by killing the virus directly and activating the immune response. At the cellular level, TPA-Py-PhMe can reduce the virus titer under preinfection prophylaxis and postinfection treatment, with its antiviral effect mainly dependent on ROS and pro-inflammatory factors. Intriguingly, when mice are injected with TPA-Py-PhMe and exposed to white light irradiation at three days post-infection, the onset of disease is delayed, and survival rates improved to some extent. Overall, this study shows that photodynamic therapy and immunotherapy open new avenues for future antiviral research.

Enhancing the Activity of an Alcohol Dehydrogenase by Using "Aromatic Residue Scanning" at Potential Plasticity Sites.

An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named "aromatic residue scanning" was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as "exploratory residues" to introduce steric hindrance or potential π-π interactions. The best variant Lk-S96Y/L199W identified with 17.2-fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2-chloro-1-(3,4-difluorophenyl)ethenone in 12 h with >99.5 % ee, giving the highest space-time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

Whole-Transcriptome Sequencing Reveals a ceRNA Regulatory Network Associated with the Process of Periodic Albinism under Low Temperature in Baiye No. 1 (Camellia sinensis).

The young shoots of the tea plant Baiye No. 1 display an albino phenotype in the early spring under low environmental temperatures, and the leaves re-green like those of common tea cultivars during the warm season. Periodic albinism is precisely regulated by a complex gene network that leads to metabolic differences and enhances the nutritional value of tea leaves. Here, we identified messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) to construct competing endogenous RNA (ceRNA) regulatory networks. We performed whole-transcriptome sequencing of 12 samples from four periods (Bud, leaves not expanded; Alb, albino leaves; Med, re-greening leaves; and Gre, green leaves) and identified a total of 6325 differentially expressed mRNAs (DEmRNAs), 667 differentially expressed miRNAs (DEmiRNAs), 1702 differentially expressed lncRNAs (DElncRNAs), and 122 differentially expressed circRNAs (DEcircRNAs). Furthermore, we constructed ceRNA networks on the basis of co-differential expression analyses which comprised 112, 35, 38, and 15 DEmRNAs, DEmiRNAs, DElncRNAs, and DEcircRNAs, respectively. Based on the regulatory networks, we identified important genes and their interactions with lncRNAs, circRNAs, and miRNAs during periodic albinism, including the ceRNA regulatory network centered on miR5021x, the GAMYB-miR159-lncRNA regulatory network, and the NAC035-miR319x-circRNA regulatory network. These regulatory networks might be involved in the response to cold stress, photosynthesis, chlorophyll synthesis, amino acid synthesis, and flavonoid accumulation. Our findings provide novel insights into ceRNA regulatory mechanisms involved in Baiye No. 1 during periodic albinism and will aid future studies of the molecular mechanisms underlying albinism mutants.

[Discrimination of cultivation modes of Dendrobium nobile based on content of mineral elements and ratios of nitrogen stable isotopes].

This study explored the feasibility of mineral element content and ratios of nitrogen isotopes to discriminate the cultivation mode of Dendrobium nobile in order to provide theoretical support for the discrimination of the cultivation mode of D. nobile. The content of 11 mineral elements(N, K, Ca, P, Mg, Na, Fe, Cu, Zn, Mn, and B) and nitrogen isotope ratios in D. nobile and its substrate samples in three cultivation methods(greenhouse cultivation, tree-attached cultivation, and stone-attached cultivation) were determined. According to the analysis of variance, principal component analysis, and stepwise discriminant analysis, the samples of different cultivation types were classified. The results showed that the nitrogen isotope ratios and the content of elements except for Zn were significantly different among different cultivation types of D. nobile(P<0.05). The results of correlation analysis showed that the nitrogen isotope ratios, mineral element content, and effective component content in D. nobile were correlated with the nitrogen isotope ratio and mineral element content in the corresponding substrate samples to varying degrees. Principal component analysis can preliminarily classify the samples of D. nobile, but some samples overlapped. Through stepwise discriminant analysis, six indicators, including δ~(15)N, K, Cu, P, Na, and Ca, were screened out, which could be used to establish the discriminant model of D. nobile cultivation methods, and the overall correct discrimination rates after back-substitution test, cross-check, and external validation were all 100%. Therefore, nitrogen isotope ratios and mineral element fingerprints combined with multivariate statistical analysis could effectively discriminate the cultivation types of D. nobile. The results of this study provide a new method for the identification of the cultivation type and production area of D. nobile and an experimental basis for the quality evaluation and quality control of D. nobile.

De novo assembly and analysis of Polygonatum cyrtonema Hua and identification of genes involved in polysaccharide and saponin biosynthesis.

BACKGROUND: The investigation of molecular mechanisms involved in polysaccharides and saponin metabolism is critical for genetic engineering of Polygonatum cyrtonema Hua to raise major active ingredient content. Up to now, the transcript sequences are available for different tissues of P. cyrtonema, a wide range scanning about temporal transcript at different ages' rhizomes was still absent in P. cyrtonema. RESULTS: Transcriptome sequencing for rhizomes at different ages was performed. Sixty-two thousand six hundred thirty-five unigenes were generated by assembling transcripts from all samples. A total of 89 unigenes encoding key enzymes involved in polysaccharide biosynthesis and 56 unigenes encoding key enzymes involved in saponin biosynthesis. The content of total polysaccharide and total saponin was positively correlated with the expression patterns of mannose-6-phosphate isomerase (MPI), GDP-L-fucose synthase (TSTA3), UDP-apiose/xylose synthase (AXS), UDP-glucose 6-dehydrogenase (UGDH), Hydroxymethylglutaryl CoA synthase (HMGS), Mevalonate kinase (MVK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (ispG), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH), Farnesyl diphosphate synthase (FPPS). Finally, a number of key genes were selected and quantitative real-time PCR were performed to validate the transcriptome analysis results. CONCLUSIONS: These results create the link between polysaccharides and saponin biosynthesis and gene expression, provide insight for underlying key active substances, and reveal novel candidate genes including TFs that are worth further exploration for their functions and values.

Screening and characterization of a nitrilase with significant nitrile hydratase activity.

PURPOSE: We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. METHODS: A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases. RESULTS: Based on the previously reported "amide-formation hotspots," we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. CONCLUSIONS: NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.

Mechanism-Guided Computational Design of ω-Transaminase by Reprograming of High-Energy-Barrier Steps.

ω-Transaminases (ω-TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism-guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps. The common features among the three high-energy-barrier steps responsible for the low catalytic efficiency were revealed using quantum mechanics (QM). Five key residues were simultaneously tailored to stabilize the rate-limiting transition state with the aid of the Rosetta design. The 14 top-ranked variants showed 16.9-143-fold improved catalytic activity. The catalytic efficiency of the best variant, M9 (Q25F/M60W/W64F/I266A), was significantly increased, with a 1660-fold increase in kcat /Km and a 1.5-26.8-fold increase in turnover number (TON) towards various indanone derivatives.

Mass Measurements of Neutron-Deficient Yb Isotopes and Nuclear Structure at the Extreme Proton-Rich Side of the N=82 Shell.

High-accuracy mass measurements of neutron-deficient Yb isotopes have been performed at TRIUMF using TITAN's multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS). For the first time, an MR-TOF-MS was used on line simultaneously as an isobar separator and as a mass spectrometer, extending the measurements to two isotopes further away from stability than otherwise possible. The ground state masses of ^{150,153}Yb and the excitation energy of ^{151}Yb^{m} were measured for the first time. As a result, the persistence of the N=82 shell with almost unmodified shell gap energies is established up to the proton drip line. Furthermore, the puzzling systematics of the h_{11/2}-excited isomeric states of the N=81 isotones are unraveled using state-of-the-art mean field calculations.

Switching the secondary and natural activity of Nitrilase from Acidovorax facilis 72 W for the efficient production of 2-picolinamide.

OBJECTIVES: Catalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases. RESULTS: In this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications. CONCLUSIONS: Nit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.

Equine-Origin Immunoglobulin Fragments Protect Nonhuman Primates from Ebola Virus Disease.

Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab')2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West African and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab')2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab')2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab')2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics.IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab')2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

Complete genome sequence analysis of canine bocavirus 1 identified for the first time in domestic cats.

In this study, we investigated the presence of canine bocaviruses (CBoVs) in fecal samples from 105 cats with diarrhea and 92 asymptomatic cats in northeast China. One fecal sample, 17CC0312, collected from an asymptomatic cat, was found to be positive for canine bocavirus 1 (CBoV1). The nearly complete genome of this virus was cloned and sequenced. The viral genome was 5,069 nucleotides (nt) in length and combined four open reading frames (ORFs) in the order 5'-NS1-ORF4-NP1-VP1/VP2-3'. The 17CC0312 virus shared more than 90.3% nucleotide sequence identity with CBoV1 reference sequences and was placed within the CBoV1 group in a phylogenetic tree based on complete genome sequences. Further phylogenetic analysis based on the deduced amino acid sequence of the VP2 gene showed that this feline CBoV1 strain belongs to CBoV1 lineage 3. These data provide the first molecular evidence of the presence of CBoV1 in a domestic cat and suggest that cats might be carriers of CBoV1.

Development of a reverse genetics system for a feline panleukopenia virus.

Feline panleukopenia virus (FPV) infects cats and can be fatal to kittens. There is evidence that canine parvovirus originated from FPV, which makes FPV important in studies of the family Parvoviridae. In the present study, the entire genome of FPV strain HH-1/86 was converted into a full-length infectious clone (pFPV). The FPV strain HH-1/86 has a 5123-nt single stranded DNA genome with a Y-shaped inverted 3' terminal repeat (ITR) and a U-shaped inverted 5' ITR. Feline kidney cells (F81) were transfected with the pFPV clone which contained a genetic marker, and a rescued virus was obtained (rFPV). The rFPV was identified by its cytopathic effects, indirect immunofluorescence, growth curve analysis, western blot assay and hemagglutination, and was indistinguishable from the parent virus. The FPV infectious clone will facilitate the study of pathogenicity and viral replication of FPV and the inter-species transmission of parvoviruses.

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2.

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein.

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response.