Comprehensive Analysis of Protein Acetylation and Glucose Metabolism in Mouse Brains Infected with Rabies Virus.

Rabies, caused by rabies virus (RABV), is a widespread zoonosis that is nearly 100% fatal. Alteration of the metabolic environment affects viral replication and the immune response during viral infection. In this study, glucose uptake was increased in mouse brains at the late stage of infection with different RABV strains (lab-attenuated CVS strain and wild-type DRV strain). To illustrate the mechanism underlying glucose metabolism alteration, comprehensive analysis of lysine acetylation and target analysis of energy metabolites in mouse brains infected with CVS and DRV strains were performed. A total of 156 acetylated sites and 115 acetylated proteins were identified as significantly different during RABV infection. Compared to CVS- and mock-infected mice, the lysine acetylation levels of glycolysis and tricarboxylic acid (TCA) cycle enzymes were decreased, and enzyme activity was upregulated in DRV-infected mouse brains. Metabolomic analysis revealed high levels of oxaloacetate (OAA) in RABV-infected mouse brains. Specifically, the OAA level in CVS-infected mouse brains was higher than that in DRV-infected mouse brains, which contributed to the enhancement of the metabolic rate at the substrate level. Finally, we confirmed that OAA could reduce excessive neuroinflammation in CVS-infected mouse brains by inhibiting JNK and P38 phosphorylation. Taken together, this study provides fresh insight into the different strategies the host adapts to regulate glucose metabolism for energy requirements after different RABV strain infections and suggests that OAA treatment is a strategy to prevent neural damage during RABV infection. IMPORTANCE Both viral replication and the host immune response are highly energy dependent. It is important to understand how the rabies virus affects energy metabolism in the brain. Glucose is the direct energy source for cell metabolism. Previous studies have revealed that there is some association between acetylation and metabolic processes. In this study, comprehensive protein acetylation and glucose metabolism analysis were conducted to compare glucose metabolism in mouse brains infected with different RABV strains. Our study demonstrates that the regulation of enzyme activity by acetylation and OAA accumulation at the substrate level are two strategies for the host to respond to energy requirements after RABV infection. Our study also indicates the role OAA could play in neuronal protection by suppressing excessive neuroinflammation.

Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8.

Infection with laboratory-attenuated rabies virus (RABV), but not wild-type (wt) RABV, can enhance the permeability of the blood-brain barrier (BBB), which is considered a key determinant for RABV pathogenicity. A previous study showed that the enhancement of BBB permeability is directly due not to RABV infection but to virus-induced inflammatory molecules. In this study, the effect of the matrix metallopeptidase (MMP) family on the permeability of the BBB during RABV infection was evaluated. We found that the expression level of MMP8 was upregulated in mice infected with lab-attenuated RABV but not with wt RABV. Lab-attenuated RABV rather than wt RABV activates inflammatory signaling pathways mediated by the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Activated NF-κB (p65) and AP-1 (c-Fos) bind to the MMP8 promoter, resulting in upregulation of its transcription. Analysis of mouse brains infected with the recombinant RABV expressing MMP8 indicated that MMP8 enhanced BBB permeability, leading to infiltration of inflammatory cells into the central nervous system (CNS). In brain-derived endothelial cells, treatment with MMP8 recombinant protein caused the degradation of tight junction (TJ) proteins, and the application of an MMP8 inhibitor inhibited the degradation of TJ proteins after RABV infection. Furthermore, an in vivo experiment using an MMP8 inhibitor during RABV infection demonstrated that BBB opening was diminished. In summary, our data suggest that the infection of lab-attenuated RABV enhances the BBB opening by upregulating MMP8. IMPORTANCE The ability to change BBB permeability was associated with the pathogenicity of RABV. BBB permeability was enhanced by infection with lab-attenuated RABV instead of wt RABV, allowing immune cells to infiltrate into the CNS. We found that MMP8 plays an important role in enhancing BBB permeability by degradation of TJ proteins during RABV infection. Using an MMP8 selective inhibitor restores the reduction of TJ proteins. We reveal that MMP8 is upregulated via the MAPK and NF-κB inflammatory pathways, activated by lab-attenuated RABV infection but not wt RABV. Our findings suggest that MMP8 has a critical role in modulating the opening of the BBB during RABV infection, which provides fresh insight into developing effective therapeutics for rabies and infection with other neurotropic viruses.

Murine Ifit3 restricts the replication of Rabies virus both in vitro and in vivo.

Rabies virus (RABV) infection can initiate the host immune defence response and induce an antiviral state characterized by the expression of interferon (IFN)-stimulated genes (ISGs), among which the family of genes of IFN-induced protein with tetratricopeptide repeats (Ifits) are prominent representatives. Herein, we demonstrated that the mRNA and protein levels of Ifit1, Ifit2 and Ifit3 were highly increased in cultured cells and mouse brains after RABV infection. Recombinant RABV expressing Ifit3, designated rRABV-Ifit3, displayed a lower pathogenicity than the parent RABV in C57BL/6 mice after intramuscular administration, and Ifit3-deficient mice exhibited higher susceptibility to RABV infection and higher mortality during RABV infection. Moreover, compared with their individual expressions, co-expression of Ifit2 and Ifit3 could more effectively inhibit RABV replication in vitro. These results indicate that murine Ifit3 plays an essential role in restricting the replication and reducing the pathogenicity of RABV. Ifit3 acts synergistically with Ifit2 to inhibit RABV replication, providing further insight into the function and complexity of the Ifit family.

Cytotoxicity of Halogenated Tyrosyl Compounds, an Emerging Class of Disinfection Byproducts.

Halogenated amino acids and peptides are an emerging class of disinfection byproducts (DBPs), having been detected in drinking water and in washed food products. However, the toxicological significance of these emerging DBPs remains unclear. In this study, the cytotoxicity of eight halogenated tyrosyl compounds was investigated in Chinese hamster ovary (CHO) cells using real-time cell analysis (RTCA). Dihalogenated tyrosyl compounds are more cytotoxic than their monohalogenated analogues. The cytotoxicity of the dihalogenated compounds is associated with their ability to induce intracellular reactive oxygen species (ROS), suggesting that oxidative stress is an important toxicity pathway of these compounds. Pearson correlation analysis of the cytotoxicity (IC50 values) of these compounds with eight physicochemical parameters showed strong associations with their lipophilicity (logP) and reactivity (polarizability, ELUMO). Finally, cytotoxicity testing of the concentrated extracts of a chloraminated mixture of eight dipeptides with bromide or iodide showed the cytotoxicity of these mixtures in the order: iodinated peptides > brominated peptides ≥ chlorinated peptides. These results demonstrate that halogenated peptide DBPs are toxicologically relevant, and further research is needed to understand the implications of long-term exposure for human health.

Formation and Occurrence of Iodinated Tyrosyl Dipeptides in Disinfected Drinking Water.

Iodinated disinfection byproducts (I-DBPs) are highly toxic, but few precursors of I-DBPs have been investigated. Tyrosine-containing biomolecules are ubiquitous in surface water. Here we investigated the formation of I-DBPs from the chloramination of seven tyrosyl dipeptides (tyrosylglycine, tyrosylalanine, tyrosylvaline, tyrosylhistidine, tyrosylglutamine, tyrosylglutamic acid, and tyrosylphenylalanine) in the presence of potassium iodide. High resolution mass spectrometry and tandem mass spectrometry (MS/MS) analyses of the benchtop reaction solutions found that all seven precursors formed both I- and Cl-substituted tyrosyl dipeptide products. Iodine substitutions occurred on the 3- and 3,5-positions of the tyrosyl-phenol ring while chlorine substituted on the free amino group. To reach the needed sensitivity to detect iodinated tyrosyl dipeptides in authentic waters, we developed a high performance liquid chromatography (HPLC)-MS/MS method with multiple reaction monitoring mode and solid phase extraction. HPLC-MS/MS analysis of tap and corresponding raw water samples, collected from three cities, identified four iodinated peptides, 3-I-/3,5-di-I-Tyr-Ala and 3-I-/3,5-di-I-Tyr-Gly, in the tap waters but not in the raw waters. The corresponding precursors, Tyr-Ala and Tyr-Gly, were also detected in the same tap and raw water samples. This study demonstrates that iodinated dipeptides exist as DBPs in drinking water.

Critical Role of K1685 and K1829 in the Large Protein of Rabies Virus in Viral Pathogenicity and Immune Evasion.

UNLABELLED: Rabies, one of the oldest infectious diseases, still presents a public health threat in most parts of the world today. Its pathogen, rabies virus (RABV), can utilize its viral proteins, such as the nucleoprotein and phosphorylation protein, to subvert the host innate immune system. For a long time, the large (L) protein was believed to be essential for RABV transcription and replication, but its role in viral pathogenicity and immune evasion was not known. Recent studies have found that the conserved K-D-K-E tetrad motif in the L protein is related to the methyltransferase (MTase) activity in the viral mRNA process. In the present study, a series of RABV mutations in this motif was constructed with the recombinant CVS-B2c (rB2c) virus. Two of these mutants, rB2c-K1685A and rB2c-K1829A, were found to be stable and displayed an attenuated phenotype in both in vitro growth and in vivo pathogenicity in adult and suckling mice. Further studies demonstrated that these two mutants were more sensitive to the expression of the interferon-stimulated gene product IFIT2 than the parent virus. Taken together, our results suggest that K1685 and K1829 in the L protein play important roles in pathogenicity and immune evasion during RABV infection. IMPORTANCE: Rabies continues to present a public health threat in most areas of the world, especially in the developing countries of Asia and Africa. The pathogenic mechanisms for rabies are not well understood. In the present study, it was found that the recombinant rabies viruses rB2c-K1685A and rB2c-K1829A, carrying mutations at the predicted MTase catalytic sites in the L protein, were highly attenuated both in vitro and in vivo. Further studies showed that these mutants were more sensitive to the expression of the interferon-stimulated gene product IFIT2 than the parent virus. These findings improve our understanding of rabies pathogenesis, which may help in developing potential therapeutics and an avirulent rabies vaccine.

Preparation of Rubber Accelerator Tetrabenzylthiuramdisulfide and Its Spectral Analysis.

In the study, rubber accelerator tetrabenzylthiuramdisulfide (TBzDT) was synthesized with two-step method with hydrogen peroxide as oxidant firstly. TBzDT was detected and characterized with XRD, FT-IR, TG-DTA. Its micro-structure was revealed. Chemical bond types into TBzDT molecule were revealed with FT-IR. TBzDT phase composition and structure were given by crystallographic data from XRD detecting such as cell parameters, crystal face index. The phase composition and qualitative identification of TBzDT structure were completed. Two kinds of information were detected with TG-DTA as to quality change and thermal effect. TBzDT phase transition and decomposition temperature were 142.5, 200.9 ℃ respectively. The decomposition temperature of TBzDT was relevtively high. It could provided reference with research on rubber vulcanizing properties by TBzDT on rubber vulcanizing machine. A little SC2 contained into TBzDT was revealed by FTIR, TG-DTA from different sides.

[Study on the Preparation of N-Tert-Butylbenzothiazole-2-Sulphenamide and Its Spectral Analysis].

In the study, N-tert-butylbenzothiazole-2-sulphenamide (TBBS) was synthesized with one-step method with hydrogen peroxide as the oxidant. TBBS was detected and characterized with FTIR，UV-Vis，XRD and TG-DSC. Its micro-structure and intrinsic regularity were revealed. Chemical bond types into TBBS molecule were revealed with FTIR. The final product was determined TBBS. Three absorption peaks were detected by UV-Vis at 228.3，281.5，298.3 nm respectively, due to n→σ*，π→π*，n→π* electronic transitions. It could provide experimental basis with enterprise to test TBBS product quality and speculate its structure. The phase composition and structure of TBBS were revealed by crystallographic data from XRD detecting such as cell parameters, crystal face index. The phase composition and qualitative identification of TBBS structure were completed. Two kinds of information were detected by TG-DSC as quality change and thermal effect. The DSC curve of TBBS was very complex. There were a small absorption peak of 53.3 ℃ as a small amount of tert-butylamine containing in TBBS was oxidized. Three exothermic peaks of 117.4，269.2，373.7 ℃, due to transition peak and decomposition phase of TBBS respectively. The decomposition temperature of TBBS was very high. It could provide reference for research on rubber vulcanizing properties by TBBS on rubber vulcanizing machine. This study could provide the basis experimental data on the enterprises to designate the working standard tracing detection of TBBS industrialized production. The performance index of TBBS was judged. The project of TBBS industry standard could be declared by the enterprises, written a draft standard.

[Preparation of Rubber Accelerator Tetraisobutylthiuram Monosulfide and Its Spectral Analysis].

In the study, rubber accelerator tetraisobutylthiuram monosulfide (TiBTM) was synthesized by two-step method with hydrogen peroxide as oxidant firstly. TiBTM was detected and characterized by FTIR, XRD, TG-DSC. Its micro-structure and intrinsic regularity were revealed. Chemical bond types into TiBTM molecule were revealed by FTIR. TiBTM phase composition and structure were given by crystallographic data from XRD detecting such as cell parameters, crystal face index. The phase composition and qualitative identification of TiBTM structure were completed. Two kinds of information were detected by TG-DSC as quality change and thermal effect. TiBTM phase transition and decomposition temperature were 75.1 and 287.0 ℃ respectively. Uhe decomposition temperature of TiBTM was very high, samples possessing with high purity, without other impurities. It could provided reference with research on rubber vulcanizing and multiple functional properties by TiBTM on rubber vulcanizing machine. This study can provide the basis experimental data on the enterprises to designate the working standard tracing detection of TiBTM industrialized production. The best technological conditions and parameters could be provided for the enterprises to adopt cleaner production process of TiBTM. Performance index of TiBTM was judged. The project of TiBTM industry standard can be declared by the enterprises, written a draft standard.

[Preparation of Zinc O,O,O',O'-Tetrabutyl Bis(Phosphorodithioate) and Its Spectral Analysis].

In the study, zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) (ZBPD) was synthesized with two-step method firstly. ZBPD was detected and characterized with UV-vis, FT-IR andTG-DSC. Its micro-structure and intrinsic regularity were revealed. Two absorption peaks were detected with UV-Vis at 212.0 and 227.0 nm respectively caused by n→σ*, π→π* electron transition. The intensity of peaks was changed with the regularity with different ZBPD concentration. It could provide the basic data with the enterprise of ZBPD product quality inspection. Chemical bond types into ZBPD molecule were revealed by FT-IR. Two kinds of information were detected by TG-DSC as quality change and thermal effect. The DSC curve of ZBPD was very complex. There were a wide absorption peak of 84.3 ℃, four exothermic peaks of 245.0, 344.3, 476.1, 344.3 ℃, due to the molecular structure of ZBPD and small amounts of impurities. The decomposition temperature of ZBPD was very high. It could provided reference with research on rubber vulcanizing properties by ZBPD on rubber vulcanizing machine. This study could provide the basis experimental data on the enterprises to designate the working standard tracing detection of ZBPD industrialized production. Performance index of ZBPD was judged. The project of ZBPD industry standard could be declared by the enterprises, written a draft standard.

[Preparation of TBSI with NS-Acetic Anhydride Method and Its Spectral Analysis].

In the study, N-t-butyl-2-benzothiazole sulfenimide (TBSI) was prepared by NS-acetic anhydride method with acetic anhydride as solvent. It was detected with FTIR, XRD, UV-Vis, TG-DTA. Micro-structure and essence disciplinarian of them were disclosed. Chemical bond types into TBSI molecule were revealed with FTIR. TBSI phase composition and structure were revealed with crystallographic data from XRD detecting such as cell parameters and crystal face index. The phase composition and qualitative identification of TBSI structure were completed. The absorption peaks were detected by UV-vis at 228.3, 281.3 and 298.3 nm, respectively caused by n→σ*, π→π*, n→π* electron transition. It could provide basic data with the enterprise of ZBPD product quality inspection. Two kinds of information were detected with TG-DTA as quality change and thermal effect. There were three absorption peaks of 46.5, 188.9, 368. 5 ℃ due to a few residual solvent volatilization in the sample, phase transition peak and decomposition peak. The decomposition temperature of TBSI was very high. It could provided reference with research on rubber vulcanizing properties with TBSI on rubber vulcanizing machine. This study could provide basis experimental data on the enterprises to designate the working standard tracing detection of TBSI industrialized production. Performance index of TBSI was judged. The project of TBSI industry standard could be declared by the enterprises, written a draft standard.

Hybrid supercapacitors using metal-organic framework derived nickel-sulfur compounds.

HYPOTHESIS: Metal-organic frameworks (MOFs) are highly suitable precursors for supercapacitor electrode materials owing to their high porosity and stable backbone structures that offer several advantages for redox reactions and rapid ion transport. EXPERIMENTS: In this study, a carbon-coated Ni9S8 composite (Ni9S8@C-5) was prepared via sulfuration at 500 ℃ using a spherical Ni-MOF as the sacrificial template. FINDING: The stable carbon skeleton derived from Ni-MOF and positive structure-activity relationship due to the multinuclear Ni9S8 components resulted in a specific capacity of 278.06 mAh·g-1 at 1 A·g-1. Additionally, the hybrid supercapacitor (HSC) constructed using Ni9S8@C-5 as the positive electrode and the laboratory-prepared coal pitch-based activated carbon (CTP-AC) as the negative electrode achieved an energy density of 69.32 Wh·kg-1 at a power density of 800.06 W·kg-1, and capacity retention of 83.06 % after 5000 cycles of charging and discharging at 5 A·g-1. The Ni-MOF sacrificial template method proposed in this study effectively addresses the challenges associated with structural collapse and agglomeration of Ni9S8 during electrochemical reactions, thus improving its electrochemical performance. Hence, a simple preparation method is demonstrated, with broad application prospects in supercapacitor electrodes.

The underlying toxicological mechanism of chemical mixtures: a case study on mixture toxicity of cyanogenic toxicants and aldehydes to Photobacterium phosphoreum.

Intracellular chemical reaction of chemical mixtures is one of the main reasons that cause synergistic or antagonistic effects. However, it still remains unclear what the influencing factors on the intracellular chemical reaction are, and how they influence on the toxicological mechanism of chemical mixtures. To reveal this underlying toxicological mechanism of chemical mixtures, a case study on mixture toxicity of cyanogenic toxicants and aldehydes to Photobacterium phosphoreum was employed, and both their joint effects and mixture toxicity were observed. Then series of two-step linear regressions were performed to describe the relationships between joint effects, the expected additive toxicities and descriptors of individual chemicals (including concentrations, binding affinity to receptors, octanol/water partition coefficients). Based on the quantitative relationships, the underlying joint toxicological mechanisms were revealed. The result shows that, for mixtures with their joint effects resulting from intracellular chemical reaction, their underlying toxicological mechanism depends on not only their interaction with target proteins, but also their transmembrane actions and their concentrations. In addition, two generic points of toxicological mechanism were proposed including the influencing factors on intracellular chemical reaction and the difference of the toxicological mechanism between single reactive chemicals and their mixtures. This study provided an insight into the understanding of the underlying toxicological mechanism for chemical mixtures with intracellular chemical reaction.

Synthesis and characterizations of iso-luminol-functionalized, tadpole-shaped, gold nanomaterials.

Iso-luminol functionalized gold nanomaterials were synthesized in high yield by a simple seeding approach, using the chemiluminescent reagent iso-luminol as reductant in the presence of HAuCl(4), AgNO(3) and cetyltrimethylammonium bromide (CTAB). The morphology of as-prepared gold nanoparticles was characterized by transmission electron microscopy and UV-vis spectroscopy, showing that gold nanotadpoles (AuNTps) were obtained. Subsequent experiments revealed that the amounts of seed colloids and AgNO(3) and the concentrations of iso-luminol and CTAB in the growth solution play critical roles in the formation of well-shaped AuNTps. The surface state of AuNTps was characterized by UV-vis spectroscopy and fluorescence spectroscopy, indicating that iso-luminol and its oxidation product, 4-aminophthalate, coexisted on the surface of AuNTps. The CL behaviour was studied by static injection CL experiments, demonstrating that AuNTps were of CL activity. Finally, the growth mechanism of AuNTps was also discussed.

Modeling Cardinality in Image Hashing.

Cardinality constraint, namely, constraining the number of nonzero outputs of models, has been widely used in structural learning. It can be used for modeling the dependencies between multidimensional labels. In hashing, the final outputs are also binary codes, which are similar to multidimensional labels. It has been validated that estimating how many 1's in a multidimensional label vector is easier than directly predicting which elements are 1 and estimating cardinality as a prior step will improve the classification performance. Hence, in this article, we incorporate cardinality constraint into the unsupervised image hashing problem. The proposed model is divided into two steps: 1) estimating the cardinalities of hashing codes and 2) then estimating which bits are 1. Unlike multidimensional labels that are known and fixed in the training phase, the hashing codes are generally learned through an iterative method and, therefore, their cardinalities are unknown and not fixed during the learning procedure. We use a neural network as a cardinality predictor and its parameters are jointly learned with the hashing code generator, which is an autoencoder in our model. The experiments demonstrate the efficiency of our proposed method.

Model of hormesis and its toxicity mechanism based on quorum sensing: a case study on the toxicity of sulfonamides to Photobacterium phosphoreum.

During the past two decades, the phenomenon of hormesis has gained increasing recognition in environmental and toxicological communities. However, the mechanistic understanding of hormesis, to date, is extremely limited. Herein is proposed a novel parametric model with a mechanistic basis and two model-based parameters for hormesis that was successfully applied to the hormetic dose-response observed in the chronic toxicity of sulfonamides on Photobacterium phosphoreum. On the basis of the methods of molecular docking and quantitative structure-activity relationships (QSARs), we proposed a mechanistic hypothesis for hormesis that introduces for the first time the concept of quorum sensing in toxicological studies and explains the mechanism at the level of the receptors. The mechanistic hypothesis stated that (1) specific target binding like interaction with LuxR may contribute to transcriptional activation leading to enhanced luciferase activity at low dose exposure of sulfonamides, and (2) as the dose of sulfonamides increases, more sulfonamides competitively bind to dihydropteroate synthase, which inhibit the biosynthesis of folic acid and thus provoke toxicity. This mechanistic hypothesis, which explains both the dose-dependent and time-dependent features of hormesis, could give new insight into the mechanistic study of hormesis.

Application of the similarity parameter (λ) to prediction of the joint effects of nonequitoxic mixtures.

Although environmental contaminants are usually encountered as nonequitoxic mixtures, most studies have investigated the toxicity of equitoxic mixtures. In the present study, a method for prediction of the toxicity of nonequitoxic mixtures was developed using the similarity parameter (λ). The joint effect of multiple contaminants at the median inhibition concentration in equitoxic ([Formula: see text]) and nonequitoxic ([Formula: see text]) binary, ternary, and quaternary mixtures was investigated using Vibrio fischeri. The observed results indicate that the concentration ratios of individual chemicals in the mixtures influenced the joint effects, and that λ could be employed to evaluate the relation between [Formula: see text] and [Formula: see text]. Prediction models for the joint effects of nonequitoxic ([Formula: see text]) mixtures were derived from a combination of [Formula: see text] and λ. The predictive capabilities of these models were validated by comparing the predicted data with the observed data for binary, ternary, and quaternary mixtures. The prediction models have promising applications in controlling environmental pollution, evaluating drug interactions, and optimizing combinations of pesticides used in agriculture.

Establishment of a model to assess mumps virus neurovirulence in neonatal Wistar rats.

Mumps virus (MuV) is highly neurotropic and neurovirulent, hence, the neurovirulence of virus seeds used in the production of mumps vaccines must be tested. The previous neurovirulence evaluation method involves measuring the area of the cavity in the Lewis neonatal rat brain caused by MuV through paraffin sectioning and hematoxylin-eosin (HE) staining. However, the processes of paraffin sectioning and HE staining are time consuming and complicated. To solve this problem, in this study, a vibratome sectioning system was first deployed to evaluate MuV neurovirulence in the rat brain instead of paraffin sectioning and HE staining. The results showed that the vibratome sectioning method could assess the neurovirulence potential of MuV more objectively and efficiently. In addition, the effects of different MuV doses and the ages of the rats in days on this evaluation method were explored. The results indicate that MuV at no less than 10 50 % cell culture infective dose (CCID50) could cause obvious cavity formation in 1-day-old rat brains. The neonatal rat model developed in this study could evaluate the neurovirulence of different MuV strains with high sensitivity and good repeatability.

A spatial and cellular distribution of rabies virus infection in the mouse brain revealed by fMOST and single-cell RNA sequencing.

BACKGROUND: Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. METHODS: In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single-cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of a recombinant RABV encoding enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional (3D) view of RABV distribution in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. RESULTS: Our fMOST data provided the 3D view of a neurotropic virus in the whole mouse brain, which indicated that the spatial distribution of RABV in the brain was influenced by the infection route. Interestingly, we provided evidence that RABV could infect multiple nuclei related to fear independent of different infection routes. More surprisingly, our scRNA-seq data revealed that besides neurons RABV could infect macrophages and the infiltrating macrophages played at least three different antiviral roles during RABV infection. CONCLUSION: This study draws a comprehensively spatial and cellular map of typical neurotropic virus infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of RABV and other neurotropic viruses.

Experimental infection of Bama miniature pigs with a highly virulent classical swine fever virus.

BACKGROUND: Currently, larger domestic pigs are only animals widely used in vaccine evaluation and pathogenicity study of classical swine fever virus (CSFV). This study was aimed to create an alternative animal experimental infection model of CSFV. RESULTS: Twenty specific-pathogen-free Bama miniature pigs were randomly divided into two groups and rooms, infected and non-infected, and the pigs in the infected group were inoculated intramuscularly with 104, 105 or 106 TCID50 (median tissue culture infective dose) CSFV Shimen strain (n = 5 × 3) or left uninoculated to serve as in-contact pigs (n = 3). The uninfected control pigs (n = 2) were housed in a separate room. Clinical signs, body temperature, viraemia, tissue antigen distribution, pathological changes and seroconversion were monitored. Clinical signs were observed as early as 2 days post-inoculation (dpi) in all infected pigs (though mild in contact pigs), but not non-infected control pigs. All inoculated pigs showed viraemia by 6 dpi. The in-contact pigs showed lower levels of viraemia. At 10 dpi, seroconversion was noted in five of the 15 inoculated pigs. All inoculated or one in-contact pigs died by 15 dpi. CONCLUSIONS: These results show that Bama miniature pigs support productive CSFV infection and display clinical signs and pathological changes consistent with CSFV infections observed in larger domestic pigs.