Investigation of oral toxicity of WS2 nanosheets to mouse intestine: Pathological injury, trace element balance, lipid profile changes, and autophagy.

The success of graphene oxides has gained extensive research interests in developing novel 2D nanomaterials (NMs). WS2 nanosheets (NSs) are novel transition metal-based 2D NMs, but their toxicity is unclear. In this study, we investigated the oral toxicity of WS2 NSs to mouse intestines. Male mice were administrated with vehicles, 1, 10, or 100 mg/kg NSs via intragastric route, once a day, for 5 days. The results indicate that the NSs did not induce pathological or ultrastructural changes in intestines. There were minimal changes of trace elements that the exposure did not induce W accumulation, and only Co levels were dose-dependently increased. Lipid droplets were observed in all groups of mice, but lipidomics data indicate that WS2 NSs only significantly decreased four lipid species, all belonging to phosphatidylcholine (PC). The levels of proteins regulating autophagic lipolysis, namely, LC3, lysosomal associated membrane protein 2 (LAMP2) and perilipin 2 (PLIN2), were increased, but it was only statistically significantly different for LC3. The results of this study suggest that repeated intragastric exposure to WS2 NSs only induced minimal influences on pathological injury, trace element balance, autophagy, and lipid profiles in mouse intestines, indicating relatively high biocompatibility of WS2 NSs to mouse intestine via oral route.

FUBP3 Degrades the Porcine Epidemic Diarrhea Virus Nucleocapsid Protein and Induces the Production of Type I Interferon.

Porcine epidemic diarrhea virus (PEDV) is the globally distributed alphacoronavirus that can cause lethal watery diarrhea in piglets, causing substantial economic damage. However, the current commercial vaccines cannot effectively the existing diseases. Thus, it is of great necessity to identify the host antiviral factors and the mechanism by which the host immune system responds against PEDV infection required to be explored. The current work demonstrated that the host protein, the far upstream element-binding protein 3 (FUBP3), could be controlled by the transcription factor TCFL5, which could suppress PEDV replication through targeting and degrading the nucleocapsid (N) protein of the virus based on selective autophagy. For the ubiquitination of the N protein, FUBP3 was found to recruit the E3 ubiquitin ligase MARCH8/MARCHF8, which was then identified, transported to, and degraded in autolysosomes via NDP52/CALCOCO2 (cargo receptors), resulting in impaired viral proliferation. Additionally, FUBP3 was found to positively regulate type-I interferon (IFN-I) signaling and activate the IFN-I signaling pathway by interacting and increasing the expression of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3). Collectively, this study showed a novel mechanism of FUBP3-mediated virus restriction, where FUBP3 was found to degrade the viral N protein and induce IFN-I production, aiming to hinder the replication of PEDV. IMPORTANCE PEDV refers to the alphacoronavirus that is found globally and has re-emerged recently, causing severe financial losses. In PEDV infection, the host activates various host restriction factors to maintain innate antiviral responses to suppress virus replication. Here, FUBP3 was detected as a new host restriction factor. FUBP3 was found to suppress PEDV replication via the degradation of the PEDV-encoded nucleocapsid (N) protein via E3 ubiquitin ligase MARCH8 as well as the cargo receptor NDP52/CALCOCO2. Additionally, FUBP3 upregulated the IFN-I signaling pathway by interacting with and increasing tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) expression. This study further demonstrated that another layer of complexity could be added to the selective autophagy and innate immune response against PEDV infection are complicated.

TARDBP Inhibits Porcine Epidemic Diarrhea Virus Replication through Degrading Viral Nucleocapsid Protein and Activating Type I Interferon Signaling.

In global infection and serious morbidity and mortality, porcine epidemic diarrhea virus (PEDV) has been regarded as a dreadful porcine pathogen, but the existing commercial vaccines are not enough to fully protect against the epidemic strains. Therefore, it is of great necessity to feature the PEDV-host interaction and develop efficient countermeasures against viral infection. As an RNA/DNA protein, the trans-active response DNA binding protein (TARDBP) plays a variety of functions in generating and processing RNA, including transcription, splicing, transport, and mRNA stability, which have been reported to regulate viral replication. The current work aimed to detect whether and how TARDBP influences PEDV replication. Our data demonstrated that PEDV replication was significantly suppressed by TARDBP, regulated by KLF16, which targeted its promoter. We observed that through the proteasomal and autophagic degradation pathway, TARDBP inhibited PEDV replication via the binding as well as degradation of PEDV-encoded nucleocapsid (N) protein. Moreover, we found that TARDBP promoted autophagic degradation of N protein via interacting with MARCHF8, an E3 ubiquitin ligase, as well as NDP52, a cargo receptor. We also showed that TARDBP promoted host antiviral innate immune response by inducing interferon (IFN) expression through the MyD88-TRAF3-IRF3 pathway during PEDV infection. In conclusion, these data revealed a new antiviral role of TARDBP, effectively suppressing PEDV replication through degrading virus N protein via the proteasomal and autophagic degradation pathway and activating type I IFN signaling via upregulating the expression of MyD88. IMPORTANCE PEDV refers to the highly contagious enteric coronavirus that has quickly spread globally and generated substantial financial damage to the global swine industry. During virus infection, the host regulates the innate immunity and autophagy process to inhibit virus infection. However, the virus has evolved plenty of strategies with the purpose of limiting IFN-I production and autophagy processes. Here, we identified that TARDBP expression was downregulated via the transcription factor KLF16 during PEDV infection. TARDBP could inhibit PEDV replication through the combination as well as degradation of PEDV-encoded nucleocapsid (N) protein via proteasomal and autophagic degradation pathways and promoted host antiviral innate immune response by inducing IFN expression through the MyD88-TRAF3-IRF3 pathway. In sum, our data identify a novel antiviral function of TARDBP and provide a better grasp of the innate immune response and protein degradation pathway against PEDV infection.

Deep Reconstruction of the NiFeSP Nanosheet Catalyst for Large Current-Density Water Oxidation.

Evidence shows that the crucial factor in achieving efficient water electrolysis for hydrogen production is the design and synthesis of electrocatalysts that exhibit both high performance and cost-effectiveness for the oxygen evolution reaction (OER). Herein, the NiFeSP nanosheets were facilely prepared on a Ni foam (NF) substrate through a cost-effective electrodeposition method. The electrode structure composed of nanosheets offers a high density of active sites and superior electrical conductivity, thereby enhancing the efficiency of the OER. In addition, the NiFeSP/NF-600 nanosheets exhibit superhydrophilic and superaerophobic characteristics, which effectively enhance the mass-transfer process by facilitating the penetration of electrolytes and enabling rapid release of gas bubbles. Consequently, NiFeSP/NF-600 demonstrates superior electrocatalytic efficacy for OER, exhibiting an overpotential of 292 mV at a high current density of 500 mA cm-2 as well as an exceptional long-term durability of 100 h. More importantly, the rapid reconstruction of FeOOH and NiOOH species from NiFeSP/NF-600 may be the true active species for OER, which is revealed utilizing in situ Raman spectroscopy in conjunction with ex situ characterization. This study not only offers an ideal "pre-catalyst" for an extremely effective OER but also offers a thorough understanding of the mechanism underlying the structural evolution of electrocatalysts and the identification of their actual active sites.

Genetic diversity of Blastocystis subtypes in the Alpine musk deer (Moschus chrysogaster) in Gansu province, northwestern China.

Blastocystis sp. is a gastrointestinal pathogen that is frequently found in humans and animals worldwide. In this study, 201 fecal samples were collected from captive Alpine musk deer (Moschus chrysogaster) at three farms in Gansu province. Blastocystis was detected and subtyped by amplifying and sequencing the small subunit ribosomal DNA gene. The overall prevalence of Blastocystis was 39.8% (80/201). Five known Blastocystis subtypes (STs), including ST1 (n = 1), ST4 (n = 12), ST10 (n = 50), ST14 (n = 6), and ST24 (n = 11) were identified using subtyping and evolutionary analysis. ST10 was the most common ST observed in each farm. This study showed the infection status and genetic characteristics of Blastocystis in M. chrysogaster. Based on the surveyed data, because various potentially zoonotic STs, such as ST1, ST4, ST10, ST14, and ST24, were detected, it is believed that the zoonotic risk of Blastocystis from the Alpine musk deer in this area cannot be ignored.

Comparative analysis of ankyrin (ANK) genes of five capripoxviruses isolate strains from Xinjiang province in China.

BACKGROUND: Sheeppox and goatpox are both economically important animal diseases in which pathogens are goatpox virus (GTPV) and sheeppox virus (SPPV). They can't cause cross-species infection between sheep and goats in general. But in recent decades, the infection of sheep by goatpox or goats by sheeppox has been reported. The literature has indicated that the occurrence of these cases has a significant and direct relationship with mutations of ankyrin genes families (ANK genes 010,138,140,141.2,145) located in two-terminal regions of capripoxvirus genomes. So it is very important to decipher these nucleotides and their coding amino acid sequences of the five genes regarded as host range and virulence factors for effective prevention and control of capripoxvirus diseases. METHODS: In this study, all the ankyrin genes of three goatpox virus, two sheeppox virus, and one GTPV vaccine strains from Nanjiang areas of Xinjiang province of China during 2010-2011 were collected, amplified, cloned and sequenced. The sequence of every ankyrin genes has been compared with not only sequences from six viruses but also all sequences from three species of capripoxvirus genus from Gene bank, and every ANK gene's mutated nucleotides and amino acids have been screened, and the relationship of genetic evolution among different virus strains has been analyzed, as well as the domain architecture of these genes was forecasted and analyzed. RESULTS: The six capripoxvirus strains can be well-distinguished GTPV and SPPV based on five ANK genes' sequence identicalness except for GTPV-SS strain, which showed higher identicalness with SPPV. The ANK gene sequence of the GTPV-SS strain was 100% identical with SPPV-M1 (ANK138,140,145) and SPPV-M2 (ANK138,145), respectively. Phylogenetically, these six capripoxvirus strains were also grouped into the same cluster of India reference strains in lineages and showed extreme identical conservative or variable regions with India capripoxvirus isolates by sequence alignment. Moreover, for the functional domains, these ANK genes of capripoxvirus except for ANK gene 145, are identical in size, and ANK genes 145 of SPPV are usually 100 bp (approximately 30 aa) longer than those of GTPV and eventually form a PRANC domain at C-terminus. CONCLUSIONS: The isolated strain of GTPV-SS may be a cross-species infection or the collected material was contaminated, and the inferred Capripox outbreak in Xinjiang in 2010 can be introduced from India. ANK genes 138,140,141.2 and 145 of capripoxvirus can be used as the target genes to identify GTPV and SPPV. Moreover, the four ANK genes determining the host range are more significant than the ANK gene 010. These ANK genes play combining roles for their function.

A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales(families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae Importantly, these findings may provide a new structural basis for antiviral drug development.

Investigating fungal community characteristics in co-composted cotton stalk and various livestock manure products.

Agricultural wastes, comprising cotton straw and livestock manure, can be effectively managed through aerobic co-composting. Nevertheless, the quality and microbial characteristics of co-composting products from different sources remain unclear. Therefore, this study utilized livestock manure from various sources in Xinjiang, China, including herbivorous sheep manure (G), omnivorous pigeon manure (Y), and pigeon-sheep mixture (GY) alongside cotton stalks, for a 40-day co-composting process. We monitored physicochemical changes, assessed compost characteristics, and investigated fungal community. The results indicate that all three composts met established composting criteria, with compost G exhibiting the fastest microbial growth and achieving the highest quality. Ascomycota emerged as the predominant taxon in three compost products. Remarkably, at the genus level, the biomarker species for G, Y, and GY are Petromyces and Cordyceps, Neurospora, and Neosartorya, respectively. Microorganisms play a pivotal role in organic matter degradation, impacting nutrient composition, demonstrating significant potential for the decomposition and transformation of compost components. Redundancy analysis indicates that potassium, total organic carbon, and C:N are key factors influencing fungal communities. This study elucidates organic matter degradation in co-composting straw and livestock manure diverse sources, optimizing treatment for efficient agricultural waste utilization and sustainable practices.

Recovery of Chlorosilane Residual Liquid to Prepare Nano-Silica via the Reverse Micro-Emulsion Process.

In this paper, nano-silica particles were prepared from chlorosilane residue liquid using an inverse micro-emulsions system formed from octylphenyl polyoxyethylene ether (TX-100)/n-hexanol/cyclohexane/ammonia. The influence of different reaction conditions on the morphology, particle size, and dispersion of nano-silica particles was investigated via single-factor analysis. When the concentration of chlorosilane residue liquid (0.08 mol/L), hydrophile-lipophilic-balance (HLB) values (10.50), and the concentration of ammonia (0.58 mol/L) were under suitable conditions, the nano-silica particles had a more uniform morphology, smaller particle size, and better dispersion, while the size of the nano-silica particles gradually increased with the increase in the molar ratio of water to surfactant (ω). The prepared nano-silica was characterized through XRD, FT-IR, N2 adsorption/desorption experiments, and TG-DSC analysis. The results showed that the prepared nano-silica was amorphous mesoporous silica, and that the BET specific surface area was 850.5 m2/g. It also had good thermal stability. When the temperature exceeded 1140 °C, the nano-silica underwent a phase transition from an amorphous form to crystalline. This method not only promoted the sustainable development of the polysilicon industry, it also provided new ideas for the protection of the ecological environment, the preparation of environmental functional materials, and the recycling of resources and energy.

Molecular detection and characterization of Enterocytozoon bieneusi in captive alpine musk deer (Moschus chrysogaster) in Central and Eastern of Gansu Province, China.

The most ubiquitously diagnosed microsporidian species in animals and humans is Enterocytozoon bieneusi (E. bieneusi). In this case, this work aimed to probe the occurrence and genotypes of this pathogen in captive alpine musk deer (Moschus chrysogaster) in Gansu Province, China. After fecal sample collection (n = 201) from three farms in Gansu, the internal transcribed spacer (ITS) region of the rRNA gene was probed by PCR for detection and genotyping of E. bieneusi. The infection rate of E. bieneusi in alpine musk deer was 6.0% (12/201), with 0% (0/36), 7.7% (5/65) and 7.0% (7/100) in farms 1 to 3, respectively. The infection rate of E. bieneusi in young alpine musk deer (3.2%; 1/31) is lower than that of adults (6.5%; 11/170), with no evident significant differences between age groups (P > 0.05). Three known genotypes D (n = 8), EbpA (n = 3) and BEB6 (n = 1) were identified by sequence analysis. This is the first such scrutiny of E. bieneusi infection in alpine musk deer in China as per our knowledge. Genotypes D and EbpA were common in humans and animals that is suggestive of the plausible zoonotic role in E. bieneusi transmission by alpine musk deer.

ATG4B hinders porcine epidemic diarrhea virus replication through interacting with TRAF3 and activating type-I IFN signaling.

Autophagy-related 4B (ATG4B) is found to exert a vital function in viral replication, although the mechanism through which ATG4B activates type-I IFN signaling to hinder viral replication remains to be explained, so far. The current work revealed that ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. In addition, ATG4B overexpression inhibited PEDV replication in both Vero cells and LLC-PK1 cells. On the contrary, ATG4B knockdown facilitated PEDV replication. Moreover, ATG4B was observed to hinder PEDV replication by activating type-I IFN signaling. Further detailed analysis revealed that the ATG4B protein targeted and upregulated the TRAF3 protein to induce IFN expression via the TRAF3-pTBK1-pIRF3 pathway. The above data revealed a novel mechanism underlying the ATG4B-mediated viral restriction, thereby providing novel possibilities for preventing and controlling PEDV.

KLF16 inhibits PEDV replication by activating the type I IFN signaling pathway.

KLF16, a member of KLFs (Krüppel-like factors), contributes to the progression of a variety of cancer types. There is, however, still uncertain regarding the role of KLF16 in viral replication and the signaling mechanism of type I IFN. It was discovered that KLF16 inhibited the replication of porcine epidemic diarrhea virus (PEDV) through the type I IFN signaling pathway. Besides, it can also be found that the expression of KLF16 was down-regulated after PEDV infection of LLC-PK1 cells. Furthermore, overexpression of KLF16 inhibited the replication of PEDV in Vero cells as well as LLC-PK1 cells, whereas the replication of PEDV was promoted by the knockdown of KLF16. KLF16 up-regulated the expression of interferons (IFNs) via the TRAF6-pTBK1-pIRF3 pathway with the aim of promoting the host antiviral innate immune response. In addition, the obtained findings proved that KLF16 plays a novel role in antiviral action, thereby offering novel possibilities for preventing and controlling PEDV.

Construction and purification of ANK gene deleted recombinant goatpox virus.

Sheeppox virus (SPPV) and goatpox virus (GTPV) are two pathogens of host specificity. Previous studies have hypothesized that ankyrin (ANK) family may play an important role in determining host range of SPPV and GTPV. In order to verify the function of ANK proteins, it is critical to generate and purify the ANK gene deleted GTPV. In this study, the GFP gene as a reporter gene was connected with two homologous arms of ANK gene by fusion PCR. The ANK gene deleted transfer vectors were generated by inserting the PCR products into PET42b, and were transfected into testicular primary cells which were infected by GTPV. The rGTPV were identified as green fluorescence positive and properly purified. The results showed that GFP gene and two homologous arms of ANK gene were connected. The sequence was inserted in PET42b to form ANK deleted transfer vector. ANK deleted rGTPV was generated successfully by transferring vector and GTPV in cells. The ANK deleted rGTPV was purified and identified in this study. The study successfully generated the ANK deleted rGTPV. It overcomes the technical barrier for future studies about the function of ANK genes.

Expression and functional analysis of Brucella outer membrane protein 25 in recombinant goat pox virus.

The Capripoxvirus (CaPV) has a large double­stranded DNA genome and a restricted host­range. At present, it is being investigated as an ideal vaccine vector. In the present study, a novel recombinant goat pox virus (rGTPV) was constructed to express Brucella outer membrane protein (OMP)25, and was validated by in vitro and in vivo immunization assays. A novel rGTPV vector was created, in which the thymidine kinase gene was used as a flanking sequence, I1L was inserted as a promoter element to enhance Brucella OMP25 expression, and p7.5 as another promoter element was used to regulate guanine phosphoribosyl­transferase as a selection maker. The rGTPV vector was transfected into sheep fetal fibroblast/lamb testis cells pre­infected with GTPV G14­STV44­55 to recombine. Brucella OMP25 protein was expressed in cells by rGTPV, and activated immune reactivity to Brucella OMP25 protein, as detected by western blotting. Furthermore, rGTPV elicited, anti­Brucella­specific immunoglobulin G responses, as determined by ELISA. Mice vaccinated with rGTPV did not exhibit pathology alterations in the kidney and liver. These results suggested that the novel rGTPV was able to efficiently drive Brucella OMP25 protein expression and activate immune reactivity, and may have applications in CaPV live vector vaccines and associated research.

Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis.

Hydrogen peroxide (H2O2) is both an exogenous and endogenous cytotoxic agent that can reliably induce apoptosis in numerous cell types for studies on apoptosis signaling pathways. However, little is known of these apoptotic processes in myocardial cells of chicken, a species prone to progressive heart failure. Sequencing of mRNA transcripts (RNA-Seq) allows for the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved, including cellular responses to exogenous and endogenous toxins. We used RNA-seq to examine genes differentially expressed during H2O2-induced apoptosis in primary cultures of embryonic chicken cardiomyocytes. Following control or H2O2 treatment, RNA was extracted and sequencing performed to identify novel transcripts up- or downregulated in the H2O2 treatment group and construct protein-protein interaction networks. Of the 19,268 known and 2,160 novel transcripts identified in both control and H2O2 treatment groups, 4,650 showed significant differential expression. Among them, 55.63% were upregulated and 44.37% downregulated. Initiation of apoptosis by H2O2 was associated with upregulation of caspase-8, caspase-9, and caspase-3, and downregulation of anti-apoptotic genes API5 and TRIA1. Many other differentially expressed genes were associated with metabolic pathways (including 'Fatty acid metabolism', 'Alanine, aspartate, and glutamate metabolism', and 'Biosynthesis of unsaturated fatty acids') and cell signaling pathways (including 'PPAR signaling pathway', 'Adipocytokine signaling pathway', 'TGF-beta signaling pathway', 'MAPK signaling pathway', and 'p53 signaling pathway'). In chicken cardiomyocytes, H2O2 alters the expression of numerous genes linked to cell signaling and metabolism as well as genes directly associated with apoptosis. In particular, H2O2 also affects the biosynthesis and processing of proteins and unsaturated fatty acids. These results highlight the value of RNA-seq for revealing unexpected molecular contributors to oxidative stress responses, thereby identifying novel potential therapeutic targets.

Identification and Comparison of Receptor Binding Characteristics of the Spike Protein of Two Porcine Epidemic Diarrhea Virus Strains.

Porcine epidemic diarrhea virus (PEDV), a member of Alphacoronavirus, has caused huge economic losses for the global pork industry recently. The spike (S) protein mediates PEDV entry into host cells. Herein, we investigated the interactions between the S protein and its receptor porcine aminopeptidase N (pAPN) or co-receptor sugars. The C-terminal domain (CTD) of the S1 domain is bound to pAPN. The prototype strain demonstrated similar receptor-binding activity compared with the variant field isolate. Three loops at the tips of the ß-barrel domains did not play crucial roles in the PEDV S-pAPN association, indicating that PEDV conforms to a different receptor recognition model compared with transmissible gastroenteritis virus (TGEV), porcine respiratory CoV (PRCV), and human coronavirus NL63 (HCoV-NL63). The N-terminal domain (NTD) of the PEDV S1 domain could bind sugar, a possible co-receptor for PEDV. The prototype strain exhibited weaker sugar-binding activity compared with the variant field isolate. Strategies targeting the receptor binding domain (RBD) may be helpful for developing vaccines or antiviral drugs for PEDV. Understanding the differences in receptor binding between the prototype and the variant strains may provide insight into PEDV pathogenesis.

Rabies virus phosphoprotein interacts with ribosomal protein L9 and affects rabies virus replication.

Rabies virus is a highly neurotropic virus that can cause fatal infection of the central nervous system in warm-blooded animals. The RABV phosphoprotein (P), an essential cofactor of the virus RNA-dependent RNA polymerase, is required for virus replication. In this study, the ribosomal protein L9, which has functions in protein translation, is identified as P-interacting cellular factor using phage display analysis. Direct binding between the L9 and P was confirmed by protein pull-down and co-immunoprecipitation analyses. It was further demonstrated that L9 translocates from the nucleus to the cytoplasm, where it colocalizes with P in cells infected with RABV or transfected with P gene. RABV replication was reduced with L9 overexpression and enhanced with L9 knockdown. Thus, we propose that during RABV infection, P binds to L9 that translocates from the nucleus to the cytoplasm, inhibiting the initial stage of RABV transcription.