A getah virus-like-particle vaccine provides complete protection from viremia and arthritis in wild-type mice.

Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 µg vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.

Activation of CTU2 expression by LXR promotes the development of hepatocellular carcinoma.

Cytosolic thiouridylase 2 (CTU2) is an enzyme modifying transfer RNAs post-transcriptionally, which has been implicated in breast cancer and melanoma development. And we found CTU2 participated in hepatocellular carcinoma (HCC) progression here. HepG2 cells as well as xenograft nude mice model were employed to investigate the role of CTU2 in HCC development in vitro and in vivo respectively. Further, we defined CTU2 as a Liver X receptor (LXR) targeted gene, with a typical LXR element in the CTU2 promoter. CTU2 expression was activated by LXR agonist and depressed by LXR knockout. Interestingly, we also found CTU2 took part in lipogenesis by directly enhancing the synthesis of lipogenic proteins, which provided a novel mechanism for LXR regulating lipid synthesis. Meanwhile, lipogenesis was active during cell proliferation, particularly in tumor cells. Reduction of CTU2 expression was related to reduced tumor burden and synergized anti-tumor effect of LXR ligands by inducing tumor cell apoptosis and inhibiting cell proliferation. Taken together, our study identified CTU2 as an LXR target gene. Inhibition of CTU2 expression could enhance the anti-tumor effect of LXR ligand in HCC, identifying CTU2 as a promising target for HCC treatment and providing a novel strategy for the application of LXR agonists in anti-tumor effect.

An adenovirus-vectored vaccine based on the N protein of feline coronavirus elicit robust protective immune responses.

Feline coronavirus (FCoV) is an unsegmented, single-stranded RNA virus belonging to the Alphacoronavirus genus. It can cause fatal feline infectious peritonitis (FIP) in cats of any ages. Currently, there are no effective prevention and control measures to against FCoV. In this study, we developed a recombinant adenovirus vaccine, AD5-N, based on the nucleocapsid(N) protein of FCoV. The immunogenicity of AD5-N was evaluated through intramuscular immunization in 6-week-old Balb/c mice and 9-12 months old cats. Compared to the control group, AD5-N specifically induced a significant increase in IgG and SIgA levels in the vaccinated mice. Furthermore, AD5-N not only effectively promoted strong cellular immune responses in cats but also induced high levels of specific SIgA, effectively helping cats resist FCoV infection. Our findings suggest that adenovirus vector vaccines based on the N gene have the potential to become candidate vaccines for the prevention and control of FCoV infection.

Performance and mechanism of various microaerobic pretreatments on anaerobic digestion of tobacco straw.

Tobacco straw is an abundant biomass in China's agricultural ecosystems, and has high potential for methane production. However, the anaerobic digestion (AD) efficiency is limited by the recalcitrant lignocellulose structure of the tobacco straw. In this study, three microaerobic pretreatments were performed for the AD of tobacco straw to increase methane production. Among them, microbial pretreatment with biogas slurry at an oxygen concentration of 4 mL/g VS resulted in the highest methane production of 349.1 mL/g VS, increasing by 19.8 % than that of untreated. During this pretreatment, the relative abundances of Enterococcus and Clostridium sensu stricto 12, which are closely related to acetic acid production and cellulose degradation, were high, and these bacteria might have an important contribution to substrate hydrolysis and the methanogenesis efficiency of the AD process. This study advances the understanding of microaerobic pretreatment processes and provides technological guidance for the efficient utilization of tobacco straw.

Pijx confers broad-spectrum seedling and panicle blast resistance by promoting the degradation of ATP ß subunit and OsRbohC-mediated ROS burst in rice.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most important diseases of rice. Utilization of blast-resistance genes is the most economical, effective, and environmentally friendly way to control the disease. However, genetic resources with broad-spectrum resistance (BSR) that is effective throughout the rice growth period are rare. In this work, using a genome-wide association study, we identify a new blast-resistance gene, Pijx, which encodes a typical CC-NBS-LRR protein. Pijx is derived from a wild rice species and confers BSR to M. oryzae at both the seedling and panicle stages. The functions of the resistant haplotypes of Pijx are confirmed by gene knockout and overexpression experiments. Mechanistically, the LRR domain in Pijx interacts with and promotes the degradation of the ATP synthase ß subunit (ATPb) via the 26S proteasome pathway. ATPb acts as a negative regulator of Pijx-mediated panicle blast resistance, and interacts with OsRbohC to promote its degradation. Consistently, loss of ATPb function causes an increase in NAPDH content and ROS burst. Remarkably, when Pijx is introgressed into two japonica rice varieties, the introgression lines show BSR and increased yields that are approximately 51.59% and 79.31% higher compared with those of their parents in a natural blast disease nursery. In addition, we generate PPLPijx Pigm and PPLPijx Piz-t pyramided lines and these lines also have higher BSR to panicle blast compared with Pigm- or Piz-t-containing rice plants. Collectively, this study demonstrates that Pijx not only confers BSR to M. oryzae but also maintains high and stable rice yield, providing new genetic resources and molecular targets for breeding rice varieties with broad-spectrum blast resistance.

Recycling different textile wastes for methane production: Morphological and microstructural changes and microbial community dynamics.

The dramatic increase of textile wastes has become a major global concern, which calls for alternative practices to alleviate severe environmental pollution and waste of resources due to their improper disposal and management. Anaerobic digestion (AD) is a cost-effective and eco-friendly technology that allows the bioconversion of organic wastes into clean energy (methane), which might be potentially useful for recycling textile wastes. In this study, AD was applied to 11 commonly available textile wastes in daily life to explore their feasibility, along with the methane production efficiency, biodegradability (BD), degradation mechanism, and microbial community dynamics during AD. The results showed that all textile wastes presented an obvious decomposition from an integrated shape to fragmented pieces within 18 days except blue denim. The highest experimental methane production (EMP) of 356.0 mL/g volatile solids (VS) and BD of 78.0 % were obtained with flax. The degradation mechanism could be concluded that predominant bacteria, especially Clostridium sensu stricto, first attached to the surface of textile waste and converted its main compositions cellulose and hemicellulose into acetate as the core intermediate. Then, acetate was utilized by the major methanogen, Methanothrix, through the acetoclastic methanogenesis pathway to produce methane. This study not only enriches the understanding of textile wastes degradation mechanisms during AD and provides very useful data on methane production from commonly available textile wastes but also proposes a promising method for efficiently recycling and utilizing the diverse range of textile wastes to reduce waste pollution and generate clean energy simultaneously.

Impact of different pretreatments on the anaerobic digestion performance of cucumber vine.

Copious amounts of cucumber vine (CV) derived from crop growing and harvesting are casually discarded in the field, posing severely negative impacts on public health and the ecological environment. Treating CV via anaerobic digestion (AD) could represent a promising approach while the recalcitrant lignocellulosic structure restricts its conversion efficiency, thus underscoring the importance of valid pretreatments. This study systematically investigated the effects of nine types of commonly applied chemical pretreatments involved H2SO4, HCl, H3PO4, NaOH, KOH, Ca(OH)2, CaO, H2O2, and alkaline hydrogen peroxide (AHP) pretreatments on methane production of CV. Results showed that alkaline and AHP pretreatments were beneficial to the methane production of CV and obtained the considerable cumulative methane yield and biodegradability of 194.3-241.5 mL·gVS-1 and 47.59-59.15%, respectively, 36.83-70.07% higher than untreated. Analyses of lignocellulosic compositions and structural characterizations revealed that alkaline and AHP pretreatments well destroyed both hemicellulose and lignin, which commendably increased the accessibility of cellulose, facilitating the methane production. The findings of this study provide not only efficient pretreatment methods for the disposal and utilization of CV during AD process but also promising alternatives for enhancing methane production performance of similar vine residues, which would be greatly valuable for industrial applications in the future.

Influences of organic loading, feed-to-inoculum ratio, and different pretreatment strategies on the methane production performance of eggplant stalk.

A large amount of eggplant stalk (ES) is incinerated after harvesting of eggplant every year, which aggravates environmental pollution and waste of resources. Converting ES into methane through anaerobic digestion (AD) technology may be a potential treatment method, considering the low environmental impact and high energy recovery. Firstly, this study explored the effects of organic loading (OL) and feed to inoculum ratio (F/I ratio) on the AD of ES by response surface methodology (RSM). In order to achieve higher AD efficiency, various pretreatments (acid, alkali, alkaline hydrogen peroxide (AHP), microwave, and ultrasound) were introduced and comprehensively assessed with regard to methane production, organic matter destruction, and kinetic parameters. Results showed that OL had a more significant impact on AD process compared to F/I ratio and methane production was enhanced remarkably when the OL and F/I ratio were 35.0 g VS/L and 3.0, respectively. XRD, FTIR, and SEM analyses of pretreated ES showed that alkali and AHP pretreatments performed better in delignification. Under optimal conditions, the ES pretreated with 1.5% AHP (adjusted by KOH) performed the maximum methane production of 262.2 mL/g VS with a biodegradability of 95.0%, which increased by 334.1% compared to untreated ES. This paper not only provides the theoretical data about methane production performance of ES but also gives practical guidance for efficient utilization of similar vegetable stalk biowastes, which is also promising for large-scale industrial applications in the future.

Effects of different microbial pretreatments on the anaerobic digestion of giant grass under anaerobic and microaerobic conditions.

Microbial pretreatment to lignocellulosic biomass for anaerobic digestion (AD) has achieved increased attention; however, the low efficiency and unclear mechanism of oxygen parameter affecting this process performance limit its practical application. In this study, five readily available microbial consortia were developed to analyze the influences of various oxygen concentrations during pretreatment process upon methane conversion efficiency and microbiota within AD of giant grass. Results found that anaerobic pretreatment by liquid or straw composting inoculant, along with microaerobic pretreatment by cow manure at 10 mL/g VS oxygen concentration, obtained 23.1%, 24.4%, and 16.0% higher methane yields (275.3, 279.8, and 265.3 mL/g VS) than corresponding untreated group, respectively. Microbial community analyses showed that microbial responses to oxygen varied significantly with microbial consortium, which consequently caused different AD performances. The findings will enrich theoretical knowledge of microbial pretreatment and provide a technological guidance for efficient utilization of giant grass and other lignocellulosic biomasses.

Zinc finger antiviral protein (ZAP) inhibits small ruminant morbillivirus replication in vitro.

Small ruminant morbillivirus (SRMV) is a highly contagious and economically important viral disease of small domestic and wild ruminants. Difficulty with its stable proliferation in ovis aries-derived cells has led to a relative lag in the study of its natural immunity and pathogenesis. Here we report the antiviral properties of ZAP against SRMV, a single-stranded negative-stranded RNA virus of the genus Morbillivirus. ZAP expression was significantly induced in sheep endometrial epithelial cells following SRMV infection. ZAP inhibited SRMV replication in cells after infection, while its overexpression in Vero-SLAM cells significantly increased their resistance to SRMV replication. The ZAP protein co-localized with SRMV RNA in the cytoplasm and ZAP-responsive elements were mapped to the 5' untranslated region of SRMV nucleocapsid, phosphoprotein, matrix, and fusion. In summary, ZAP confers resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication. Our findings further extend the ranges of viral targets of ZAP and help elucidate the mechanism of SRMV replication.

Hemoglobin subunit beta interacts with the capsid, RdRp and VPg proteins, and antagonizes the replication of rabbit hemorrhagic disease virus.

Rabbit hemorrhagic disease virus (RHDV) causes a highly contagious disease in rabbits that is associated with high mortality. Because of the lack of a suitable cell culture system for RHDV, its pathogenic mechanism and replication remain unclear. This study found that the expression level of host protein rabbit hemoglobin subunit beta (HBB) was significantly downregulated in RHDV-infected cells. To investigate the role of HBB in RHDV replication, small interfering RNAs for HBB and HBB eukaryotic expression plasmids were used to change the expression level of HBB in RK-13 cells and the results showed that the RHDV replication level was negatively correlated with the expression level of HBB. It was also verified that HBB inhibited RHDV replication using constructed HBB stable overexpression cell lines and HBB knockout cell lines. The interaction of HBB with viral capsid protein VP60, replicase RdRp, and VPg protein was confirmed, as was the activation of the expression of interferon γ by HBB. The results of this study indicated that HBB may be an important host protein in host resistance to RHDV infection.

Caprine MAVS Is a RIG-I Interacting Type I Interferon Inducer Downregulated by Peste des Petits Ruminants Virus Infection.

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-ß promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.

pH regulation of the first phase could enhance the energy recovery from two-phase anaerobic digestion of food waste.

The effect of pH regulation in phase I on hydrolysis and acidogenesis rate, metabolites production, microbial community, and the overall energy recovery efficiency during two-phase anaerobic digestion (AD) of food waste (FW) was investigated. pH strongly affected the acidogenesis rate and the yield of the fermentation products. The highest acidogenesis efficiency (60.4%) and total volatile fatty acids (VFA)/ethanol concentration (12.4 g/L) were obtained at pH 8 during phase I. Microbial community analysis revealed that Clostridium IV was enriched at pH 8, relating to the accumulation of butyrate. Also, Clostridium sensu stricto played a crucial role in hydrogen production and was abundant at pH 6, resulting in the highest hydrogen yield (212.2 ml/g VS). In phase II, the highest cumulative methane yield (412.6 ml/g VS) was obtained at pH 8. By considering the hydrogen and methane production stages, the highest energy yield (22.8 kJ/g VS, corresponding to a 76.4% recovery efficiency) was generated at pH 8, which indicates that pH 8 was optimal for energy recovery during two-phase AD of FW. Overall, the results demonstrated the possibility of increasing the energy recovery from FW by regulating the pH in the hydrolysis/acidogenesis phase based on the two-phase AD system. PRACTITIONER POINTS: pH 8 was suitable for hydrolysis, acidogenesis, and methanogenesis. High hydrogen yields were obtained at pH 5-8 (about 200 ml/d). Clostridium sensu stricto might have played a crucial role in hydrogen production. High methane production (about 400 ml/g VS) was obtained at pH 7-9. pH 8 was optimal for energy recovery from FW with an efficiency of 76.4% (22.8 kJ/g VS).

Contrast-enhanced ultrasound evaluation of the prostate before transrectal ultrasound-guided biopsy can improve diagnostic sensitivity: A STARD-compliant article.

To conduct a STARD-compliant validity that the contrast-enhanced ultrasound (CEUS) evaluation of prostate for the improvement of positive rate of biopsy and diagnostic efficiency of prostate carcinoma (PCa).Data of 137 patients with suspected PCa who underwent relevant examinations and treatment were reviewed, and 82 of 137 patients were finally included. The patients consisted of Group 1 (26 patients) and Group 2 (42 patients) according to which they underwent transrectal ultrasound (TRUS) biopsy selected from CEUS evaluation of the prostate and who underwent TRUS-guided biopsy directly. A systematic 12-core biopsy was performed at first, and additional 1 to 2 cores biopsy was made in the suspected target area where CEUS had showed abnormal enhancement. The assumed diagnoses were compared with pathological findings.There were 37 patients with PCa and 31 patients with benign lesions; and 14 patients without biopsy after CEUS did not find PCa emerging in follow-up (18-47 months). The positive rates of biopsy-malignant lesions were 73.1% and 42.8% in Group 1 and Group 2, respectively. The positive rate of biopsy in Group 1 was significantly higher than that in Group 2 (P = .024). The sensitivity and accuracy of TRUS biopsy and a combination of TRUS biopsy after transrectal CEUS for the evaluation of prostate benign and malignant lesion were 60% and 66.7% (P=0.0139), and 94.4% and 88.5% (P=0.0453), respectively.CEUS evaluation of the prostate of PSA-elevated patient before biopsy can help select target patient with high risk of PCa, reduce unnecessary biopsy, increase detection rate of PCa, and improve diagnostic sensitivity and accuracy.

Construction and immunogenicity of novel bivalent virus-like particles bearing VP60 genes of classic RHDV(GI.1) and RHDV2(GI.2).

Rabbit hemorrhagic disease (RHD) is an acute, inflammatory, septic, and devastating infectious disease caused by Rabbit hemorrhagic disease virus (RHDV), which poses a serious threat to the rabbit industry. RHDV2 (GI.2/RHDVb), a recently reported new variant could cause RHD in wild populations, but also RHDV-vaccinated rabbits. For now, both RHDV and RHDV2 are the main causes of RHD. To develop a new subunit vaccine that could protect rabbits against both classic RHDV and RHDV2 infections, we constructed a recombinant baculovirus (Bac-classic RHDV VP60-RHDV2 VP60) containing the VP60 genes of classic RHDV and RHDV2. Both VP60 genes were well expressed simultaneously in Spodoptera frugiperda cells (Sf9) after infection with the recombinant baculovirus. Transmission electron microscopy showed that the recombinant VP60 self-assembled into virus-like particles (VLPs). The antigenicity and immunogenicity of the bivalent VLPs vaccine were examined with animal experiments. Our results demonstrated that both the humoral and cellular immune responses were efficiently induced in rabbits by a subunit vaccine based on the recombinant baculovirus. In addition, all rabbits immunized with the bivalent VLPs vaccine survived after challenged with classic RHDV, and showed no clinical signs of RHD, whereas all the rabbits in the negative control group died from classic RHDV infection and showed typical clinical signs of RHD. In summary, our results indicated that the recombinant baculovirus carrying two VP60 genes is a candidate construct from which to develop a bivalent VLPs vaccine against both classic RHDV and RHDV2 infections.

Effect of organic loading, feed-to-inoculum ratio, and pretreatment on the anaerobic digestion of tobacco stalks.

This work firstly investigated the suitable organic loading (OL) and feed to inoculum (F/I) ratio of three kinds of tobacco stalks (TS116, TS99, and TS85) during anaerobic digestion (AD) via response surface methodology (RSM). The highest experimental methane yield (EMY) of 148.1 mL/g VS was achieved from TS116 at OL of 20.2 g VS/L and F/I ratio of 1.1. To further increase EMY, various pretreatments including alkaline hydrogen peroxide (AHP), NaOH, KOH, Ca(OH)2, HCl, and oxalic acid (H2C2O4) were implemented on TS116. Results showed that AHP was most effective, and the maximal EMY of 350.7 mL/g VS and biodegradability (Bd) of 81.4% were obtained from 7% AHP pretreated TS116, which increased by 105.6% than untreated. XRD, FTIR, and SEM analyses evidenced that the structure of AHP pretreated TS116 was strongly disrupted. This study lays the foundation for applying this waste into AD in future applications.

Steam explosion pretreatment of rice straw to improve structural carbohydrates anaerobic digestibility for biomethanation.

Effectiveness of steam explosion (SE) pretreatment for deconstructing the complex structural carbohydrates (SC) and lignin recalcitrance properties of rice straw (RS) for conjunctive improvement of biofuel yield and waste valorization was evaluated. This work exhibited successful pretreatment of RS at a different pressure (1.2, 1.5, and 1.8 MPa) and retention (3, 6, 9, and 12 min) for enhancement of SC contribution to biomethane production. Regression analysis demonstrated that SE pretreatment efficiency improved at high-temperature and short-retention time for biodegradation of RS. Maximum cumulative methane yield (EMY) achieved 254.8 mL/gvs at 1.2 MPa (3 min) of SE-treated RS with 62.7% of very significant improvement compared with untreated RS (156.6 mL/gvs). Furthermore, solid fraction of xylose, arabinose, cellobiose, glucose, and acid-soluble lignin in SE-treated RS of 1.2 MPa (3 min) were biodegraded by 27.4%, 46.4%, 100%, 48.8%, and 14.1%, respectively, after anaerobic digestion. Therefore, SE pretreatment was an encouraging approach for enhancing SC conversion to biomethane and waste resource to circular economy.

Anaerobic digestion of tobacco stalk: biomethane production performance and kinetic analysis.

Tobacco stalk, a common agricultural waste derived from the harvest of tobacco, caused serious environmental pollution in China. In this study, the performance of biomethane production and characteristics of four varieties of tobacco stalk were investigated for the first time. The results showed that the highest cumulative methane yield of 130.2 mL/g-VS was obtained from Nicotiana tabacum L., Yunyan114, which had lower lignin content than other varieties of tobacco stalk. Moreover, different kinetic models were used to describe the biomethane production process, and it was found that the modified Gompertz model was more suitable to simulate the anaerobic digestion (AD) of tobacco stalk. The findings of this study not only showed a feasible method for minimizing the pollution issues of tobacco stalk waste but also gave fundamental information for future AD application.

A recombinant adenovirus expressing the E protein of duck Tembusu virus induces protective immunity in duck.

Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50=10-2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.

Nucleolin mediates the internalization of rabbit hemorrhagic disease virus through clathrin-dependent endocytosis.

Rabbit hemorrhagic disease virus (RHDV) is an important member of the Caliciviridae family and a highly lethal pathogen in rabbits. Although the cell receptor of RHDV has been identified, the mechanism underlying RHDV internalization remains unknown. In this study, the entry and post-internalization of RHDV into host cells were investigated using several biochemical inhibitors and RNA interference. Our data demonstrate that rabbit nucleolin (NCL) plays a key role in RHDV internalization. Further study revealed that NCL specifically interacts with the RHDV capsid protein (VP60) through its N-terminal residues (aa 285-318), and the exact position of the VP60 protein for the interaction with NCL is located in a highly conserved region (472Asp-Val-Asn474; DVN motif). Following competitive blocking of the interaction between NCL and VP60 with an artificial DVN peptide (RRTGDVNAAAGSTNGTQ), the internalization efficiency of the virus was markedly reduced. Moreover, NCL also interacts with the C-terminal residues of clathrin light chain A, which is an important component in clathrin-dependent endocytosis. In addition, the results of animal experiments also demonstrated that artificial DVN peptides protected most rabbits from RHDV infection. These findings demonstrate that NCL is involved in RHDV internalization through clathrin-dependent endocytosis.