Self-standing bacterial cellulose-reinforced poly(3,4-ethylenedioxythiophene)/poly(4-styrenesulfonate) doped with graphene oxide composite electrodes for high-performance ionic electroactive soft actuators.

Flexible electrode films with good film-forming properties, large deformation ability, high conductivity, and strong charge and discharge capabilities are crucial for ionic electroactive polymer soft actuators. However, there are still challenges in preparing high-quality electrode films that can combine well with the intermediate polyelectrolyte to form high-performance soft actuators. Herein, we propose an advanced sandwich ionic electroactive actuator utilizing self-standing bacterial cellulose (BC) reinforced poly(3,4-ethylenedioxythiophene)/poly(4-styrenesulfonate) (PP) doped with graphene oxide (GO) conductive composite electrodes and a Nafion ion-exchange membrane via a hot-pressing method. The prepared BC-PP-GO electrodes have good film-forming properties with a Young's modulus of 1360 MPa and a high conductivity of 150 S cm-1. The hot-pressed BC-PP-GO/Nafion ionic actuator exhibited a large bending displacement of 6.2 mm (1 V, 0.1 Hz) with a long-term actuation stability up to 95% over 360 cycles without degradation. Furthermore, we introduced the actuator's potential applications including bionic grippers, flies, and fish, providing more opportunities for the development of next-generation micromanipulators and biomimetic microrobots in cm-scale space.

Classification of genotypes based on the VP1 gene of feline calicivirus and study of cross-protection between different genotypes.

Feline calicivirus (FCV) causes upper respiratory tract diseases and even death in cats, thereby acting as a great threat to feline animals. Currently, FCV prevention is mainly achieved through vaccination, but the effectiveness of vaccination is limited. In this study, 105 FCV strain VP1 sequences with clear backgrounds were downloaded from the NCBI and subjected to a maximum likelihood method for systematic evolutionary analysis. Based on the genetic analysis results, FCV-positive sera were prepared using SPF mice and Chinese field cats as target animals, followed by a cross-neutralization assay conducted on the different genotype strains and in vivo challenge tests were carried out to further verify with the strain with best cross-protection effect. The results revealed that FCV was mainly divided into two genotypes: GI and GII. The GI genotype strains are prevalent worldwide, but all GII genotype strains were isolated from Asia, indicating a clear geographical feature. This may form resistance to FCV prevention in Asia. The in vitro neutralization assay conducted using murine serum demonstrated that the cross-protection effect varied among strains. A strain with broad-spectrum neutralization properties, DL39, was screened. This strain could produce neutralizing titers (10 × 23.08-10 × 20.25) against all strains used in this study. The antibody titers against the GI strains were 10 × 23.08-10 × 20.5 and those against the GII strains were 10 × 20.75-10 × 20.25. Preliminary evidence suggested that the antibody titer of the DL39 strain against GI was higher than that against GII. Subsequent cross-neutralization assays with cat serum prepared with the DL39 strain and each strain simultaneously yielded results similar to those described above. In vivo challenge tests revealed that the DL39 strain-immunized cats outperformed the positive controls in all measures. The results of several trials demonstrated that strain DL39 can potentially be used as a vaccine strain. The study attempted to combine the genetic diversity and phylogenetic analysis of FCV with the discovery of potential vaccines, which is crucial for developing highly effective FCV vaccines.

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.

RPS5 interacts with the rabbit hemorrhagic disease virus 3' extremities region and plays a role in virus replication.

Rabbit hemorrhagic disease virus (RHDV), a member of Caliciviridae family, causes a highly contagious disease in rabbits. The RHDV replication mechanism is poorly understood due to the lack of a suitable culture system in vitro. This study identified RHDV 5' and 3' extremities (Ex) RNA binding proteins from the rabbit kidney cell line RK-13 based on a pull-down assay by applying a tRNA scaffold streptavidin aptamer. Using mass spectrometry (MS), several host proteins were discovered which interact with RHDV 5' and 3' Ex RNA. The ribosomal protein S5 (RPS5) was shown to interact with RHDV 3' Ex RNA directly by RNA-pulldown and confocal microscopy. To further investigate the role of RPS5 in RHDV replication, small interfering RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5. It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5 knockdown cell lines. In summary, it has been identified that RPS5 interacted with the RHDV 3' Ex RNA region and played a role in virus replication. These results will help to understand the mechanism of RHDV replication.

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.

Nucleolin (NCL) inhibits the growth of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants that is caused by peste des petits ruminants virus (PPRV). To date, the molecular mechanism of PPRV infection is still unclear. It is well known that host proteins might be involved in the pathogenesis process for many viruses. In this study, we first proved that nucleolin (NCL), a highly conserved host factor, interacts with the core domain of PPRV N protein through its C terminus and co-locates with the N protein in the nucleus of cells. To investigate the role of NCL in PPRV infection, the expression level of NCL was inhibited with small interfering RNAs of NCL, and the results showed that PPRV growth was improved. However, the proliferation of PPRV was inhibited when the expression level of NCL was improved. Further analysis indicated that the inhibitory effect of NCL on the PPRV was caused by stimulating the interferon (IFN) pathways in host cells. In summary, our results will help us to understand the mechanism of PPRV infection.

Bioinformatics analysis of capsid protein of different subtypes rabbit hemorrhagic disease virus.

BACKGROUND: Rabbit Hemorrhagic Disease Virus (RHDV) belongs to the Caliciviridae family, is a highly lethal pathogen to rabbits. Increasing numbers of studies have demonstrated the existence of antigenic variation in RHDV, leading to the emergence of a new RHDV isolate (RHDVb). However, the underlying factors determining the emergence of the new RHDV and its unpredictable epidemiology remain unclear. To investigate these issues, we selected more than 184 partial and/or complete genome sequences of RHDV from GenBank and analyzed their phylogenetic relationships, divergence, and predicted protein modification sites. RESULTS: Phylogenetic analysis showed that classic RHDV isolates, RHDVa, and RHDVb formed different clades. It's interesting to note that RHDVa being more closely related to classic RHDV than RHDVb, while RHDVb had a closer genetic relationship to Rabbit Calicivirus (RCV) than to classic RHDV isolates. Moreover, divergence analysis suggested that the accumulation of amino acid (aa) changes might be a consequence of adaptive diversification of capsid protein (VP60) during the division between classical RHDV, RHDVa, RHDVb, and RCV. Notably, the prediction of N-glycosylation sites suggested that RHDVb subtypes had two unique N-glycosylation sites (aa 301, 362) but lacked three other N-glycosylation sites (aa 45, 308, 474) displayed in classic RHDV and RHDVa VP60 implying this divergence of N-glycosylation sites in RHDV might affect viral virulence. Analysis of phosphorylation sites also indicated that some phosphorylation sites in RHDVa and RHDVb differed from those in classic RHDV, potentially related to antigenic variation in RHDV. CONCLUSION: The genetic relationship between RHDVb and RCV was closer than classic RHDV isolates. Moreover, compared to RHDV and RHDVa, RHDVb had two unique N-glycosylation sites but lacked three sites, which might affect the virulence of RHDV. These results may provide new clues for further investigations of the origin of new types of RHDV and the mechanisms of genetic variation in RHDV.

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.