Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Strain localisation and failure at twin-boundary complexions in nickel-based superalloys.

Twin boundaries (TBs) in Ni-based superalloys are vulnerable sites for failure in demanding environments, and a current lack of mechanistic understanding hampers the reliable lifetime prediction and performance optimisation of these alloys. Here we report the discovery of an unexpected γ″ precipitation mechanism at TBs that takes the responsibility for alloy failure in demanding environments. Using multiscale microstructural and mechanical characterisations (from millimetre down to atomic level) and DFT calculations, we demonstrate that abnormal γ″ precipitation along TBs accounts for the premature dislocation activities and pronounced strain localisation associated with TBs during mechanical loading, which serves as a precursor for crack initiation. We clarify the physical origin of the TBs-related cracking at the atomic level of γ″-strengthened Ni-based superalloys in a hydrogen containing environment, and provide practical methods to mitigate the adverse effect of TBs on the performance of these alloys.

Env diversity-dependent protection of the attenuated equine infectious anaemia virus vaccine.

Lentiviruses harbour high genetic variability for efficient evasion from host immunity. An attenuated equine infectious anaemia (EIA) vaccine was developed decades ago in China and presented remarkably robust protection against EIA. The vaccine was recently proven to have high genomic diversity, particular in env. However, how and to what extent the high env diversity relates to immune protection remains unclear. In this study, we compared immune protections and responses of three groups of horses stimulated by the high-diversity vaccine EIAV_HD, a single molecular clone of the vaccine EIAV_LD with low env diversity, as well as a constructed vaccine strain EIAV_MD with moderate env diversity. The disparity of virus-host interactions between three env diversity-varied groups (5 horses in each group) was evaluated using clinical manifestation, pathological scores, and env-specific antibody. We found the highest titres of env antibodies (Abs) or neutralizing Abs (nAbs) in the EIAV_HD group, followed by the EIAV_MD group, and the lowest titres in the EIAV_LD group (P<0.05). The occurrence of disease/death was different between EIAV_HD group (1/0), EIAV_MD (2/2), and EIAV_LD group (4/2). A similar env diversity-related linear relationship was observed in the clinical manifestations and pathological changes. This diversity-dependent disparity in changes between the three groups was more distinct after immunosuppression, suggesting that env diversity plays an important role in protection under low host immunocompetence. In summary, inoculation with vaccines with higher genetic diversity could present broader and more efficient protection. Our findings strongly suggest that an abundance of Env antigens are required for efficient protection against lentiviruses.

The N-glycosylation of Equine Tetherin Affects Antiviral Activity by Regulating Its Subcellular Localization.

Tetherin is an interferon-inducible type II transmembrane glycoprotein which inhibits the release of viruses, including retroviruses, through a "physical tethering" model. However, the role that the glycosylation of tetherin plays in its antiviral activity remains controversial. In this study, we found that mutation of N-glycosylation sites resulted in an attenuation of the antiviral activity of equine tetherin (eqTHN), as well as a reduction in the expression of eqTHN at the plasma membrane (PM). In addition, eqTHN N-glycosylation mutants colocalize obviously with ER, CD63, LAMP1 and endosomes, while WT eqTHN do not. Furthermore, we also found that N-glycosylation impacts the transport of eqTHN in the cell not by affecting the endocytosis, but rather by influencing the anterograde trafficking of the protein. These results suggest that the N-glycosylation of eqTHN is important for the antiviral activity of the protein through regulating its normal subcellular localization. This finding will enhance our understanding of the function of this important restriction factor.

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.

Isolation and molecular characterization of a virulent systemic feline calicivirus isolated in China.

In this study, a virulent systemic (VS) feline calicivirus (FCV) strain, SH, was isolated from a household cat with severe systemic clinical signs, and its full-length genome was determined and analyzed. Through immunofluorescence assays (IFA) and western blotting assays, we found that FCV SH strain, like other isolates, can stably proliferate in Crandell feline kidney (CRFK) cells. Moreover, the typical morphology of FCV particles, with a diameter of about 35 nm, was observed using electron microscopy. The full-length genome of FCV strain SH was sequenced and determined to be 7704 nucleotides (nt) in length with a 5'-terminal untranslated region (UTR) of 19 nt and a 3'-terminal UTR of 67 nt. Three open reading frames (ORF1, ORF2, and ORF 3) were found within the genome, coding for a polypeptide, a capsid precursor (VP1) and a minor structural protein (VP2), respectively. Amino acid sequence comparison revealed diversity (from 82.2% to 88.5% homology) between the VP1 protein sequences of the SH/14 isolate and those of 33 reference isolates from different regions. Phylogenetic analyses using alignments of VP1 protein sequences showed that the SH/14 isolate shares the highest sequence homology with the reported VS-FCV George strain (88.5%), and is located in the same clade as other reported VS-FCV isolates, indicating that the FCV SH/14 strain is a VS-FCV isolate. However, the SH/14 strain does not belong to the same lineage as most other Chinese FCV isolates, suggesting that, in China, a very large geographical entity, the virulent systemic FCV might has emerged.

A Facile Strategy to Prepare an Enzyme-Responsive Mussel Mimetic Coating for Drug Delivery Based on Mesoporous Silica Nanoparticles.

Surface functional mesoporous silica nanoparticles (MSNs) have been widely used as promosing materials for drug delivery. Herein, we reported a facile strategy to construct MSNs coated by enzyme-resposive polylysine-dopamine (PLDA) films through self-polymerization of dopamine derivative lysine-dopamine, in which the drug could be loaded and delivered efficiently. In detail, RhB or DOX was used as a drug model and loaded in functional MSNs via a one-pot procedure among MSNs, drug, and lysine-dopamine (LDA) under basic conditions. Owing to the fact that the peptide bonds between lysine and dopamine can be cleaved under triggering by pepsin, the resulting RhB/DOX@PLDA-MSNs exibit enzyme-responsive characterization. After the DOX@PLDA-MSNs enter into the cancer cells, the drug can be released effectively through degradation of peptide bonds under the influence of enzyme in cancer cells, which shows marked anticancer activity in vitro. This facile strategy may provide a new platform to construct enzyme-responsive controlled drug delivery systems.

New serotypes of porcine teschovirus identified in Shanghai, China.

Teschoviruses are widely endemic and commonly found in pig fecal samples. In this study, we collected fecal specimens from various pig herds and genotyped them based on the VP1 gene. Of 322 samples, 276 were positive, giving a PTV infectivity rate of 85.7 %. PTV4 was the most common serotype found in Shanghai, followed by PTV8 and PTV10. Interestingly, Some Shanghai strains belonging to a new PTV serotype were also isolated. In phylogenetic analysis, PTV SH8 did not correspond to any known serotype. PTV4 and PTV6 showed similar levels of sequence identity to PTV SH8. These data suggest that PTV SH8 is a new serotype, distinct from the new serotype PTV wild boar/WB2C-TV/2011/HUN, which clusters with PTV SH2, SH10, and SH25.

HEV-ORF3 Encoding Phosphoprotein Interacts With Hepsin.

BACKGROUND: Hepatitis E virus (HEV) is a major causative agent of acute clinical hepatitis in adults through much of Asia, the Middle East and Africa. Open reading frame 3 (ORF3) encodes around 120 amino acids of phosphorylation protein that associates with the cytoskeleton, while its precise biological function is still unknown. OBJECTIVES: In order to understand the function of ORF3 protein (pORF3) in depth, HEV ORF3 interacting proteins were screened in human hepatocytes cDNA library using two-hybrid system techniques and further verification of the interactions were carried out through co-immunoprecipitation (Co-IP). MATERIALS AND METHODS: The Cyto-Trap two-hybrid system technology, a classical method for analyzing protein interactions, was used to screen the pORF3 interacting proteins from human hepatocytes cDNA library. RESULTS: Through the Cyto-Trap two-hybrid system, eight proteins interacting with pORF3 were winnowed. The Co-IP results confirmed that hepsin which is reported to function as the inhibitor of several tumors reacted with pORF3. CONCLUSIONS: Out of eight screened proteins interacting with pORF3, hepsin was confirmed to have specific interactions with pORF3.

Depolymerization of cytokeratin intermediate filaments facilitates intracellular infection of HeLa cells by Bartonella henselae.

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.