A Natural Hydrogel with Prohealing Properties Enhances Tendon Regeneration.

Tendon regeneration and reduction of peritendinous adhesion remain major clinical challenges. This study addresses these challenges by adopting a unique hydrogel derived from the skin secretion of Andrias davidianus (SSAD) and taking advantage of its biological effects, adhesiveness, and controllable microstructures. The SSAD-derived hydrogel contains many cytokines, which could promote tendon healing. In vitro, leach liquid of SSAD powder could promote tendon stem/progenitor cells migration. In vivo, the SSAD-derived hydrogel featuring double layers possesses strong adhesiveness and could reconnect ruptured Achilles tendons of Sprague-Dawley rats without suturing. The intimal SSAD-derived hydrogel, with a pore size of 241.7 ± 21.0 µm, forms the first layer of the hydrogel to promote tendon healing, and the outer layer SSAD-derived hydrogel, with a pore size of 3.3 ± 1.4 µm, reducing peritendinous adhesion by serving as a dense barrier. Additionally, the SSAD-derived hydrogel exhibits antioxidant and antibacterial characteristics, which further contribute to the reduction of peritendinous adhesion. In vivo studies suggest that the SSAD-derived hydrogel reduces peritendinous adhesion, increases collagen fiber deposition, promotes cell proliferation, and improves the biomechanical properties of the regenerated tendons, indicating better functional restoration. The SSAD-derived bilayer hydrogel may be a feasible biomaterial for tendon repair in the future.

A Bioinspired Hemostatic Powder Derived from the Skin Secretion of Andrias davidianus for Rapid Hemostasis and Intraoral Wound Healing.

High-performance hemostasis has become increasingly essential in treating various traumas. However, available topical hemostats still have various drawbacks and side-effects. Herein, hemostatic powders derived from the skin secretion of Andrias davidianus (SSAD) with controllable particle size are prepared using feasible frozen-ball milling following lyophilization for hemorrhage-control. Scanning electron microscopy, rheometry, and Brunauer-Emmett-Teller test are used to characterize the coagulation-promoting surface topography, rheological properties, and porous structure of the SSAD particles. The blood-coagulation assays showed that the SSAD powders can induce blood-absorption in a particle size-dependent manner. Particle sizes of the SSAD powders larger than 200 µm and smaller than 800 µm greatly affect the blood-clotting rate. Associated with the thromboelastography (TEG) and amino acid/protein composition analyses, the accessibility and diffusion of blood are mainly dependent on the wettability, adhesivity, and clotting factors of the SSAD particles. Rapid hemostasis in vivo further involves three hemorrhage models (liver, femoral artery, and tail) as well as an oral wound model, which suggest favorable hemostatic and simultaneous regenerative effects of the SSAD hemostatic powder. Considering its degradability and good biocompatibility, SSAD can be an optimal candidate for a new class of inexpensive, natural, and promising hemostatic and wound-dressing agent.

Newcastle disease virus V protein inhibits apoptosis in DF-1 cells by downregulating TXNL1.

Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.

Dynamic distribution and tissue tropism of avian encephalomyelitis virus isolate XY/Q-1410 in experimentally infected Korean quail.

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). However, to date, the dynamic distribution of AEV in quails has not been well described. Quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) assays were used to investigate the dynamic distribution and tissue tropism of AEV in experimentally infected Korean quail. AEV was detected in the cerebrum, cerebellum, proventriculus, intestine, liver, pancreas, spleen, bursa, lung and kidney as early as 3 days post-infection (dpi). The viral loads in the proventriculus, intestine, spleen and bursa were relatively higher than in other tissues. According to the qPCR results, AEV XY/Q-1410 infection lasted for at least 60 days in infected Korean quail. Immunohistochemistry-positive staining signals of AEV antigen were analysed by Image-Pro Plus software. A positive correlation between qPCR and IHC results was identified in most tissues. Our results provide an insight into the dynamic distribution of AEV in various tissues after infection. The distinct dynamic distribution of the viral genome in Korean quail in the early and late stages of infection suggests that AEV replication is affected by antibody levels and the maturity of the immune system of the host.

Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus.

BACKGROUND: Newcastle disease (ND), which is caused by the Newcastle disease virus (NDV), is one of the most important avian diseases in poultry. Since its discovery in 1926, ND has caused great economic losses to the world poultry industry and remains a threat to chickens and wild birds. Although a stringent vaccination policy is widely adopted to control ND, ND outbreaks still occur, and virulent NDV is sporadically isolated from chickens and wild birds. To study the pathogenesis of ND and provide tools to prevent its prevalence, novel antibody fragments should be developed. The variable domains of the heavy chain of the heavy-chain antibodies (VHH) are the smallest naturally occurring antibodies derived from camelid heavy-chain antibodies. The comparatively small size, high affinity, high solubility, low immunogenicity and ability to bind epitopes inaccessible to conventional antibodies of VHH make them ideal candidates for a considerable number of therapeutic and biotechnological applications. However, an anti-NDV VHH has not been reported to date. RESULTS: In this study, a VHH yeast two-hybrid library was constructed from NDV vaccine immunized C. bactrianus, and seven VHH fragments to the haemagglutinin-neuraminidase (HN) protein of NDV were successfully screened and characterized for the first time. These selected VHH clones were all expressed as soluble protein in E. coli. ELISA, dot blot, immunocytochemistry and pull down results showed that the screened VHHs could interact with NDV virion, among which five had neutralizing activity. In addition, the seven VHHs could inhibit the haemagglutination activity of different NDV strains. CONCLUSIONS: We constructed an NDV-immunized VHH yeast two-hybrid library and screened and characterized seven VHHs targeting NDV HN protein for the first time. The seven VHHs may have great potential for NDV diagnosis, pathogenesis and therapeutics.

Pathogenic Providencia alcalifaciens strain that causes fatal hemorrhagic pneumonia in piglets.

Investigation of a serious pig disease with high mortality and typical lung lesions yielded a bacterial isolate identified as Providencia alcalifaciens based on the 16S ribosomal DNA sequence analysis. The pathogenicity of this bacterial isolate was confirmed in piglets and mice. The bacterial strain caused the typical illness in piglets, which suffered serious dyspnea and hemorrhagic pneumonia. The drug resistance spectrum of the bacterium was also determined. The results indicated that the isolate is resistant to 12 antibiotics and intermediately resistant to 10 antibiotics out of the 34 antibiotics tested. The current study is the first to report a serious lung disease in piglets caused by a multidrug resistant P. alcalifaciens isolate, which should be given more attention during surveillance and diagnostics.

A Nature-Derived, Hetero-Structured, Pro-Healing Bioadhesive Patch for High-Performance Sealing of Wet Tissues.

Tissue adhesives are promising alternatives to sutures and staples to achieve wound closure and hemostasis. However, they often do not work well on tissues that are soaked in blood or other biological fluids, and organs that are typically exposed to a variety of harsh environments such as different pH values, nonhomogeneous distortions, continuous expansions and contractions, or high pressures. In this study, a nature-derived multilayered hetero-bioadhesive patch (skin secretion of Andrias davidianus (SSAD)-Patch) based on hydrophilic/hydrophobic pro-healing bioadhesives derived from the SSAD is developed, which is designed to form pressure-triggered strong adhesion with wet tissues. The SSAD-Patch is successfully applied for the sealing and healing of tissue defects within 10 s in diverse extreme injury scenarios in vivo including rat stomach perforation, small intestine perforation, fetal membrane defect, porcine carotid artery incision, and lung lobe laceration. The findings reveal a promising new type of self-adhesive regenerative SSAD-Patch, which is potentially adaptable to broad applications (under different pH values and air or liquid pressures) in sutureless wound sealing and healing.

Antigenic variation in hemagglutinin-neuraminidase of Newcastle disease virus isolated from Tibet, China.

Vaccines are widely used to prevent Newcastle disease virus (NDV). Under the pressure of immunization, NDVs with mutations among epitopes of F and HN protein were isolated, which indicates that the efficiency of vaccine may decrease in terms of preventing emerged NDV. However, the lack of evidences to support whether these mutations contribute to antigenic mutation and immune escape in NDV leading to the controversy that the matched vaccine is more effective than the mismatched vaccine. In this study, a genotype VII velogenic NDV strain (C22) was isolated from a vaccinated farm in Tibet, China. We found that this strain was close to NDV from east China, but it had a specific mutation (K138R) in one epitope (131DYIGGIGKE139) of HN protein. This mutation might change the interaction between amino acids in stalk-head link region of HN protein and then induce the specific antibody to worse recognize the C22 strain, but it did not alter viral virulence and growth ability. Then, the C22 strain was attenuated via modification of the F protein cleavage site to generate a matched vaccine. Comparing to a mismatched vaccine (LaSota), this matched vaccine showed advantages in inhibiting viral shedding and tissue damage. However, both vaccines induced chicken to generate similar level of neutralizing antibodies against C22, C22mut (R138K) and LaSota. These results suggest that the epitope mutation is insufficient to help NDV escaping neutralizing antibodies of vaccinated chicken, supporting that the merits of NDV matched vaccine are not totally related to humoral immunity.

Newcastle disease virus forms inclusion bodies with features of liquid-liquid phase separation.

Formation of inclusion bodies (IBs) is a hallmark of infections with negative-strand RNA viruses. Although the Newcastle disease virus (NDV) IBs had been observed in the 1950s, the characteristics of NDV IBs remained largely unknown. Here, we show that NDV infection triggers the formation of IBs that contain newly synthesized viral RNA. The structures of NDV IBs, observed by electron microscopy, were not membrane-bound. Fluorescence recovery after photobleaching a region of NDV IBs occurred rapidly, and IBs were dissolved by 1,6-hexanediol treatment, demonstrating they exhibited properties consistent with liquid-liquid phase separation (LLPS). We find the nucleoprotein (NP) and phosphoprotein (P) are sufficient to generate IB-like puncta, with the N arm domain and N core region of NP and the C terminus of P playing important roles in this process. In summary, our findings suggest that NDV forms IBs containing viral RNA, and provide insights into the formation of NDV IBs.

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains.

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.

The canonical Wnt/ß-catenin signaling pathway facilitates pseudorabies virus proliferation and enhances virus-induced autophagy.

Pseudorabies virus (PRV) is a swine herpesvirus with a broad host range that causes significant economic losses worldwide. The Wnt/ß-catenin signaling pathway is reportedly involved in multiple viruses' proliferation. In this study, we demonstrated that PRV infection significantly activated the Wnt/ß-catenin signaling and promoted the nuclear translocation of ß-catenin. Applying specific chemical inhibitors (FH535 and iCRT14) caused a remarkable decrease in PRV titers in various cell lines. Knockdown of ß-catenin by siRNA also reduced the proliferation of PRV. On the contrary, treatment with lithium chloride (LiCl), an inhibitor of GSK3ß, stimulated the Wnt/ß-catenin signaling pathway and enhanced the PRV proliferation. Similarly, overexpression of ß-catenin promoted PRV proliferation and reversed the antiviral effect of FH535. Moreover, LiCl promoted PRV-induced autophagy, whereas FH535 and iCRT14 showed converse effects. These findings suggest that PRV infection stimulates the canonical Wnt/ß-catenin signaling pathway, facilitating PRV proliferation and regulating virus-induced autophagy. These data also provide potential targets for developing antiviral agents against PRV.

Evasion of Host Antiviral Innate Immunity by Paramyxovirus Accessory Proteins.

For efficient replication, viruses have developed multiple strategies to evade host antiviral innate immunity. Paramyxoviruses are a large family of enveloped RNA viruses that comprises diverse human and animal pathogens which jeopardize global public health and the economy. The accessory proteins expressed from the P gene by RNA editing or overlapping open reading frames (ORFs) are major viral immune evasion factors antagonizing type I interferon (IFN-I) production and other antiviral innate immune responses. However, the antagonistic mechanisms against antiviral innate immunity by accessory proteins differ among viruses. Here, we summarize the current understandings of immune evasion mechanisms by paramyxovirus accessory proteins, specifically how accessory proteins directly or indirectly target the adaptors in the antiviral innate immune signaling pathway to facilitate virus replication. Additionally, some cellular responses, which are also involved in viral replication, will be briefly summarized.

Comparison of Antimicrobial Resistance, Virulence Genes, Phylogroups, and Biofilm Formation of Escherichia coli Isolated From Intensive Farming and Free-Range Sheep.

Pathogenic E. coli are among the most frequently isolated bacterial pathogens on large-scale sheep farms in China. Antibiotic use in wool sheep production is a risk factor for promoting the emergence of resistant E. coli. To reveal the differences of E. coli populations in sheep from different farming systems the antimicrobial resistance, virulence genes, biofilm formation, and phylogroups of 500 E. coli isolates obtained between September 2019 and December 2020 in northwest China from diarrheic infections of intensive farming and free-range sheep were analyzed. The antimicrobial susceptibility test for 12 classes of antimicrobial agents was determined using the broth microdilution susceptibility method, and PCR was used to detect the differences in virulence genes and phylogroups. Additionally, biofilm formation was determined using microtiter plate and slide agglutination methods. Among the 500 E. coli isolates, the majority of the isolates were multidrug resistant (75.4%) and carried at least one virulence gene (94.8%). We observed that 412 (82.4%), 360 (72.0%), and 266 (53.2%) are found to be resistant to sulfisoxazole, florfenicol, and tetracyclines, respectively. Resistance was also observed to mequindox (46.8%), ampicillin (43.6%), spectinomycin (38.6%), enrofloxacin (34.2%), ceftiofur (21.0%), gentamycin (20.4%), ceftazidime (17.8%), and polymyxin B (7.8%) but no resistance was found to meropenem. These results showed that strains from free-range subjects had fewer antibiotic resistance strains rather than sheep that were intensively farmed (P < 0.05). We observed fifteen virulence genes, of which etrA (n = 401, 80.2%) is the most common. In addition, EAEC (86.4%) is dominant among free-range sheep and EHEC (80.1%) is dominant among intensive farming. Among all virulence genes, the strongest correlation was found between etrA and papC gene (P < 0.001, OR = 455.68). Similarly, the strongest correlation was also found between eltA and sulfisoxazole (P < 0.001, OR = 877). Furthermore, the majority of the E. coli isolates belonged to phylogroup B1 (50.6%), followed by phylogroup C (20.6%), A (7.4%), E (7.4%), D (5.8%), B2 (1.6%), and F (1%). Interestingly, phylogroup B2 and D were all distributed in intensive farms. In addition, 33 (6.6%), 373 (74.6%), and 94 (18.8%) showed moderate, weak, and no connection biofilm formation ability, respectively. These data uncovered that wool sheep serve as a reservoir of pathogenic E. coli harboring multiple resistance phenotypes and virulence genes. The overlapping virulence-associated traits between IPEC and ExPEC indicated the zoonotic potential and safety threats of sheep food products. It is urgent to improve the proper use of antimicrobials in China as well as other countries.

Characterization of Five Escherichia coli Isolates Co-expressing ESBL and MCR-1 Resistance Mechanisms From Different Origins in China.

Present study characterized five Escherichia coli co-expressing ESBL and MCR-1 recovered from food, food-producing animals, and companion animals in China. Antimicrobial susceptibility tests, conjugation experiments, and plasmid typing were performed. Whole genome sequencing (WGS) was undertaken for all five isolates using either PacBio RS II or Illumina HiSeq 2500 platforms. The cefotaxime and colistin resistance encoded by bla CTX-M and mcr-1 genes, respectively, was transferable by conjugation either together or separately for all five strains. Interestingly, the ESBL and mcr-1 genes could be co-selected by cefotaxime, while the colistin only selected the mcr-1-carrying plasmids during the conjugation experiments. Five E. coli sequence types (ST88, ST93, ST602, ST162, and ST457) were detected. Although diverse plasmid profiles were identified, IncI2, IncFIB, and IncFII plasmid types were predominant. These five clonally unrelated isolates harbored the mcr-1 gene located on similar plasmid backbones, which showed high nucleotide similarity to plasmid pHNSHP45. The mcr-1 gene can be co-transmitted with bla CTX-M genes through IncI2 plasmids with or without ISApl1 in our study. Characterization of these co-existence ESBL and mcr-1 isolates extends our understanding on the dissemination of these resistance markers among bacteria of diverse origins.

Ivermectin inhibits DNA polymerase UL42 of pseudorabies virus entrance into the nucleus and proliferation of the virus in vitro and vivo.

Pseudorabies virus (PRV) is an important viral pathogen of pigs that causes huge losses in pig herds worldwide. Ivermectin is a specific inhibitor of importin-α/ß-dependent nuclear transport and shows antiviral potential against several RNA viruses by blocking the nuclear localization of viral proteins. Since the replication of DNA viruses is in the nucleus, ivermectin may be functional against DNA virus infections if the DNA polymerase or other important viral proteins enter the nucleus via the importin-α/ß-mediated pathway. Here, we determined whether ivermectin suppresses PRV replication in hamster kidney BHK-21 cells and investigated the effect of ivermectin on the subcellular localization of the PRV UL42 protein, the accessory subunit of PRV DNA polymerase. Also, an in vivo anti-PRV assay was conducted in mice. Our data demonstrate that ivermectin treatment inhibits PRV infection in cells in a dose-dependent manner. Treatment of PRV-infected cells with ivermectin significantly suppressed viral DNA synthesis and progeny virus production. Ivermectin disrupted the nuclear localization of UL42 by targeting the nuclear localization signal of the protein in transfected cells. Ivermectin treatment increased the survival rates of mice infected with PRV and relieved infection as indicated by lower clinical scores and fewer gross lesions in the brain. Together, our results suggest that ivermectin may be a therapeutic or preventative agent against PRV infection.

Immune protection efficacy of FAdV-4 surface proteins fiber-1, fiber-2, hexon and penton base.

The spread of hydropericardium syndrome has recently become serious in China since 2015. There is, therefore, an urgent need for new, safe and effective vaccines that prevent the disease. Here, the immune protection induced by Escherichia coli-expressed capsid proteins of fowl adenovirus serotype 4, including fiber-1, fiber-2, penton base and hexon (loop-1 region) were compared in chickens at different inoculation amounts. According to challenge mortalities and tissue gross/micro lesion results, fiber-2 induced the best protection, followed by fiber-1 and hexon. Fiber-1 and fiber-2 provided complete protection against 105.5 TCID50 viral load challenge with 100 or 50µg doses per chicken, respectively. Penton could induce effective protection only at the high dosage of 200µg per chicken. The immunoprotective characteristics of these FAdV-4 capsid proteins may prove useful for developing subunit vaccines to control hydropericardium syndrome.

Re-evaluation the immune efficacy of Newcastle disease virus vaccine in commercial laying chickens.

Newcastle disease virus (NDV) infection causes serious problems in laying chickens, like reducing egg production, increasing rate of abnormal eggs in spite of strict vaccination in layer farms program. A new evaluation system is needed to show complete protection of the immunization in laying chickens based on the egg-laying performance, rather than clinical signs of the disease. In this study, laying chickens with different anti-NDV HI (hemagglutination-inhibition) antibody titer after vaccination were divided into different groups. These chickens were then challenged with field isolated highly virulent NDV strains. Results showed that the chickens in low HI titers group (5log2 to 8log2) and medium HI titers group (9log2 to 11log2) had atypical symptoms, produced abnormal eggs, and shed virus. Whereas, with HI titers≥12log2, the chickens were completely protected, and did not show symptoms, or produce abnormal eggs or shed virus. Morbidity, positive viral shedding rate and abnormal egg-rate decreased with increase in pre-challenge HI antibody titer. Our result suggested that 12log2 is the threshold of the HI antibody in providing complete protection to laying chickens under field condition, and protective efficacy is correlated with HI antibody titer. This study provides a valuable reference for the vaccination and control of ND in poultry.

Antigenic characteristics of glycosylated protein 3 of highly pathogenic porcine reproductive and respiratory syndrome virus.

Highly pathogenic (HP)-porcine reproductive and respiratory syndrome virus (PRRSV) emerged in 2006 and has now become a global threat to pig farms. Despite extensive characterization of HP-PRRSV proteins by direct analysis and comparison with typical PRRSV, immune recognition remain poorly understood. Glycosylated protein 3 (GP3) has an important function in inducing protective immune response. To analyze the antigenic character of HP-PRRSV GP3, a total of 217 peptides were printed on a chip and used to react with HP-PRRSV specific serum. The reactions of these peptides to HP-PRRSV specific pig serum were scanned and quantified using the software PepSlide Analyzer by fluorescence intensity. The intensity plots showed various reactions in different parts of GP3. The highest reaction intensity value reached 29,184.5 with the peptide sequence of CSENDHDELGFMVPP. Conversely, 88 peptides showed no reaction with 0 florescence intensity. A further analysis based on the result of the peptide microarray revealed an antigen reaction active region (AR) from Y(51) to S(106) in GP3. The AR had four parts of variation that may be a significant mutation of the typical PRRSV to HP-PRRSV. Acquired data may be useful for understanding HP-PRRSV variation and its GP3 immune recognition.

Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

Oral administration of attenuated Salmonella typhimurium containing a DNA vaccine against rabbit haemorrhagic disease.

The use of attenuated Salmonella typhimurium as a bactofection vehicle for the oral delivery of a DNA vaccine against rabbit haemorrhagic disease virus (RHDV) was investigated. The DNA vaccine plasmid pcDNA3.1-VP60, which encodes the viral capsid protein VP60, was transformed into the attenuated S. typhimurium strain SL7207. The resulting recombinant bacteria, named as SL/pcDNA3.1-VP60, were orally used to immunise rabbits. The successful delivery of the DNA plasmid was confirmed by the detected VP60 transcription in the rabbit intestines through the reverse transcription polymerase chain reaction. In addition, the RHDV-specific humoral and cell-mediated immune response that was induced by SL/pcDNA3.1-VP60 was detected by the enzyme-linked immunosorbent assay as well as the assays for T lymphocyte proliferation and cytokines secretion. The significant protection of immunised rabbits against the RHDV strain XA/China/2010 at 42 d post-immunisation was demonstrated. This study is the first report about the efficient usage of attenuated Salmonella as a live vector for the oral delivery of a DNA vaccine against RHDV.