A novel CpG ODN compound adjuvant enhances immune response to spike subunit vaccines of porcine epidemic diarrhea virus.

CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs have been extensively utilized in human vaccines. In our study, we evaluated five B-type CpG ODNs that have stimulatory effects on pigs by measuring the proliferation of porcine peripheral blood mononuclear cells (PBMCs) and assessing interferon gamma (IFN-γ) secretion. Furthermore, this study examined the immunoenhancing effects of the MF59 and CpG ODNs compound adjuvant in mouse and piglet models of porcine epidemic diarrhea virus (PEDV) subunit vaccine administration. The in vitro screening revealed that the CpG ODN named CpG5 significantly stimulated the proliferation of porcine PBMCs and elevated IFN-γ secretion levels. In the mouse vaccination model, CpG5 compound adjuvant significantly bolstered the humoral and cellular immune responses to the PEDV subunit vaccines, leading to Th1 immune responses characterized by increased IFN-γ and IgG2a levels. In piglets, the neutralizing antibody titer was significantly enhanced with CpG5 compound adjuvant, alongside a considerable increase in CD8+ T lymphocytes proportion. The combination of MF59 adjuvant and CpG5 exhibits a synergistic effect, resulting in an earlier, more intense, and long-lasting immune response in subunit vaccines for PEDV. This combination holds significant promise as a robust candidate for the development of vaccine adjuvant.

Rapid Construction of an Infectious Clone of Fowl Adenovirus Serotype 4 Isolate.

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis-hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors.

Nanoparticle-Based Bivalent Swine Influenza Virus Vaccine Induces Enhanced Immunity and Effective Protection against Drifted H1N1 and H3N2 Viruses in Mice.

Swine influenza virus (SIV) circulates worldwide, posing substantial economic loss and disease burden to humans and animals. Vaccination remains the most effective way to prevent SIV infection and transmission. In this study, we evaluated the protective efficacy of a recombinant, baculovirus-insect cell system-expressed bivalent nanoparticle SIV vaccine in mice challenged with drifted swine influenza H1N1 and H3N2 viruses. After a prime-boost immunization, the bivalent nanoparticle vaccine (BNV) induced high levels of hemagglutination inhibition (HAI) antibodies, virus-neutralization (VN) antibodies, and antigen-specific IgG antibodies in mice, as well as more efficient cytokine levels. The MF59 and CPG1 adjuvant could significantly promote both humoral and cellular immunity of BNV. The MF59 adjuvant showed a balanced Th1/Th2 immune response, and the CPG1 adjuvant tended to show a Th1-favored response. The BALB/c challenge test showed that BNV could significantly reduce lung viral loads and feces viral shedding, and showed fewer lung pathological lesions than those in PBS and inactivated vaccine groups. These results suggest that this novel bivalent nanoparticle swine influenza vaccine can be used as an efficacious vaccine candidate to induce robust immunity and provide broad protection against drifted subtypes in mice. Immune efficacy in pigs needs to be further evaluated.

Incorporation of a truncated form of flagellin (TFlg) into porcine circovirus type 2 virus-like particles enhances immune responses in mice.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important pathogen in the swine industry worldwide. Vaccination remains the principal tool to control PCV2-associated diseases (PCVADs). Current vaccines do not eliminate viral shedding in the environment. To enhance the efficacy of PCV2 vaccines, recombinant virus-like particles (VLPs) of PCV2 were generated by fusing a truncated form of flagellin FliC (TFlg: 85-111aa) with the PCV2 capsid protein (Cap). RESULTS: The recombinant proteins were expressed in Escherichia coli and detected using Western blotting. The abilities of the recombinant proteins to assemble into VLPs were observed under transmission electron microscopy (TEM). The protective immune responses of recombinant VLPs were further evaluated by immunization of mice. The results showed that insertion of TFlg into C terminal of the Cap protein did not affect the formation of VLPs and boosted both humoral and cellular immune responses in mice. After a challenge with PCV2, in the Cap-TFlg vaccinated group, viremia was milder and viral loads were lower as compared with those in the Cap vaccinated group. CONCLUSION: These results suggest that recombinant VLPs of PCV2 containing a TFlg adjuvant can be used as a promising PCV2 vaccine candidate.

Generation of infectious clone of bovine adenovirus type I expressing a visible marker gene.

BACKGROUND AND OBJECTIVE: Bovine adenovirus type 3 (BAdV3) has been widely used as a vector for vaccine research and development, whereas BAdV1 biology and BAdV1-based vectored vaccine have been less frequently reported. We aimed to construct an infectious BAdV1 clone and explore the functions of BAdV1 genes. METHODS: First, the infectious clone of pUCBAdV1 containing the full-length BAdV1 DNA and the recombinant plasmid pUCBAV1-EYFP expressing the marker gene EYFP were constructed. Then, the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued. The stability of the exogenous EYFP gene was analyzed by continuous passage, PCR, and western blotting. Finally, the virus neutralization titer of the rescued viruses was evaluated. RESULTS: The infectious clones of pUCBAdV1 and pUCBAV1-EYFP were constructed and the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued successfully. Moreover, the results showed that the EYFP gene could be expressed continuously. In addition, the replication of rBAdV1-EYFP was less efficient than that of the wild-type virus wtBAdV1 in vitro, while the efficacy of BAdV101 replication was almost the same as that of wtBAdV1. Furthermore, the neutralization test showed that the neutralization titer of rBAdV1-EYFP was consistent with that of wtBAdV1. CONCLUSION: To our knowledge, the infectious genome of pUCBAV1-EYFP expressing a visible marker gene EYFP was constructed for the first time, and the finding forms a basis for the development of BAdV1-based efficient vectored vaccine.

A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.

Full-Length Genome Sequences of Two Chinese Porcine Circovirus Type 3 Strains, NWHEB21 and NWHUN2.

Two porcine circovirus 3 (PCV3) strains, named NWHEB21 and NWHUN2, were identified in heart and brain tissues of aborted piglets. Their complete genome sequences were sequenced and analyzed to further characterize PCV3 in China and worldwide.

Melatonin Relieves Busulfan-Induced Spermatogonial Stem Cell Apoptosis of Mouse Testis by Inhibiting Endoplasmic Reticulum Stress.

BACKGROUND/AIMS: Busulfan is commonly used for cancer chemotherapy. Although it has the advantage of increasing the survival rate of patients, it can cause male infertility via damaging the testes and reducing sperm counts. Therefore, the underlying mechanism should be explored, and new agents should be developed to protect the male reproductive system from busulfan-induced damage. Endoplasmic reticulum stress (ERS) is considered a key contributor to numerous pathologies. Despite several studies linking ERS to toxicants, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage. Melatonin is a well-known broad-spectrum antioxidant, anti-inflammatory and antitumour agent, but the effects of melatonin on busulfan-induced ERS in mouse testes damage are less documented. METHODS: The effects of melatonin were measured by immunofluorescence staining, Western blot, qRT-PCR analysis and flow cytometry assay. The underlying mechanism was investigated by measuring ERS. RESULTS: We found that ERS was strongly activated in mouse testes (in vivo) and the C18-4 cell line (in vitro) after busulfan administration. ERS-related apoptosis proteins such as caspase-12, CHOP and caspase-3 were activated, and the expression of apoptotic proteins such as P53 and PUMA were upregulated. Furthermore, we investigated whether melatonin reduced the extent of damage to mouse testes and improved the survival rates of busulfan-treated mice. When exploring the underlying mechanisms, we found melatonin could counteract ERS by decreasing the expression levels of the ERS markers GRP78, ATF6, pIRE1 and XBP1 in mouse testes and mouse SSCs (C18-4 cells). Moreover, it blocked the activation of ERS-related apoptosis proteins caspase-12, CHOP and caspase-3 and suppressed P53 and PUMA expression stimulated by busulfan both in vivo and in vitro. CONCLUSION: Our results demonstrate that ERS is an important mediator for busulfan-induced apoptosis. The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage. Thus, this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment.

Surface displaying of swine IgG1 Fc enhances baculovirus-vectored vaccine efficacy by facilitating viral complement escape and mammalian cell transduction.

Baculovirus-mediated gene transfer has been developed as a vaccine design strategy against a number of diseases without apparent viral replication. However, it has been hampered by complement-dependent inactivation, thus hindering the in vivo application of baculovirus. A variety of approaches have been exploited to bypass the complement system in the serum. In this study, we constructed and screened a series of baculovirus vectors displaying complement interfering factors, of which a baculovirus vector displaying swine IgG1 Fc (pFc) showed the highest complement antagonism (75.6%). Flow cytometry analysis of transduced cells demonstrated that the baculovirus display of pFc had a significant increase in transduction efficiency and transgene expression of reporter genes. On this basis, a VSV-G-pseudotyped with swine IgG1 Fc surface displayed baculovirus vector was developed to express the classical swine fever virus (CSFV) E2 gene. The translational enhancers Syn21 and P10UTR were incorporated to improve the antigen expression. The E2 gene was efficiently expressed in both insect and mammalian cells. Pigs immunized with this recombinant baculovirus developed high levels of E2-specific antibody, CSFV-specific neutralizing antibody and IFN-γ-secreting cellular immune responses. These results demonstrate that the strategy of surface-displaying swine IgG1 Fc has a great potential to improve the efficiency of baculovirus-vectored vaccine for CSFV and other swine pathogens.

Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles.

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene (vp7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg, respectively), ii) DH5α-BG/pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine (DNA dose ranged from 2.5 to 5 µg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA-vp7 group than that of naked pcDNA-vp7 (42.22%) at the highest DNA dose (5 µg) after 14 days of post infection. Moreover, the level of pcDNA-vp7 plasmid was higher in DH5α-BG/pcDNA-vp7 groups than naked pcDNA-vp7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection.

Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX.

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

Enhanced protective immunity of the chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever in pigs by a Salmonella bacterial ghost adjuvant.

Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 10(5) median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 10(10) colony forming units (CFU) BG, 10(6) or 10(5) TCID50 rAdV-SFV-E2 alone or 10(10) CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 10(5) TCID50 rAdV-SFV-E2 plus BG or 10(6) TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 10(5) TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines.

Preparation of a Chicken scFv to Analyze Gentamicin Residue in Animal Derived Food Products.

Chicken is an ideal model for simplified recombinant antibody library generation. It has been rarely been reported to apply chicken single-chain variable fragments (scFvs) in immunoassays for the detection of antibiotic and chemical contaminants in animal food products. In this study, the scFvs (S-1 and S-5) were isolated from a phage display library derived from a hyperimmunized chicken. The checker board titration revealed that the optimum concentrations of S-1 and S-5 were 0.78 µg/mL and 0.44 µg/mL respectively, to obtain OD450 around 1.0 at 5 µg/mL of Gent-OVA coating concentration. Both S-1 and S-5 exhibited negligible cross reactivity with kanamycin and amikacin. The 50% inhibitory concentration (IC50) of S-1 and S-5 were 12.418 ng/mL and 14.674 ng/mL respectively. In the indirect competitive ELISA (ic-ELISA), the limits of detection for S-1 and S-5 were 0.147 ng/mL and 0.219 ng/mL respectively. The mean recovery for Gent ranged from 60.91% to 118.09% with no more than 10.35% relative standard deviation (RSD) between the intra-assay and the inter-assay. These results indicate the chicken scFv based ic-ELISA method is suitable for the detection of Gent residue in animal derived edible tissues and milk.

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.

Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues.

Gentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5(th) booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL(-1) of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC50 value of anti-Gent IgY was 2.69 ng mL(-1) and regression curve equation was y = -16.27x + 56.97 (R(2) = 0.95, n = 3), confirming that, the detection limit (LOD, IC10 value) was 0.01 ng mL(-1). Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples.

Functional analysis of RNAi suppressor P19 on improving baculovirus yield and transgene expression in Sf9 cells.

OBJECTIVE: To investigate whether RNA interference suppressor P19 derived from tombusvirus can enhance baculovirus yield and transgene expression. RESULTS: A number of recombinant baculoviruses with P19 under the control of white spot syndrome virus immediate-early promoter, baculovirus early-to-late promoter, or P10 late promoter were constructed The budded virus titer and the expression levels of eGFP and luciferase were determined. P19 was clearly functional in Sf9 cells and could enhance baculovirus yield, eGFP and luciferase expression levels up to 6.8-, 1.8-, and 2.1-fold respectively, at 72 h post infection. CONCLUSION: P19 enhanced baculovirus production and transgene expression, and thus has potential applications in baculovirus-based gene therapy and vaccine studies.

A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620 ± 49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

Enhanced production of porcine circovirus type 2 (PCV2) virus-like particles in Sf9 cells by translational enhancers.

OBJECTIVE: To investigate the effect of three translational enhancers for enhancing transgene expression in baculovirus expression vector system using GFP as a reporter gene and selected translational enhancers to increase porcine circovirus type 2 (PCV2) VLPs production. RESULTS: P10UTR (the 3'-untranslated region from the baculovirus p10 gene), Syn21 (a synthetic AT-rich 21-bp sequence) and P10UTR/Syn21 increased the GFP yield by 1.4-, 4- and 4.8-fold, respectively. While IVS (intron from Drosophila myosin heavy chain gene) decreased the GFP yield by 65%. Moreover, the synergy of P10UTR/Syn21 increased the yield of PCV2 VLPs by 4.1 fold (45 µg/10(6) cells) compared with standard baculovirus vector. CONCLUSION: The synergy of P10UTR/Syn21 is a potential strategy to improve the recombinant vaccine production besides PCV2 VLPs in BEVS.

Genomic characterisation of two virulent Newcastle disease viruses isolated from crested ibis (Nipponia nippon) in China.

This paper describes the complete genomic sequences of two virulent Newcastle disease virus (NDV) isolates, Shaanxi06 (prevalent genotype VIId) and Shaanxi10 (novel sub-genotype VIi), from sick crested ibises. The genomes of both isolates were 15,192 nt long and consisted of six genes in the order of 3'-NP-P-M-F-HN-L-5'. The genomes of the two isolates were highly similar to other reference NDV strains. However, some unique features were found in the HN protein of Shaanxi06 and the F gene end of Shaanxi10. Shaanxi06 and Shaanxi10 shared the same virulent motif (112-)R-R-Q-K-R-F(-117) at the F protein cleavage site, which coincided with previous pathogenicity test results. Phylogenetic analysis revealed that both isolates were clustered within class II NDV, with Shaanxi06 in genotype VII and Shaanxi10 in genotype VI. Both isolates shared high homology with the prevalent genotype NDV strains that circulate in fowls and waterfowls. This study is the first to provide genomic information about a novel sub-genotype VIi NDV strain and another genotype VIId virus, which will be useful for subsequent investigations.

Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells.

CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells.