A colloidal gold test strip assay for the detection of African swine fever virus based on two monoclonal antibodies against P30.

African swine fever (ASF), caused by African swine fever virus (ASFV), was first reported in Kenya in 1921, but an effective vaccine or antiviral drug is still not available for ASFV control. Rapid and effective diagnostics are key steps in managing ASF. We generated two monoclonal antibodies (MAbs) against the ASFV phosphoprotein P30 and designated these as 3H7A7 and 6H9A10. Epitope mapping revealed that MAb 3H7A7 and 6H9A10 recognized aa 144-154 and aa 12-18 of P30, respectively. A signal-amplified sandwich colloidal gold test strip for rapid detection of ASFV was developed based using these MAbs. Sensitivity and specificity analysis showed that the detection limit of the strip was 2.16 ng of P30. The strip only reacted with ASFV and did not react with other common porcine viruses. In detection tests using 153 clinical field samples including sera, plasma, anticoagulant-treated blood, and tissue, the strip had 95.42% concordance with real-time PCR. The new MAbs specific for P30 and the rapid colloidal gold test strip helped to reveal novel B cell epitopes in P30 and provide an efficient diagnostic test for on-site clinical detection of ASF.

MicroRNA-138 promotes neuroblastoma SH-SY5Y cell apoptosis by directly targeting DEK in Alzheimer's disease cell model.

BACKGROUND: Alzheimer's disease (AD) is a progressive neuro-degenerative disease with a major manifestation of dementia. MicroRNAs were reported to regulate the transcript expression in patients with Alzheimer's disease (AD). In this study, we investigated the roles of miR-138, a brain-enriched miRNA, in the AD cell model. METHODS: The targets of miRNA-138 was predicted by bioinformatic analysis. The expression levels of DEK at both mRNA and protein levels were determined by qRT-PCR and Western blot, respectively. Luciferase assays were carried out to examine cell viabilities. Hoechst 33258 staining was used to detect cell apoptosis. RESULTS: Our results demonstrated that the expression levels of miR-138 were increased in AD model, and DEK was a target of miR-138. Overexpression of miR-138 in SH-SY5Y cells obviously down-regulated the expression of DEK in SH-SY5Y cells, resulting in the inactivation of AKT and increased expression levels of proapoptotic caspase-3. MiR-138 mediated-suppression of DEK increased the susceptibility of cell apoptosis. CONCLUSIONS: MicroRNA-138 promotes cell apoptosis of SH-SY5Y by targeting DEK in SH-SY5Y AD cell model. The regulation of miR-138 may contribute to AD via down-regulation of the DEK/AKT pathway.

Generation and immunogenicity assessment of ELPylated virus-like particles of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). METHODS: The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. RESULTS: ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. CONCLUSION: A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.

Half-life extension of porcine interferon-α by fusion to the IgG-binding domain of streptococcal G protein.

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.

A novel method for concentration of porcine reproductive and respiratory syndrome virus from the environmental samples using self-aggregating peptide-tagged CD151-binding capture.

Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance.

A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease.

Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.

Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.

A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples.

Inhibition of porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages with the 3' UTR-targeted amiRNA.

Objective: To study the inhibitory effect of 3' untranslated region (UTR)-targeted artificial microRNA (amiRNA) against porcine reproductive and respiratory syndrome virus (PRRSV) replication in porcine alveolar macrophages (PAM). Methods: Recombinant adenovirus (rAd) expressing the 3' UTR-targeted or control amiRNA and green fluorescent protein (GFP) reporter gene were generated by transfecting AAV-293 cells with the transfer vector. The expression of sequence-specific amiRNA was detected by quantitative RT-PCR. The anti-PRRSV effect of amiRNA was detected by quantitative RT-PCR, Western blotting and viral titration assay. Results: Two rAds, namely rAd-amiR3UTR-GFP and rAd-amiRcon-GFP, were generated. Both primary PAM and 3D4/163 cells could be transduced by rAd with different transduction efficiencies. The amiR3UTR was expressed in dose-and timedependent manners in rAd-transduced PAM cells. The amiR3UTR, but not amiRcon, had significant and stable inhibitory effects against replication of three different PRRSV strains in a dose-dependent manner. Conclusion: The rAd-delivered amiR3UTR had strong anti-PRRSV effect against different PRRSV strains and rAd-amiR3UTR-GFP could be explored further as the alternative strategy against PRRS.

The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese dairy herd are different from the previously reported, and that the herd- or farm-based diagnosis of staphylococcal bovine mastitis is required.

Construction and in vivo evaluation of a mammary gland-specific expression vector for human lysozyme.

A mammary gland-specific expression vector p205C3 was constructed with the 5'- and 3'-flanking regions of ß-lactoglobulin gene and the first intron of ß-casein gene of Chinese dairy goat as regulatory sequences. Human lysozyme (hLYZ) cDNA from mammary gland was cloned into p205C3 and the recombinant vector was used to generate transgenic mice by microinjection. Based on the lysoplate assay, four female offspring of one male founder were detected expressing recombinant hLYZ in their milk at the levels of 5-200 mg/l, and the expressed protein had the same molecular weight as that of normal hLYZ. Besides mammary glands, ectopic expressions were also found in the spleens and the small intestines of the transgenic mice. Among the offspring, the female transgenic mice maintained and expressed the transgene stably with a highest expression level of 750 mg/l. Therefore, p205C3 could be used to develop animal mammary gland bioreactors expressing hLYZ.

Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

BACKGROUND: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described. METHODS: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration. RESULTS: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains. CONCLUSION: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.

Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human ß defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

Efficient inhibition of porcine reproductive and respiratory syndrome virus replication by artificial microRNAs targeting the untranslated regions.

A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5' or 3' UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID(50) (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV.

[Establishment of a porcine CD151 transgenic PK-15 cell line susceptible to porcine reproductive and respiratory syndrome virus].

OBJECTIVE: In order to study the role of porcine CD151 in infection of porcine cells by porcine reproductive and respiratory syndrome virus (PRRSV), we established a porcine CD151 transgenic PK-15 cell line. METHODS: The full-length complementary DNA (cDNA) for porcine CD151 was amplified from porcine alveolar macrophages by reverse transcription-polymerase chain reaction (RT-PCR), and subcloned into eukaryotic expression vector pcDNA3. The recombinant vector pcDNA-CD151 was transfected into PK-15 cells and the transgenic cell line was generated after G418 selection. Transcription of the CD151 cDNA in transgenic cell line was detected by RT-PCR and immunofluorescence. The cell line, together with control cell lines PK-15, 3D4-CD163 and MARC-145, was infected with PRRSV, and the viral RNA genome or antigens in the infected cells was detected by RT-PCR or immunofluorescence. At different time points post-infection, the virus was harvested and titrated on MARC-145 cells. RESULTS: The expected size of porcine CD151 cDNA was amplified with a sequence identity of 99.7% to the published sequence. From the pcDNA-CD151-transfected cell culture, a transgenic cell line PK15-CD151 was generated and correct expression of porcine CD151 was confirmed. After PRRSV infection, the viral RNA genome and antigens were detected in the cell-line. Although apparent cytopathic effect was not observed in the virally infected cell line, the infectious virus with a high viral title was detected. The cell line had been passed for more than 30 generations and no significant difference in viral title was observed among generations 10, 20 and 30 after PRRSV infection. CONCLUSION: Transfection of non-permissive PK-15 cells with porcine CD151 cDNA conferred the susceptibility to PRRSV infection, indicating an important role of the porcine CD151 in PRRSV infection of porcine cells.

Inhibition of infectious bursal disease virus infection by artificial microRNAs targeting chicken heat-shock protein 90.

Infectious bursal disease virus (IBDV) causes an important disease in young chickens. Chicken heat-shock protein 90 (cHsp90) has been shown to be a functional component of the cellular receptor complex for IBDV infection. This study demonstrates the inhibitory effect of vector-expressed anti-cHsp90α microRNA (miRNA) on IBDV infection. The reporter vectors pcHsp90α-EGFP and pcHsp90ß-EGFP were constructed to facilitate effective miRNA selection. Two anti-cHsp90α and one anti-cHsp90ß miRNA-expression vectors were constructed for a stable transfection study. Poly(A)-tailed RT-PCR detected sequence-specific miRNA transcription in transfected cells. Semiquantitative RT-PCR showed inhibition of cHsp90 transcription in transfected cells. A virus-titration assay showed that the anti-cHsp90α miRNA, but not the anti-cHsp90ß miRNA, had inhibitory effects on IBDV infection. These results suggest that cHsp90α is a functional component of the cellular receptor complex for IBDV infection, and that anti-cHsp90α miRNA could be used as an anti-IBDV reagent.

Partial antiviral activities of the Asn631 chicken Mx against newcastle disease virus and vesicular stomatitis virus.

Conflicting data existed for the antiviral potential of the chicken Mx protein and the importance of the Asn631 polymorphism in determination of the antiviral activity. In this study we modified the chicken Mx cDNA from the Ser631 to Asn631 genotype and transfected them into COS-I cells, chicken embryonic fibroblast (CEF) or NIH 3T3 cells. The Mx protein was mainly located at the cytoplasm. The transfected cell cultures were challenged with newcastle disease virus (NDV) or vesicular stomatitis virus (VSV), cytopathic affect (CPE) inhibition assay showed that the times for development of visible and full CPE were significantly postponed by the Asn631 cDNA transfection at 48 h transfection, but not by the Ser631 cDNA transfection. Viral titration assay showed that the virus titers were significantly reduced before 72 h postinfection. CEF cells was incubated by the cell lysates extracted from the COS-I cells transfected with pcDNA-Mx/Asn631, could resist and delayed NDV infection. These data suggested the importance of the Asn631 polymorphism of the chicken Mx in determination of the antiviral activities against NDV and VSV at early stage of viral infection, which were relatively weak and not sufficient to inhibit the viral replication at late stage of viral infection.

Protection of Ducklings from Duck Hepatitis A Virus Infection with ELPylated Duck Interferon-α.

Duck viral hepatitis type I (DVH I) is a lethal disease in ducklings caused by duck hepatitis A virus (DHAV). Although the commercial vaccine is available for vaccination of one-day-old ducklings or breeder ducks, the disease is still prevalent due to the delayed immune response in ducklings and variable maternal antibody levels in breeder duck flocks. To explore the feasibility of duck interferon-α (DuIFN-α) for control of DVH I, DuIFN-α was expressed as an elastin-like polypeptide (ELP) fusion protein (ELP-DuIFN-α) in E. coli and purified by inverse phase transition cycling (ITC). After detection of its cytotoxicity, bioactivity, plasma stability and serum half-life, the protective efficacy of ELP-DuIFN-α against DHAV-1 infection of embryos or ducklings was evaluated using different treatment routes at different infection times. The results show that ELP-DuIFN-α was correctly expressed and purified to more than 90% purity after two cycles of ITC. The purified fusion protein had a specific anti-DHAV-1 activity of 6.0 × 104 IU/mg protein, significantly extended plasma stability and serum half-life without overt cytotoxicity. After allantoic injection with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 5/5, 5/5 or 4/5 embryos survived from the virus challenge. After intramuscular injection or oral administration with ELP-DuIFN-α, 3/5 or 4/5 ducklings survived from co-infection with DHAV-1. After oral administration with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 3/5, 4/5 or 4/5 ducklings survived from the virus challenge, and the relative transcription levels of interferon-stimulated genes were significantly higher than the normal control group and virus challenge control group (p < 0.01). These experimental data suggest that ELP-DuIFN-α can be used as a long-lasting anti-DHAV-1 reagent.

Enhancing half-life and cytotoxicity of porcine respiratory and reproductive syndrome virus soluble receptors by taming their Fc domains.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen. Although tremendous effort has been made for the vaccine development, only modified live vaccines are widely used with arguably limited efficacy. Our previous study showed that the Fc-fused first four Ig-like domains of Sn (Sn4D-Fc) and the SRCR domains 5-9 of CD163 (SRCR59-Fc) can act as PRRSV soluble receptors (VSRs). In this study, we improved the VSR-based anti-PRRSV strategy by taming their Fc domains. Sequence alignment showed that the CH3 domain of pig IgG1 contained five putative amino acids involved in the interaction with the neonatal Fc receptor (FcRn). The M455L/N461S variant of SRCR59-Fc/Sn4D-Fc was created for the higher affinity of FcRn binding. Both rBac-SRCR59-lsFc/Sn4D-lsFc and rBac-SRCR59-Fc/Sn4D-Fc expressing the mutated or wild-type VSRs were generated for conceptual validation. Both immunofluorescence and Western blotting analysis showed that the two rBac vectors could express the encoded VSRs in cells with similar expression levels and anti-PRRSV effects. In the rBac-injected mice, the expression of SRCR59-lsFc/Sn4D-lsFc was significantly prolonged than that of SRCR59-Fc/Sn4D-Fc. Both plasma stability and serum half-life of the purified SRCR59-lsFc/Sn4D-lsFc were significantly improved than that of SRCR59-Fc/Sn4D-Fc. SRCR59-lsFc/Sn4D-lsFc-treated peripheral blood mononuclear cells showed significantly stronger cytotoxicity on PRRSV-infected primary alveolar macrophages than SRCR59-Fc/Sn4D-Fc-treated cells. For the first time, we demonstrated that both half-life and effector function of pig IgG Fc-fused proteins could be significantly improved by taming their CH3 domains. The rBac-SRCR59-lsFc/Sn4D-lsFc could be further developed as a novel anti-PRRSV reagent.

Comparison of mucosal immune responses to African swine fever virus antigens intranasally delivered with two different viral vectors.

Transmission of African swine fever virus (ASFV) in domestic swine occurs mainly via contact with mucosal surfaces. In this study, we constructed a pseudotyped surface-displaying BacMam-F1 vector expressing ASFV CD2v-p30-p54 fusion antigen, and compared its mucosal responses in pigs with that of rAd-F1 vector expressing the same antigen. From day 21 after intranasal immunization, the antigen-specific IgG and intranasal secretory IgA (S-IgA) antibody responses induced by BacMam-F1 were significantly stronger than that by rAd-F1. The significantly different S-IgA antibody responses were also detected in their tracheal washes and lung lavages. After stimulation with ASFV antigens, 4/6 S-IgA-promoting cytokine responses in porcine alveolar macrophages (PAMs) from BacMam-F1-immunized pigs were significantly stronger than that from rAd-F1-immunized pigs. The similar expression patterns of S-IgA-promoting cytokines were also detected in their lung lavages. After pretreating ASFV with different samples from immunized pigs, significant inhibitory effects were detected in tracheal washes, lung lavages and PAM cultures, but not serum samples with slight inter-group difference. These data suggest that the pseudotyped surface-displaying BacMam vector is more suitable for swine mucosal immunization.

Gene Cloning, Tissue Expression Profiles and Antiviral Activities of Interferon-ß from Two Chinese Miniature Pig Breeds.

The porcine interferon (PoIFN) complex represents an ideal model for studying IFN evolution which has resulted from viral pressure during domestication. Bama and Banna miniature pigs are the two Chinese miniature pig breeds that have been developed as laboratory animal models for studying virus infection, pathogenesis, and vaccine evaluation. However, the PoIFN complex of such miniature pig breeds remains to be studied. In the present study, we cloned PoIFN-ß genes from Bama and Banna miniature pigs, detected their PoIFN-ß tissue expression profiles, prepared recombinant PoIFN-ß (rPoIFN-ß) using the E. coli expression system, and measured their antiviral activities against three different pig viruses. At the amino acid sequence level, PoIFN-ßs of the two miniature pig breeds were identical, which shared 100% identity with that of Congjiang Xiang pigs, 99.4-100% identity with that of domestic pigs, and 99.5% identity with that of three species of African wild boars. The tissue expression profiles of PoIFN-ß mRNA differed not only between the two miniature pig breeds but between miniature pigs and domestic pigs as well. The four promoter domains of PoIFN-ß of the two miniature pig breeds were identical with that of humans, domestic pigs, and three species of African wild boars. The recombinant PoIFN-ß prepared from the two miniature pig breeds showed dose-dependent pre-infection and post-infection antiviral activities against vesicular stomatitis virus, porcine respiratory and reproductive syndrome virus, and pig pseudorabies virus. This study provided evidence for the high sequence conservation of PoIFN-ß genes within the Suidae family with different tissue expression profiles and antiviral activities.