Orf virus as an adjuvant enhances the immune response to a PCV2 subunit vaccine.

Orf virus (ORFV), a member of the genus Parapoxvirus, possesses an excellent immune activation capability, which makes it a promising immunomodulation agent. In this study, we evaluated ORFV as a novel adjuvant to enhance the immune response of mice to a subunit vaccine using porcine circovirus type 2 (PCV2) capsid (Cap) protein as a model. Our results showed that both inactivated and live attenuated ORFV activated mouse bone marrow-derived dendritic cells and increased expression of immune-related cytokines interleukin (IL)-1ß, IL-6, and TNF-α. Enhanced humoral and cellular immune responses were induced in mice immunized with PCV2 Cap protein combined with inactivated or live attenuated ORFV adjuvant compared with the aluminum adjuvant. Increased secretion of Th1 and Th2 cytokines by splenic lymphocytes in immunized mice further indicated that the ORFV adjuvant promoted a mixed Th1/Th2 immune response. Moreover, addition of the ORFV adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the spleen and lungs of PCV2-challenged mice and prevented pathological changes in lungs. This study demonstrates that ORFV enhances the immunogenicity of a PCV2 subunit vaccine by improving the adaptive immune response, suggesting the potential application of ORFV as a novel adjuvant.

PCV2 Induced Endothelial Derived IL-8 Affects MoDCs Maturation Mainly via NF-κB Signaling Pathway.

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Novel Porcine Circoviruses in View of Lessons Learned from Porcine Circovirus Type 2-Epidemiology and Threat to Pigs and Other Species.

Porcine circovirus type 2 (PCV2) plays a key role in PCV2-associated disease (PCVAD) etiology and has yielded significant losses in the pig husbandry in the last 20 years. However, the impact of two recently described species of porcine circoviruses, PCV3 and PCV4, on the pork industry remains unknown. The presence of PCV3 has been associated with several clinical presentations in pigs. Reproductive failure and multisystemic inflammation have been reported most consistently. The clinical symptoms, anatomopathological changes and interaction with other pathogens during PCV3 infection in pigs indicate that PCV3 might be pathogenic for these animals and can cause economic losses in the swine industry similar to PCV2, which makes PCV3 worth including in the differential list as a cause of clinical disorders in reproductive swine herds. Moreover, subsequent studies indicate interspecies transmission and worldwide spreading of PCV3. To date, research related to PCV3 and PCV4 vaccine design is at early stage, and numerous aspects regarding immune response and virus characteristics remain unknown.

Porcine TRIM21 Enhances Porcine Circovirus 2 Infection and Host Immune Responses, But Inhibits Apoptosis of PCV2-Infected Cells.

Tripartite motif protein 21 (TRIM21) is an interferon-inducible E3 ligase, containing one RING finger domain, one B-box motif, one coiled-coil domain at the N-terminal, as well as one PRY domain and one SPRY domain at the C-terminal. TRIM21 is expressed in many tissues and plays an important role in systemic autoimmunity. However, TRIM21 plays different roles in different virus infections. In this study, we evaluate the relationship between porcine TRIM21 and PCV2 infection as well as host immune responses. We found that PCV2 infection modulated the expression of porcine TRIM21. TRIM21 can enhance interferons and proinflammatory factors and decrease cellular apoptosis in PCV2-infected cells. These results indicate that porcine TRIM21 plays a critical role in enhancing PCV2 infection, which is a promising target for controlling and developing the treatment of PCV2 infection.

The construction and immunogenicity analyses of a recombinant pseudorabies virus with porcine circovirus type 3 capsid protein co-expression.

Porcine circovirus-associated diseases (PCVADs) and pseudorabies (PR) are highly contagious and economically significant diseases of swine in China. Porcine circovirus type 3 (PCV3) is an emerging swine pathogen of PCVAD. Currently, no PCV3 vaccine is commercially available, and the epidemic caused by it is still spreading worldwide. In this study, we used the PRV variant strain HNX as the parental virus to construct recombinant PRV with TK/gE gene deletion and capsid (Cap) protein co-expression, named HNX-ΔTK/ΔgE-ORF2. The results revealed that PCV3 Cap protein can be detected in HNX-ΔTK/ΔgE-ORF2-infected PK-15 cells by both western blotting and immunofluorescence assays. Vaccination with HNX-ΔTK/ΔgE-ORF2 did not cause pruritus, ruffled fur, systemic infection, or inflammation (without high expression of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) in plasma). Furthermore, HNX-ΔTK/ΔgE-ORF2 immunization induced an anti-Cap specific antibody, activated a PRV-specific cellular immune response, and provided 100 % protection to mice against the challenge of the virulent HNX strain. Thus, HNX-ΔTK/ΔgE-ORF2 appears to be a promising vaccine candidate against PRV and PCV3 for the control of the PRV variant and PCV3.

NOD2 Genotypes Affect the Symptoms and Mortality in the Porcine Circovirus 2-Spreading Pig Population.

The nucleotide oligomerization domain (NOD)-like receptor 2 (NOD2) is an intracellular pattern recognition receptor that detects components of peptidoglycans from bacterial cell walls. NOD2 regulates bowel microorganisms, provides resistance against infections such as diarrhea, and reduces the risk of inflammatory bowel diseases in humans and mice. We previously demonstrated that a specific porcine NOD2 polymorphism (NOD2-2197A > C) augments the recognition of peptidoglycan components. In this study, the relationships between porcine NOD2-2197A/C genotypes affecting molecular functions and symptoms in a porcine circovirus 2b (PCV2b)-spreading Duroc pig population were investigated. The NOD2 allele (NOD2-2197A) with reduced recognition of the peptidoglycan components augmented the mortality of pigs at the growing stage in the PCV2b-spreading population. Comparison of NOD2 allele frequencies in the piglets before and after invasion of PCV2b indicated that the ratio of NOD2-2197A decreased in the population after the PCV2b epidemic. This data indicated that functional differences caused by NOD2-2197 polymorphisms have a marked impact on pig health and livestock productivity. We suggest that NOD2-2197CC is a PCV2 disease resistant polymorphism, which is useful for selective breeding by reducing mortality and increasing productivity.

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection.

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-ß induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-ß induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Pathogenic Characterization of a Porcine Circovirus Type 3 Isolate from Heilongjiang, China.

The clinical outcome of porcine circovirus 3 (PCV3) infection is still controversial. Herein, a novel PCV3 isolate (PCV3-China/DB-1/2017) with the molecular characterization of 24A and 27K in the Cap protein was used to inoculate three-week-old cesarean-derived, colostrum-deprived piglets. The nine PCV3 DB-1 inoculated piglets exhibited no obvious clinical symptoms or macroscopic lesions. PCV3 displayed a broad histotropism, including the heart, liver, spleen, lung, kidney, brain, lymph nodes, and tonsil, and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs. From 7 days after PCV3 DB-1 inoculation, the piglets showed obvious IgG antibody responses against PCV3 rCap-VLPs. The cumulative results demonstrated that PCV3 trend to low pathogenicity.

Porcine circovirus type 3 capsid protein induces NF-κB activation and upregulates pro-inflammatory cytokine expression in HEK-293T cells.

Porcine circovirus type 3 (PCV3) has been widely detected throughout the world since it was first discovered on pig farms in 2015. PCV3 is closely associated with cardiac and multisystem inflammation, respiratory disease, congenital tremors, myocarditis, diarrhea, encephalitis and neurologic disease, and periarteritis. However, there have been few reports on the relationship between PCV3 and inflammatory pathways. The NF-κB signaling pathway plays an important role in the defense against viral infection. Here, we demonstrate that the capsid protein (Cap) of PCV3 plays a key role in the activation of NF-κB signaling in HEK-293T cells. Furthermore, PCV3 Cap promotes the mRNA expression of the pro-inflammatory cytokines IL6 and TNFα. In addition, PCV3 Cap promotes RIG-I and MDA5 mRNA expression in RIG-like receptor (RLR) signaling and MyD88 mRNA expression in Toll-like receptor (TLR) signaling but does not influence TRIF mRNA expression in TLR signaling. These results show that PCV3 Cap activates NF-κB signaling, possibly through the RLR and the TLR signaling pathways. This work illustrates that PCV3 Cap activates NF-κB signaling and thus may provide a basis for the pathogenesis of PCV3 and the innate immunity of the host.

Multi-walled carbon nanotube polysaccharide modified Hericium erinaceus polysaccharide as an adjuvant to extend immune responses.

In recent years, the utilization of CS-MWCNT as targeted drug carriers has attracted considerable attention. Hericium erinaceus polysaccharide (HEP) has been reported as an immunostimulant to improve immune responses. This study was focussed on developing CS-MWCNT encapsulating HEP (CS-MWCNT-HEP). Using in mice peritoneal macrophages, we found the immune response could be effectively regulated by CS-MWCNT-HEP, promoted the expression of the MHCII, CD86, F4/80 and gp38. Moreover, the mice immunized with CS-MWCNT-HEP nanoparticles significantly extended PCV2-specific IgG immune response and the levels of cytokines. The results demonstrated that CS-MWCNT-HEP may be a promising drug delivery system for immuno-enhancement.

A comparative efficacy test of 1 versus 2 doses of CIRCOQ PCV2 subunit vaccine against naturally occurring PCV2-type d in piglets with high maternally derived antibodies (MDAs) on a Vietnamese swine farm.

The aim of this study was to evaluate the protective efficacy of the CIRCOQ porcine circovirus type 2 (PCV2) subunit vaccine in piglets with high maternally derived antibodies (MDAs) against disease caused by natural infection with PCV2d. A total of 130 weaned, 21-day-old healthy pigs was allocated into 3 trial groups. The signs of respiratory disorder were higher in unvaccinated pigs than in vaccinated pigs at 13 to 17 weeks old (P < 0.05), 18 to 22 weeks old (P < 0.001), and 23 to 27 weeks old (P < 0.01). The unvaccinated pigs had an early rate of dermatitis at 8 to 12 weeks old (10.0%), 13 to 17 weeks old (30.0%), 18 to 22 weeks old (46.7%), and 23 to 27 weeks old (33.3%), while there were no cases of dermatitis in vaccinated pigs. There was a significant difference (P < 0.05) in the mortality of pigs in the unvaccinated group and the 2-dosed vaccinated group. PCV2 viremia was detected in the blood and peaked at 105 days old in both unvaccinated pigs (Ct-adj = 8.40) and pigs vaccinated with 1 dose (Ct-adj = 6.37), while no detectable PCV2 virus was found in the blood of pigs vaccinated with 2 doses. At 77 and 105 days old, the PCV2 viremia load (Ct-adj) of unvaccinated pigs and those vaccinated with 1 dose was significantly higher (P < 0.05) than that of the 2-dosed vaccinated pigs. The body weight (BW), average weight gain (AWG), and average daily gain (ADG) in both groups of vaccinated pigs were significantly higher (P < 0.05) than those of unvaccinated pigs. The study vaccine was significantly efficacious in protecting vaccinated pigs against clinical symptoms, blood viral load, and mortality, as well as improving productivity, compared with unvaccinated pigs.

Le but de la présente étude était d'évaluer l'efficacité protectrice du vaccin sous-unitaire CIRCOQ du circovirus porcin de type 2 (PCV2) chez les porcelets ayant une grande quantité d'anticorps d'origine maternelle (MDA) contre la maladie causée par une infection naturelle par le PCV2d. Un total de 130 porcs sains sevrés âgés de 21 jours a été réparti dans trois groupes d'essai. Les signes de troubles respiratoires étaient plus élevés chez les porcs non vaccinés que chez les porcs vaccinés âgés de 13 à 17 semaines (P < 0,05), de 18 à 22 semaines (P < 0,001) et de 23 à 27 semaines (P < 0,01). Les porcs non vaccinés avaient un taux précoce de dermatite entre 8 et 12 semaines (10,0 %), 13 à 17 semaines (30,0 %), 18 à 22 semaines (46,7 %) et 23 à 27 semaines (33,3 %), alors qu'il n'y a eu aucun cas de dermatite chez les porcs vaccinés. Il y avait une différence significative (P < 0,05) dans la mortalité des porcs dans le groupe non vacciné et le groupe vacciné à deux doses. La virémie du PCV2 a été détectée dans le sang et a atteint un pic à 105 jours chez les porcs non vaccinés (Ct-adj = 8,40) et les porcs vaccinés avec une dose (Ct-adj = 6,37), tandis qu'aucun virus PCV2 détectable n'a été détecté dans le sang des porcs vacciné avec deux doses. À 77 et 105 jours, la charge de virémie PCV2 (Ct-adj) des porcs non vaccinés et de ceux vaccinés avec une dose était significativement plus élevée (P < 0,05) que celle des porcs vaccinés à deux doses. Le poids corporel (BW), le gain de poids moyen (AWG) et le gain quotidien moyen (ADG) dans les deux groupes de porcs vaccinés étaient significativement plus élevés (P < 0,05) que ceux des porcs non vaccinés. Le vaccin de l'étude s'est avéré significativement efficace pour protéger les porcs vaccinés contre les symptômes cliniques, la charge virale sanguine et la mortalité, ainsi que pour améliorer la productivité, par rapport aux porcs non vaccinés.(Traduit par Docteur Serge Messier).

The Mink Circovirus Capsid Subunit Expressed by Recombinant Baculovirus Protects Minks against Refractory Diarrhea in Field.

Mink refractory diarrhea is a seasonal disease that occurs in many mink farms in China. Mink circovirus (MiCV) has been recognized as the causative agent of the disease. The aim of the study was to develop a subunit vaccine against mink refractory diarrhea. A recombinant baculovirus strain expressing the capsid protein was constructed using the baculovirus expression vector system (BEVS). A subunit vaccine was developed based on the capsid protein with appropriate adjuvant. Then, a field trial was carried out in two districts in order to evaluate the efficiency of the subunit vaccine. The field trial indicated that in total, only 1.8% of the minks developed typical diarrhea in the vaccinated group compared with 74.5% in the control group. The vaccination could significantly reduce the infection rate of MiCV among the mink herds and could restrain the virus' shedding from feces. Furthermore, the vaccinated group had a higher average litter size in the following year compared to the control group. Collectively, the results indicated that the subunit vaccine based on the capsid protein can provide reliable protection against MiCV infection.

A new strategy to develop pseudorabies virus-based bivalent vaccine with high immunogenicity of porcine circovirus type 2.

Herpesvirus based multivalent vaccines have been extensively studied, whereas few of them have been successfully used in clinic and animal husbandry industry due to the low expression of foreign immunogens in herpesvirus. In this study, we developed a new strategy to construct herpesvirus based bivalent vaccine with high-level expression of foreign immunogen, by which the ORF2 gene encoding the major antigen protein Cap of porcine circovirus type 2 (PCV2), was highly expressed in pseudorabies virus (PRV). To obtain the high expression of PCV2 immunogen, tandem repeats of PCV2 ORF2 gene were firstly linked by protein quantitation ratioing (PQR) linker to reach equal expression of each ORF2 gene. Then, the multiple copies of ORF2 gene were respectively inserted into the gE and gG sites of PRV using CRISPR/Cas9 system, in which the expression of ORF2 gene was driven by endogenous strong promoters of PRV. Through this way, the highest yield of Cap protein was achieved in two copies of quadruple ORF2 gene insertion. Finally, in mice and pigs immunized with the bivalent vaccine candidate, we detected high titer of specific antibodies for PRV and neutralized antibodies for PCV2, and observed protective effect of the bivalent vaccine candidate against PRV challenge in immunized pigs, suggesting a potential clinical application of the bivalent vaccine candidate we constructed. Together, our strategy could be extensively applied to the generation of other multivalent vaccines, and will pave the way to construct herpesvirus based multivalent vaccines to effectively reduce the cost of vaccine.

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this study was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

Expression and evaluation of porcine circovirus type 2 capsid protein mediated by recombinant adeno-associated virus 8.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. OBJECTIVES: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. METHODS: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. RESULTS: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. CONCLUSIONS: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2.

BACKGROUND: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)­horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. RESULTS: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15­HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti­PCV2 antibodies. The cut­off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. CONCLUSIONS: In brief, the newly developed cELISA based PCV2-Nb15­HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

A 14 bp indel polymorphism in the promoter region is associated with different responses to porcine circovirus type 2 infection by regulating MRC1 transcription.

Mannose receptor, C type 1 (MRC1) is a key factor in regulating the body's immune response to resist pathogen invasions. In this study, mRNA expressions of MRC1 gene in nine porcine organs/tissues were compared between Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs prior to and post PCV2 infection. We found that, for pigs uninfected with PCV2, MRC1 mRNA expressions in the lung, spleen, large intestine, small intestine and mesenteric lymph node tissues of LW were significantly higher than those of YL pigs (P < 0.05). After PCV2 infection, MRC1 mRNA levels in the liver, kidney and mesenteric lymph node were significantly increased in LW pigs (P < 0.05); while, significantly decreased in the heart and lung tissues of YL pigs (P < 0.05). The transcriptional activity of porcine MRC1 promoter was further analyzed to investigate the molecular mechanism underlying these expressional differences in response to PCV2 infection. Luciferase assay indicated that a 14 bp indel polymorphism "GTTTTTTTTTTTTT" at the site -864 of MRC1 promoter contributed to the transcriptional activity. The frequency of 14 bp insertion in LW and Dapulian pigs, generally resistant to PCV2 infection, was higher than that in Duroc, Landrace and Yorkshire pigs, which were sensitive to PCV2 infection. The promoter with 14 bp insertion displayed higher MRC1 transcription level both prior to and post PCV2 infection compared with that carrying no insertion in PK15 cells (P < 0.01). The results suggest that this 14 bp indel polymorphism is associated with different responses to PCV2 infection by regulating MRC1 transcription.