Cytotoxicity effect and transcriptome analysis of PCV3-infected cells revealed potential viral pathogenic mechanisms.

Porcine circovirus type 3 (PCV3) has become an important pathogen in the global swine industry and poses a threat to pig health, but its pathogenic mechanism remains unknown. In this study, we constructed an innovative, linear infectious clone of PCV3 for rescuing the virus, and explored the transcriptome of infected cells to gain insights into its pathogenic mechanisms. Subsequently, an in vivo experiment was conducted to evaluate the pathogenicity of the rescued virus in pig. PCV3 nucleic acid was distributed across various organs, indicating systemic circulation via the bloodstream and viremia. Immunohistochemical staining also revealed a significant presence of PCV3 antigens in the spleen, lungs, and lymph nodes, indicating that PCV3 had tropism for these organs. Transcriptome analysis of infected ST cells revealed differential expression of genes associated with apoptosis, immune responses, and cellular metabolism. Notably, upregulation of genes related to the hypoxia-inducible factor-1 pathway, glycolysis, and the AGE/RAGE pathway suggests activation of inflammatory responses, ultimately leading to onset of disease. These findings have expanded our understanding of PCV3 pathogenesis, and the interplay between PCV3 and host factors.

Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein.

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120-134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131-143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120-134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.

A prospective CSFV-PCV2 bivalent vaccine effectively protects against classical swine fever virus and porcine circovirus type 2 dual challenge and prevents horizontal transmission.

Classical swine fever virus (CSFV) infection leading to CSF outbreaks is among the most devastating swine diseases in the pig industry. Porcine circovirus type 2 (PCV2) infection, resulting in porcine circovirus-associated disease (PCVAD), is also a highly contagious disease affecting pig health worldwide. To prevent and control disease occurrence, multiple-vaccine immunization is necessary in contaminated areas or countries. In this study, a novel CSFV-PCV2 bivalent vaccine was constructed and demonstrated to be capable of eliciting humoral and cellular immune responses against CSFV and PCV2, respectively. Moreover, a CSFV-PCV2 dual-challenge trial was conducted on specific-pathogen-free (SPF) pigs to evaluate vaccine efficacy. All of the vaccinated pigs survived and showed no clinical signs of infection throughout the experimental period. In contrast, placebo-vaccinated pigs exhibited severe clinical signs of infection and steeply increased viremia levels of CSFV and PCV2 after virus challenge. Additionally, neither clinical signs nor viral detections were noted in the sentinel pigs when cohabitated with vaccinated-challenged pigs at three days post-inoculation of CSFV, indicating that the CSFV-PCV2 bivalent vaccine completely prevents horizontal transmission of CSFV. Furthermore, conventional pigs were utilized to evaluate the application of the CSFV-PCV2 bivalent vaccine in field farms. An adequate CSFV antibody response and a significant decrease in PCV2 viral load in the peripheral lymph nodes were observed in immunized conventional pigs, suggesting its potential for clinical application. Overall, this study demonstrated that the CSFV-PCV2 bivalent vaccine effectively elicited protective immune responses and the ability to prevent horizontal transmission, which could be a prospective strategy for controlling both CSF and PCVAD in commercial herds.

Evaluation of classical swine fever E2 (CSF-E2) subunit vaccine efficacy in the prevention of virus transmission and impact of maternal derived antibody interference in field farm applications.

BACKGROUND: Classical swine fever (CSF) is one of the most devastating pig diseases that affect the swine industry worldwide. Besides stamping out policy for eradication, immunization with vaccines of live attenuated CSF or the CSF-E2 subunit is an efficacious measure of disease control. However, after decades of efforts, it is still hard to eliminate CSF from endemically affected regions and reemerging areas. Most of previous studies demonstrated the efficacy of different CSF vaccines in laboratories under high containment conditions, which may not represent the practical performance in field farms. The inadequate vaccine efficacy induced by unrestrained factors may lead to chronic or persistent CSF infection in animals that develop a major source for virus shedding among pig populations. In this study, a vaccination-challenge-cohabitation trial on specific-pathogen-free (SPF) pigs and long-term monitoring of conventional sows and their offspring were used to evaluate the efficacy and the impact of maternally derived antibody (MDA) interference on CSF vaccines in farm applications. RESULTS: The trials demonstrated higher neutralizing antibody (NA) titers with no clinical symptoms and significant pathological changes in the CSF-E2 subunit vaccine immunized group after CSFV challenge. Additionally, none of the sentinel pigs were infected during cohabitation indicating that the CSF-E2 subunit vaccine could provoke adequately acquired immunity to prevent horizontal transmission. In field farm applications, sows immunized with CSF-E2 subunit vaccine revealed an average of higher and consistent antibody level with significant reduction of CSF viral RNA detection via saliva monitoring in contrast to those of live attenuated CSF vaccine immunized sows possessing diverse antibody titer distributions and higher viral loads. Furthermore, early application of the CSF-E2 subunit vaccine in 3-week-old piglets illustrated no MDA interference on primary immunization and could elicit consistent and long-lasting adequate antibody response suggesting the flexibility of CSF-E2 subunit vaccine on vaccination program determination. CONCLUSIONS: The CSF-E2 subunit vaccine demonstrated significant efficacy and no MDA interference for immunization in both pregnant sows and piglets. These advantages provide a novel approach to avoid possible virus shedding in sow population and MDA interference in piglets for control of CSF in field farm applications.

Detection and phylogenetic analysis of porcine circovirus type 3 in Taiwan.

Porcine circovirus type 3 (PCV3) is a newly emerging porcine circovirus that infects pig populations worldwide. In this study, we investigated the prevalence of PCV3 in Taiwan and analyzed the phylogenetic relationships between the Taiwanese PCV3 strains and those from other countries. A total of 463 clinical specimens from sick pigs were collected in 2016-2019 and analyzed for PCV3 by PCR. The positivity rate for PCV3 was 10.6% in 2016, increasing markedly to 34.78% in 2019. A phylogenetic analysis based on full-length genomic sequences of PCV3 divided the PCV3 strains into three clades, with the Taiwanese strains in clade 1.

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein.

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity.

Evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV is characterized by rapid mutation rates and extensive genetic divergences. It is divided into two genotypes, which are composed of several distinct sub-lineages. The purpose of the present study was to evaluate the cross-protective efficacy of Fostera PRRS MLV, an attenuated lineage 8 strain, against the heterologous challenge of a lineage 3 isolate. Eighteen pigs were randomly divided into mock, MLV and unvaccinated (UnV) groups. The pigs in the MLV group were administered Fostera PRRS vaccine at 3 weeks of age and both the MLV and UnV groups were inoculated with a virulent PRRSV isolate at 7 weeks. Clinically, the MLV group showed a shorter duration and a lower magnitude of respiratory distress than the UnV group. The average days of fever in the MLV group was 3.0 ± 0.5, which was significantly lower than the 6.2 ± 0.5 days of the UnV group (P < 0.001). The average daily weight gains of the mock, MLV and UnV groups were 781 ± 31, 550 ± 44 and 405 ± 26 g/day, respectively, during the post-challenge phase. The pathological examinations revealed that the severity of interstitial pneumonia in the MLV group was milder compared to the UnV group. Furthermore, PRRSV viremia titers in the MLV pigs were consistently lower (101-101.5 genomic copies) than those of the UnV pigs from 4 to 14 DPC. In conclusion, vaccination with Fostera PRRS MLV confers partial cross-protection against heterologous challenge of a virulent lineage 3 PRRSV isolate.

The impact of porcine circovirus associated diseases on live attenuated classical swine fever vaccine in field farm applications.

Porcine circovirus associated diseases (PCVADs) are among the most important diseases affecting the worldwide swine industry. Vaccination against porcine circovirus type 2 (PCV2) infection has been utilized for disease control and effectively reduces clinical signs of PCVADs. To evaluate the efficacy of the PCV2 vaccine in field farms, we conducted a trial using conventional pigs immunized with the subunit PCV2 vaccine followed by PCV2 challenge. Immunized pigs demonstrated lower serum viral loads, less viral antigen staining in lymph nodes, and higher average daily weight gain, confirming the protective efficacy of the vaccine. However, low levels of PCV2 infection were still detected in vaccinated pigs after challenge, suggesting that the PCV2 vaccine was unable to eradicate the virus, which could lead to asymptomatic PCV2 subclinical infection (PCV2-SI) in pig farms. Additionally, PCV2 infection is a risk factor for impaired pig immune response development during the weaning to growth stages, which is a crucial period to receive vaccines against classical swine fever (CSF). Therefore, the impact of PCV2-SI or PCV2-systemic disease (PCV2-SD) on live attenuated CSF vaccine was investigated. After PCV2 challenge, there was no difference in levels of classical swine fever virus (CSFV) neutralizing antibodies (NA) between pigs with PCV2-SD and PCV2-SI, suggesting that the efficacy of CSF vaccine was compromised. Moreover, results of long-term monitoring of CSFV NA titers in PCV2-SI pigs with minimized interference by maternally-derived antibodies suggested that serum PCV2 viral loads greater than 102 copies/mL may compromise the efficacy of CSF vaccine. Overall, a conventional pig model was established to demonstrate the impaired efficacy of the subunit PCV2 vaccine and its impact on the CSF vaccine in vaccination-challenge trials. Additionally, the impaired efficacy of the PCV2 vaccine resulted in increased PCV2-SI, eventually leading to compromised the live attenuated CSF vaccine induced NA response in field farm applications.

N-terminus of Classical swine fever virus strain TD96 glycoprotein Erns contains a potential heparin-binding domain.

Classical swine fever virus (CSFV) envelope glycoprotein Erns has been shown to bind to cell surface sulphated-heparin-like glycosaminoglycans (GAGs), which participate in cell attachment of the virus. In this study, the CSFV Erns gene was codon optimized for expression in the yeast Pichia pastoris. A C-terminally truncated Erns recombinant protein lacking the previously identified heparin-binding domain (HBD) bound to heparin column, suggesting the presence of another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed a common potential N-terminal HBD at residues 301－311. Site-directed mutagenesis of the basic amino acids at K303 and K306 significantly reduced the heparin-binding affinity of the protein. Further mutations of both T310 and H311 had little effect. Thus, a novel potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been detected, and the two basic amino acids K303 and K306 are crucial for binding activity to heparin matrix and cell-surface GAGs.

Growth properties and vaccine efficacy of recombinant pseudorabies virus defective in glycoprotein E and thymidine kinase genes.

Pseudorabies virus (PRV) is an alphaherpesvirus that causes pseudorabies (PR), an economically important viral disease of pigs. Marker vaccines were widely used in PR prevention and eradication programs. The purpose of this study was to construct a novel recombinant virus with deletions at defined regions in the glycoprotein E (gE) and thymine kinase (TK) genes by homologous recombination. This study also evaluated the safety and efficacy of the virus for a live attenuated marker vaccine. No significant difference was observed in virus replication between gE gene-deleted (gE(-)), gE/TK double gene-deleted (gE(-)TK(-)), and wild-type PRV by growth curve analysis. However, gE(-)TK(-) PRV was completely attenuated in mice. To evaluate the immunogenicity of gE(-)TK(-) PRV, four 12-week-old specific-pathogen-free pigs per group were immunized intramuscularly with viral titers of 1×10(4), 1×10(5), or 1×10(6) TCID50, followed by intranasal challenge infection with virulent PRV (1×10(8) TCID50) at 3 weeks post vaccination. The gE(-)TK(-) PRV-vaccinated pigs displayed no general adverse effects after immunization and had protective immune responses after PRV challenge. Thus, gE(-)TK(-) PRV was safe and efficacious and might be a potential candidate for a live attenuated marker vaccine against PRV.

Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli.

Porcine circovirus type 2 (PCV2) capsid (Cap) protein has been successfully used as a vaccine to control porcine circovirus associated disease (PCVAD). Most PCV2 subunit vaccines are recombinant Cap protein expressed in baculovirus/insect cell expression system, but using this eukaryotic system is laborious and expensive. In our previous study, full-length of PCV2Cap protein expressed in Escherichia coli formed virus-like particles (VLPs). This expression system has the advantages of being relatively simple and inexpensive. In this study, we constructed a recombinant plasmid containing the full-length codon-optimized cap (ORF2) gene to improve high-level expression of recombinant Cap protein (rCap) with no changed amino acids. The highly water-soluble rCap protein was purified by a single-column, high-throughput fractionation procedure based on size exclusion chromatography. Yield was 10mg per 200ml bacterial culture. The rCap protein self-assembled into VLPs of diameter 25-30nm that contained exogenous nucleic acids. The immunogenicity of PCV2 VLPs was analyzed by immunizing mice. VLP-immunized mice mounted specific immune responses to PCV2. Thus, expression of rCap in E. coli was feasible for large-scale production of PCV2 VLPs, which could potentially be used for a VLP-based PCV2 vaccine.

The preparation of porcine circovirus type 2 (PCV2) virus-like particles using a recombinant pseudorabies virus and its application to vaccine development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of an economically important swine disease, now known as porcine-associated disease (PCVAD). The only structural protein of viral capsid, Cap has become the major target for development of PCV2 subunit vaccines. The purpose of this study is to express Cap of PCV2 using a recombinant pseudorabies virus (PRV) that is gE gene deficient, which is a widely used PRV marker vaccine. The recombinant PRV, gE(-)/PCV2cap(+)PRV, was constructed using homologous recombination techniques, in order to replace the upstream of the gE gene with the PCV2 cap gene. The expression of Cap during virus replication was confirmed using immunofluorescence and Western blotting analysis. The expressed Cap protein self-assembled into virus-like particles (VLPs), which was demonstrated using electromicrography. The immunization of mice or guinea pigs with purified VLPs could induce significant, specific antibody responses to PCV2 Cap. These results demonstrate an alternative to PCV2 for the development of a VLP-based subunit vaccine.

Kidney stone distribution caused by melamine and cyanuric acid in rats.

BACKGROUND: Melamine (M), which is composed of multi-amine, has been used as a food additive to falsely increase protein contents. Furthermore, cyanuric acid (CA) is a derivative of melamine. It is known that these mixtures can cause renal toxicity. METHODS: The objective of this study was to investigate the possible target cells during acute renal toxicity of melamine and cyanuric acid (MCA) mixture crystals in vivo. Rats were provided with a lethal dose of MCA (1:1; 400mg/kg) and observed after 0.5, 1, 3, 12, 24, and 48-h intervals. RESULTS: MCA caused degeneration/necrosis in the proximal tubules starting at 12h and increased at 24 and 48 h. A small number of yellow-green crystals were observed in the dilated distal renal tubules at 48 h post-treatment. Ultrastructurally, pyknosis, mitochondrial vesicles, and cellular swelling were found in the proximal tubular cells at 0.5h. Small needle-like crystals in the cytoplasm and large crystals in the lumen of tubules indicated physical damage to the renal cells. CONCLUSION: These results clearly reveal that in the MCA-induced renal toxicity model, crystals are distributed to both the proximal and distal tubules in rats. The proximal tubular cells may be initially injured and subsequently block the distal tubules with MCA crystals during early acute intoxication.

Enhancing expression of the classical swine fever virus glycoprotein E2 in yeast and its application to a blocking ELISA.

Classical swine fever virus (CSFV) infection is a severe swine disease, often causing large economic losses. A Pichia pastoris yeast-expressed CSFV glycoprotein E2 (yE2) has been shown to induce a protective immune response against the virus. To improve the expression level of yE2, the first codon of E2 gene, Arg (CGG), which is the least used in P. pastoris, was optimized to the most favorite codon AGA. The yield of E2 protein was remarkably increased in the codon optimized strain (N342). Three truncated E2 subunits encoding the N-terminal 330 (N330), 301 (N301), and 190 (N190) residues, respectively, were also constructed. The immunogenicity of each recombinant E2 subunits was confirmed by immunization of pigs, and all immunized groups demonstrated high neutralizing antibody titers after boost immunization, which lasted for a long period of time. In addition, a monoclonal antibody (MAb), 1B6, specific to yE2, was generated and shown to recognize CSFV-infected cells. A panel of swine sera were tested by peroxidase-conjugated MAb 1B6-based blocking enzyme-linked immunosorbent assay (ELISA) using N330 as coated antigen, and the assay demonstrated high sensitivity and specificity. The recombinant yE2 subunits may provide potential subunit vaccine candidates and useful diagnostic reagents for CSFV with easy manipulation and low cost.

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10.

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin- resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.

A low-pH medium in vitro or the environment within a macrophage decreases the transcriptional levels of fimA, fimZ and lrp in Salmonella enterica serovar Typhimurium.

Many Salmonella Typhimurium isolates produce type 1 fimbriae and exhibit fimbrial phase variation in vitro. Static broth culture favours the production of fimbriae, while solid agar medium inhibits the generation of these appendages. Little information is available regarding whether S. Typhimurium continues to produce type 1 fimbriae during in vivo growth. We used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCRwhen the pH of static brothmediumwas shifted frompH 7 to amore acidic pH 4. A fimA-deleted strain continued to multiply within macrophages as did the parental strain. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. We propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA.

Characterization of the swine U6 promoter for short hairpin RNA expression and its application to inhibition of virus replication.

Expression of short hairpin RNAs (shRNAs) by the RNA polymerase type III U6 promoter is an effective and widely used strategy for RNA interference (RNAi) which is a sequence-specific gene silencing mechanism. The U6 promoters from human, mouse, and swine were cloned, respectively for constructing various shRNA expression vectors. The transcription efficiency of each U6 promoter was analyzed for its activity to drive expression of shRNA targeting enhanced green fluorescent protein (EGFP) mRNA in different mammalian cells. All three U6 promoters were functional and the swine U6 promoter demonstrated the most efficient knockdown of EGFP synthesis in all these three species of cell lines including porcine kidney (PK-15), human embryonic kidney (HEK293T), and mouse fibroblast (LM) cells. Furthermore, the antiviral effect of shRNA targeting the classical swine fever virus (CSFV) NS5B driven by the swine U6 promoter was confirmed by the significant reduction of virus replication.

Effects of sodium citrate on melamine-cyanuric acid mixture-induced urolithiasis in rats.

BACKGROUND: When melamine is used as an additive in infant formula, it may cause acute nephrotoxicity in humans as well as in other animals. This study was designed to examine the effects of a melamine-cyanuric acid mixture on cytotoxicity in vitro and rat-acute nephrotoxicity. METHODS: In the in vitro study, crystal formations created by the melamine-cyanuric acid mixture were evaluated in media with differing pH conditions over a 24-h period and co-treatment with sodium citrate to observe the crystal formation. In the animal study, rats were exposed to a melamine-cyanuric acid mixture (400 mg/kg, 1:1) via oral gavage 14 days and co-treated with sodium citrate to observe the crystal formation in rats. RESULTS: Melamine-cyanuric acid mixture-induced crystal was pH dependent in a conditioned medium, and sodium citrate could decrease the crystal formation. Melamine-cyanuric acid-treated rats showed marked kidney swelling, vacuolization and necrosis in the proximal tubules, and numerous polarizable crystals were located in the distal segments, causing increases in kidney weight, serum BUN and creatinine. After co-treatment with sodium citrate, these increases can all be reversed. Moreover, the degrees of nephrotoxicity, proliferating of cell nuclear antigen protein and urolithiasis-related osteopontin were also decreased in the kidneys. CONCLUSION: Sodium citrate could decrease melamine-cyanuric acid mixture-induced crystal formation that leads to urolithiasis and nephrotoxicity in rats. These results may provide a strategy for melamine-cyanuric acid-intoxication therapy in animal.

Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.

Yeast expressed classical swine fever E2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission.

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E(rns) and E2 are major targets for eliciting antibodies against CSFV in infected animals. A Pichia pastoris yeast expressed E2 protein (yE2) has been shown to induce a protective immune response against CSFV challenge. The purpose of this study is to determine the optimal dose of yE2 and its efficacy on the prevention of virus horizontal transmission. A yeast-expressed E(rns) (yE(rns)) protein was also included to evaluate its immunogenicity. The yE(rns) vaccinated pigs seroconverted to CSFV-E(rns)-specific antibody but no neutralizing antibody was detected and none survived after challenge infection, suggesting yE(rns) and yE2 retain correct immunogenicity but only the yE2 is able to induce a protective immune response. All three doses of yE2 (200, 300, and 400µg) could elicit high titers of neutralizing antibodies and protective responses after challenge. The yE2/200 group demonstrated a mild fever response but recovered soon, and none of the yE2/300 and yE2/400 pigs became febrile. The optimal dose of yE2 was recommended to be 300µg of the total amount of secreted proteins. In addition, the yE2 vaccine could cross-protect from all three genotypes of viruses. Further, the yE2 vaccine efficacy in preventing virus horizontal transmission was evaluated by cohabitation of unimmunized sentinels 3 days after challenge infection. All the sentinel pigs were alive and had no clinical symptoms confirming yE2 vaccine could confer a protective immune response and prevent horizontal transmission of CSFV.