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.

The Association between Serum Creatinine/Cystatin C Ratio and Cardiovascular Morbidity and Mortality: Insights from NHANES.

Background: The Serum creatinine/cystatin C ratio (Cr/CysC ratio) is an emerging alternative index for muscle mass loss, a risk factor for cardiovascular diseases (CVDs). However, the association between the Cr/CysC ratio and CVD morbidity and mortality remains unknown. Methods: A total of 11,150 participants of the National Health and Nutrition Examination Survey (NHANES) were included in this study. Univariable and multivariable logistic regression models were employed to assess the association between the Cr/CysC ratio and self-reported CVD morbidity. Cox proportional hazard models were used to estimate the hazard ratio (HR) and 95% confidence interval (CI) of the Cr/CysC ratio for CVD mortality. Results: At baseline, 1181 (7.90%) participants had self-reported CVDs. Lower Cr/CysC ratios were found in participants with CVDs (1.18 ± 0.30 vs. 1.05 ± 0.23, p < 0.001). In the multivariable logistic regression model, the Cr/CysC ratio was inversely linked to CVD morbidity (odds ratio: 0.65, 95% CI: 0.52-0.81, p < 0.001, per standard deviation [SD] increase). 997 (8.94%) CVD deaths were documented during a median follow-up of 16.9 years. A higher Cr/CysC ratio was associated with a decreasing risk of CVD mortality (adjusted HR: 0.54, 95% CI: 0.46-0.65, p < 0.001, per SD increase). Conclusions: In NHANES participants, the Cr/CysC ratio had an inverse correlation with CVD morbidity and mortality.

Complete genome and pathogenesis of a novel recombinant Senecavirus A isolate in P.R. China.

Senecavirus A (SVA), formerly called Seneca Valley virus (SVV) was first isolated from the USA in 2002. This study isolated an SVA strain from a pig herd in Shandong Province, PR China and designated it SVA-CH-SDGT-2017. The full-length genome, excluding the poly(A) tails of the SVA isolates, was 7280 nucleotides long, with the genomic organization resembling and sharing high nucleotide identities of 90.7-96.9 % with other previously reported SVA isolates. To investigate the pathogenicity of the SVA isolates, experimental infections of pigs were performed. The SVA strains successfully infected the pigs, as evidenced by the presence of virus shedding and robust serum neutralizing antibody responses. In addition, the contact-exposed experiment showed that the virus shedding of the contact-exposed pigs was approximately a 100-fold reduced compared to that of the inoculated group, indicating that the virus is capable of transmission to pigs. Our findings provide useful data for studying the pathogenesis and transmission of SVA in pigs.

A chimeric PCV rescued virus with the immunogenic cap gene of PCV3 cloned into the genomic backbone of the nonpathogenic PCV1 induces specific antibodies but with no pathogenic in pigs.

A chimeric PCV called PCV1-3 with the immunogenic Cap gene of pathogenic PCV type 3(PCV3) cloned into the genomic skeleton of the nonpathogenic PCV1 was rescued and inoculated into PCV3 negative piglets. The results of fluorescence quantitative PCR showed that the PCV1-3 DNA detected in serum and tissues was negative. The pathogenicity of piglets showed that PCV 1-3 did not cause the clinical characteristics and pathological changes. The viral neutralization assay revealed that infected pigs could produce antibodies and neutralize the viral activity. All results showed that chimeric virus induced specific antibodies but with no pathogenic in pigs, which provided new candidate strains for the development of PCV3 vaccine.

Mutation of the sixth amino acid of the Rep protein has no effect on porcine circovirus 2b but enhances porcine circovirus 2d replication in vitro.

Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication.

The correlation between the mutual deletions of amino acids within porcine circovirus rep protein and the discrepancy of replication.

Porcine circovirus (PCV) has two potential open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA. In some studies, PCV1 replicated more efficiently in PK-15 cells than PCV2 was elucidated. PCV1 compared with PCV2; there is some amino acids' deficiency on Rep protein. To identify whether the above amino acids deletion affects the replication of PCV1 and PCV2, we constructed three double copy clones by overlap extension PCR. The 2PCV2(vV) clone deleted the valine of Rep protein in the backbone of PCV2 genome. The 2PCV2(dSGR) clone inserted serine, glycine and arginine of Rep protein successively in the backbone of PCV2 genome. The 2PCV2(dSGR&vV) clone inserted serine, glycine and arginine as well as deleted the valine of Rep protein in the backbone of PCV2 genome. These clones we constructed with amino acid mutations and parental DNA clones were all transfected in PK-15 cells that free of PCV contamination, and their growth characteristics in vitro were determined and compared, to evaluating the replication of the mutant infectious DNA clones. Our results showed that the double copy infectious clones with amino acid mutations could be rescued in vitro. The 2PCV2(vV) replicated more efficiently than parental viruses 2PCV2 and 2PCV1 but the replicated ability of 2PCV2(dSGR) and 2PCV2(dSGR&vV) is attenuated than parental viruses 2PCV2 and 2PCV1. We can determine the valine is the important amino acid that cause PCV1 replicated more efficiently in PK-15 cells than PCV2 primarily. These findings are benefit for exploring the mechanisms of viral replication in pigs and important implications for PCV2 vaccine development.

Identifying domains of EFHC1 involved in ciliary localization, ciliogenesis, and the regulation of Wnt signaling.

EFHC1 encodes a ciliary protein that has been linked to Juvenile Myoclonic Epilepsy. In ectodermal explants, derived from Xenopus laevis embryos, the morpholino-mediated down-regulation of EFHC1b inhibited multiciliated cell formation. In those ciliated cells that did form, axoneme but not basal body formation was inhibited. EFHC1b morphant embryos displayed defects in central nervous system (CNS) and neural crest patterning that were rescued by a EFHC1b-GFP chimera. EFHC1b-GFP localized to ciliary axonemes in epidermal, gastrocoele roof plate, and neural tube cells. In X. laevis there is a link between Wnt signaling and multiciliated cell formation. While down-regulation of EFHC1b led to a ~2-fold increase in the activity of the ß-catenin/Wnt-responsive TOPFLASH reporter, EFHC1b-GFP did not inhibit ß-catenin activation of TOPFLASH. Wnt8a RNA levels were increased in EFHC1b morphant ectodermal explants and intact embryos, analyzed prior to the on-set of ciliogenesis. Rescue of the EFHC1b MO's ciliary axonemal phenotypes required the entire protein; in contrast, the EFHC1b morpholino's Wnt8a, CNS, and neural crest phenotypes were rescued by a truncated form of EFHC1b. The EFHC1b morpholino's Wnt8a phenotype was also rescued by the injection of RNAs encoding secreted Wnt inhibitors, suggesting that these phenotypes are due to effects on Wnt signaling, rather than the loss of cilia, an observation of potential relevance to understanding EFHC1's role in human neural development.

The integrity of PRRSV nucleocapsid protein is necessary for up-regulation of optimal interleukin-10 through NF-κB and p38 MAPK pathways in porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease, has been constantly causing huge economic losses all over the world. PRRS virus (PRRSV) infection results in immunosuppression and IL-10 up-regulation. The relationship between them is still in dispute. Previous studies demonstrated the protein of PRRSV nucleocapsid (N) protein is able to up-regulate IL-10, yet the underlying molecular mechanisms remain unknown. In this study, the expression kinetics of IL-10 up-regulation induced by PRRSV N protein were analyzed in immortalized porcine alveolar macrophages (PAMs). N protein induced IL-10 expression in a time- and dose-dependent manner. Inhibition experiments of signaling pathways suggested NF-κB and p38 MAPK pathways are both involved in N protein-induced IL-10 up-regulation. Besides, the integrity of N protein is essential for significant IL-10 up-regulation. This research is beneficial for further understanding of the interplay between PRRSV and host immune system.

Immunogenicity of porcine circovirus type 2 nucleic acid vaccine containing CpG motif for mice.

BACKGROUND: This study aimed at reseaching the immune effect of porcine circovirus type 2 (PCV2) DNA vaccine containing CpG motif on mice. METHODS: A total of 40 6-week-old female BALB/c mice were randomly divided into four groups which were immunized by 18CpG-pVAX1-ORF2, pVAX1-ORF2, pVAX1 and PBS, respectively, and immunized again 2 weeks later. All mice were challenged with 0.2 mL PCV2 cells virulent strain SD (106.0 TCID50/mL) after 4 weeks. Average daily gain, blood antibody levels, microscopic changes and viremia were detected to estimate the effect of DNA vaccine. RESULTS AND DISCUSSION: The results showed that compared to those of the control mice, groups immunized with pVAX1-ORF2 and 18CpG-pVAX1-ORF2 could induce PCV2-specific antibodies. The PCV2-specific antibodies level of 18 CpG-pVAX1-ORF2 groups was higher significantly than other groups and decreased slowly along with time. There was no distinct pathological damage and viremia occurring in mice that inoculated with CpG motif DNA vaccines. The results demonstrated that the DNA vaccine containing 18 CpG could build up resistibility immunity and reduce immune organ damage on mice.

Chibby functions in Xenopus ciliary assembly, embryonic development, and the regulation of gene expression.

Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates ß-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby's embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates ß-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby's role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning.

Phylogenetic analysis of 32 porcine circovirus type 2 isolates from Shandong, China.

Porcine circovirus type 2 (PCV2), the essential causative agent of postweaning multisystemic wasting syndrome (PMWS), can be divided into distinct genotypes. Thirty-two PCV2 isolates obtained made from pigs in Shandong Province between 2005 and 2013. Complete genome sequences were obtained in three replicates for each virus isolate, and the sequences were submitted to the NCBI database. The ORF1-encoded amino acid sequences had 98.4 %-100 % identity among the 32 isolates, and there were no significant differences among the three potential glycosylation positions: aa 23-25 (NPS), aa 256-258 (NQT) and aa 286-288 (NAT). The amino acid sequences of ORF2 had 88 %-100 % identity among the 32 isolates and the potential glycosylation position in the cap protein, aa 143-145 (NYS), had no variation. Phylogenetic analysis revealed that the PCV2b/1C genetic lineage was prevalent in swine populations in Shandong Province. It also suggested that selection pressure has made the PCV2 isolates more genetically distant from current vaccine strains.

Snail2 controls mesodermal BMP/Wnt induction of neural crest.

The neural crest is an induced tissue that is unique to vertebrates. In the clawed frog Xenopus laevis, neural crest induction depends on signals secreted from the prospective dorsolateral mesodermal zone during gastrulation. The transcription factors Snail2 (Slug), Snail1 and Twist1 are expressed in this region. It is known that Snail2 and Twist1 are required for both mesoderm formation and neural crest induction. Using targeted blastomere injection, morpholino-based loss of function and explant studies, we show that: (1) Snail1 is also required for mesoderm and neural crest formation; (2) loss of snail1, snail2 or twist1 function in the C2/C3 lineage of 32-cell embryos blocks mesoderm formation, but neural crest is lost only in the case of snail2 loss of function; (3) snail2 mutant loss of neural crest involves mesoderm-derived secreted factors and can be rescued synergistically by bmp4 and wnt8 RNAs; and (4) loss of snail2 activity leads to changes in the RNA levels of a number of BMP and Wnt agonists and antagonists. Taken together, these results identify Snail2 as a key regulator of the signals involved in mesodermal induction of neural crest.

Identification of putative virulence-associated genes among Haemophilus parasuis strains and the virulence difference of different serovars.

This study was aimed at determining virulence-associated genes among Haemophilus parasuis (H. parasuis) strains, and supplying for the Kielstein-Rapp-Gabrielson serotyping scheme. The subtractive fragments, obtained through suppression subtractive hybridization and reverse Southern blot hybridization, were found to encode genes representative of 7 different functions. PCR was used to investigate the distribution of these fragments in H. parasuis strains isolated from different infection sites in pigs. Mice challenge was then used to analyze the correlationship between subtractive fragments, infection sites and bacterial virulence. Eight weeks old female BALB/c mice (10 mice/group) were inoculated intraperitoneally with 3.0 × 10(9) CFU suspension (0.5 ml/mouse) of H. parasuis strains in PBS. Results indicated that H. parasuis possessed varied virulence even among the same serovar strains. Transcription units hsdR, hsdS, gpT and ompP2, identified from the subtractive fragments, were uniformly expressed in highly virulent strains, while absent in weakly virulent strains, and demonstrated variable degrees of expression in moderately virulent strains. Moreover, H. parasuis strains, isolated from pericardium and heart blood, were all highly virulent strains, while from nasal cavity and joint were moderately or weakly virulent strains. This study indicated that fragments hsdR, hsdS, gpT and ompP2 were associated with the virulence of H. parasuis. The virulence of H. parasuis strains isolated from different infection sites was different. The current research provides a new reference for determining bacterial virulence in different H. parasuis strains.

Complete genome sequence of recombinant porcine circovirus type 2 strain SD-3.

We report here the genome sequence of a recombinant porcine circovirus type 2 strain SD-3, isolated from a commercial swine farm with an outbreak of postweaning multisystemic wasting syndrome (PMWS) in pigs in Shandong Province of China. The complete circular genome of this isolate is 1,767 nucleotides in length. This recombinant isolate has the ORF1 regions from PCV2a viruses and ORF2 regions from PCV2b. The findings will help us to understand the molecular evolution of porcine circovirus type 2 and the relationship between porcine circovirus type 2 and disease.

Complete genome sequence of porcine circovirus 2b strain Shandong.

Shandong is a porcine circovirus 2b (PCV2b) strain that was isolated and purified from tissue samples from pigs with postweaning multisystemic wasting syndrome (PMWS) in the Shandong Province of China. Here, we report the complete genome sequence of strain Shandong, which may aid in understanding the molecular characteristics of this strain.

Protective effect of polysaccharides on the stability of parenteral emulsions.

The main purpose of this study is to investigate the influence of two polysaccharides (dextran, hydroxyethyl starch) on the stability of parenteral emulsions. All parenteral emulsions were prepared by high-pressure homogenization. The influence of polysaccharides concentration was studied. The stabilities of autoclaving sterilization, centrifugation and freeze-thawing process were investigated extensively. Following the addition of polysaccharides, the stabilities of the parenteral emulsions were improved. A high-concentration polysaccharides solution (13%, w/v) produced better protection than a low one (1.3%, w/v), especially during freeze-thawing process. The protective mechanisms of polysaccharides were attributed to increasing systematic viscosity, non-frozen water absorbed by polysaccharides, formation of a linear bead-like structure and thicker mixed emulsifier film. Overall, polysaccharides can offer greatly increased protection for parenteral emulsions, and represent a novel protective strategy for improving the stability of this delivery system.

METTL14 and FTO mediated m6A modification regulate PCV2 replication by affecting miR-30a-5p maturity.

The epigenetic modification of the N6-methyladenosine (m6A) methylation plays an important role in virus infection and replication. However, its role in Porcine circovirus type 2 (PCV2) replication has not been well studied. Here, we demonstrated that m6A modifications are increased in PK-15 cells after PCV2 infection. In particular, PCV2 infection could increase the expression of methyltransferase METTL14 and demethylase FTO. Moreover, interfering with METTL14 accumulation reduced the m6A methylation level and virus reproduction, whereas depleting the FTO demethylase enhanced the m6A methylation level and stimulated virus reproduction. Besides, we showed that METTL14 and FTO regulate PCV2 replication by affecting the process of miRNA maturity, especially the miRNA-30a-5p. Taken together, our results demonstrated that the m6A modification positively affects PCV2 replication and the role of m6A modification in the replication mechanism of the PCV2 virus provides a new idea for the prevention and control of the PCV2.

Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon α/γ in immediate and long-lasting protection against HP-PRRSV challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pork industry. Current vaccination strategies only provide a limited protective efficacy. In this study, a DNA vaccine, pVAX1(©)-α-γ-GP35, co-expressing GP3 and GP5 of PRRSV with interferon α/γ was constructed, and its immediate and long-lasting protection against highly pathogenic PRRSV (HP-PRRSV) challenge were examined in pigs. For immediate protection, the results showed that pVAX1(©)-α-γ-GP35 could provide partially protective efficacy, which was similar to the pVAX1(©)-α-γ (expressing interferon α/γ). For long-lasting protection, pigs inoculated with pVAX1(©)-α-γ-GP35 developed significantly higher PRRSV-specific antibody response, T cell proliferation, IFN-γ, and IL-4, than those vaccinated with pVAX1(©)-GP35 (expressing GP3 and GP5 of PRRSV). Following homologous challenge with HP-PRRSV strain SD-JN, pigs inoculated with pVAX1(©)-α-γ-GP35 showed almost no clinical signs, no lung lesions, and significantly lower viremia, as compared to those in pVAX1(©)-GP35 group. It indicated that pVAX1(©)-α-γ-GP35 could induce enhanced immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs. The DNA vaccine pVAX1(©)-α-γ-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections.

Treatment of Preschool Children With Autism Spectrum Disorder: A Trial to Evaluate a Learning Style Profile Intervention Program in China.

Objective: To investigate whether the provision of learning style profile (LSP) training improves development in preschool children with autism spectrum disorder (ASD) in China and to describe the characteristics of children who benefit from the intervention. Methods: Eighty-one children aged 36 to 72 months who were diagnosed with ASD for the first time were recruited for the intervention group. All of them received 24 weeks of LSP training, consisting of hospital- and home-based training. Twenty-one children with ASD of the same age in the control group had never received any intervention after diagnosis but underwent an assessment. Assessments were conducted at baseline and 24 weeks later. Differences in the developmental level and severity of ASD symptoms over time and between groups were analyzed by repeated standardized measures. Secondary analyses examined age effects among the 36- 48-, 48- 60-, and 60-72-month age groups. Results: Within-group comparison of the intervention group revealed significant treatment effects after the intervention, according to: language, social and adaptive developmental quotients (DQs) of the China Developmental Scale; total Childhood Autism Rating Scale (CARS) score; and hyperactivity, peer problems, total difficulties, and prosocial behavior scores of the Strengths and Difficulties Questionnaire (SDQ). Similar gains were observed in gross and fine motor DQs of the China Developmental Scale and emotional symptoms and conduct problems scores of the SDQ; however, the differences between these pre- and postintervention scores did not reach statistical significance. Comparisons among the three age groups in the intervention groups demonstrated a significant age effect on adaptive DQs of the China Developmental Scale; total CARS score; hyperactivity, peer problems and total difficulties scores of the SDQ. Comparison between the intervention and control groups revealed significant treatment effects on language, social and adaptive DQs of the China Developmental Scale; total CARS score; and emotional symptoms, conduct problems, hyperactivity, peer problems, total difficulties, and prosocial behavior scores of the SDQ after the intervention. Similar gains were observed in gross and fine motor DQs of the China Developmental Scale, although differences between the two groups did not reach statistical significance. Conclusion: Our findings suggest that LSP training can effectively improve social behavior and reduce the severity of ASD symptoms in children with ASD. Our data also highlight the importance of early intervention.

CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy.

RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-DicerΔ- and BHK-Ago2Δ-. Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.