Porcine Circovirus Research Articles

277 Effect of yeast probiotics in lactation and nursery diets on progeny lifetime growth performance, immune response, and carcass characteristics

Abstract Mixed parity sows (n = 28; Line 241; DNA) and their offspring were used in a farrow-to-finish study to evaluate the effect of live yeast supplementation during lactation with or without yeast extract supplementation during the nursery period on sow and litter performance and lifetime growth performance, antibody titers, and carcass characteristics. Sows were blocked by parity and body weight (BW) on d 110 of gestation and allotted to 1 of 2 dietary treatments with 14 replications per treatment which consisted of a standard corn-soybean meal lactation diet with or without a yeast-based probiotic (0.10% Actisaf Sc 47 HR+; Phileo by Lesaffre, Milwaukee, WI). Following weaning, pigs (n = 350; 241×600, DNA; initial BW = 6.1 ± 0.02 kg) were randomly assigned in a 2 × 2 factorial design within sow treatment to 1 of 2 nursery diets which consisted of a control diet or a diet with pre-probiotic combination (0.10% MS309; Phileo by Lesaffre, Milwaukee, WI) fed for 42 d after weaning followed by common diets until marketing. Two nursery pens were combined in the finisher, such that there were 5 and 10 pigs/pen with 18 and 9 replications/treatment during the nursery and finishing periods, respectively. There were no significant effects of live yeast supplementation on lactation performance (P > 0.079). There was a sow × nursery diet interaction (P = 0.024) during the nursery period where pigs from sows fed Actisaf had increased average daily gain (ADG) when fed control nursery diets compared with pigs from control sows fed control nursery diets (Table). Pigs fed MS309 in the nursery from either sow treatment were intermediate. Pigs from sows fed Actisaf tended to be heavier at marketing (P = 0.067) with heavier hot carcass weight (HCW; P = 0.101), but there were no differences in overall finishing growth performance with the inclusion of yeast in lactation diets or nursery diets (P > 0.10). A subset of pigs were sampled at 7 time points to determine serum porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae antibody sample-to-positive (S/P) ratios and at 5 time points to ascertain percent inhibition of Lawsonia intracellularis. For all 3 immunological criteria, there were no sow diet × nursery diet × day interactions. There was a sow diet × day interaction for PCV2 S/P ratio (P = 0.097) where offspring from Actisaf sows tended to have greater PCV2 S/P ratios at 101 d of age compared with offspring from control sows (P = 0.046). There was a PCV2 S/P ratio nursery diet × day interaction (P = 0.036) where pigs fed MS309 had reduced PCV2 S/P ratios at 66, 78, and 162 d of age (P ≤ 0.022). In conclusion, feeding a pre/probiotic combination in the nursery did not affect performance or immune criteria. Conversely, feeding a live yeast probiotic during lactation resulted in tendencies for increased nursery ADG, final BW, and HCW.

Molecular detection and genetic characteristics of porcine circovirus 3 and porcine circovirus 4 in central China.

Porcine circoviruses (PCVs) are a significant cause of concern for swine health, with four genotypes currently recognized. Two of these, PCV3 and PCV4, have been detected in pigs across all age groups, in both healthy and diseased animals. These viruses have been associated with various clinical manifestations, including porcine dermatitis and nephropathy syndrome (PDNS) and respiratory and enteric signs. In this study, we detected PCV3 and PCV4 in central China between January 2022 and February 2023. We tested fecal swabs and tissue samples from growing-finishing and suckling pigs with or without respiratory and systemic manifestations and found the prevalence of PCV3 to be 15.15% (15/99) and that of PCV3/PCV4 coinfection to be 4.04% (4/99). This relatively low prevalence might be attributed to the fact that most of the clinical samples were collected from pigs exhibiting respiratory signs, with only a few samples having been obtained from pigs with diarrhea. In some cases, PCV2 was also detected, and the coinfection rates of PCV2/3, PCV2/4, and PCV2/3/4 were 6.06% (6/99), 5.05% (5/99), and 3.03% (3/99), respectively. The complete genomic sequences of four PCV3 and two PCV4 isolates were determined. All four of the PCV3 isolates were of subtype PCV3b, and the two PCV4 isolates were of subtype PCV4b. Two mutations (A24V and R27K) were found in antibody recognition domains of PCV3, suggesting that they might be associated with immune escape. This study provides valuable insights into the molecular epidemiology and evolution of PCV3 and PCV4 that will be useful in future investigations of genotyping, immunogenicity, and immune evasion strategies.

Porcine circovirus 2d associated with stillborn mummified foetus of pig

Recurrent occurrence of stillborn piglets was noticed along with live births during farrowing in pigs in an organized pig farm of Uttar Pradesh. On detailed post mortem examination, externally, the foetus was found to be partially dehydrated and dark brown in colour with shrunken eyes. Internally, visceral organs including lungs, heart, kidneys, liver, spleen and intestine were partially dehydrated and dark brown in colour. Upon molecular investigation, the pooled tissue samples were found positive for porcine circovirus 2 (PCV2) infection using PCR technique and further confirmed the PCV2d genotype by DNA sequencing. Other most possible infections including porcine parvoviral infection (PPV), PCV3 and porcine respiratory and reproductive syndrome virus infection (PRRS) were found negative using PCR and RT-PCR techniques, respectively. This result is suggestive of role of PCV2d as a possible etiological agent to cause stillbirth and mummification of porcine foetuses.

Development and preliminary application of a quadruplex real-time PCR assay for differential detection of porcine circovirus 1-4 in Chengdu, China.

Porcine circovirus (PCV) typically causes severe immune suppression in pigs, leading to mixed clinical infections with various pathogens that can cause significant harm to the pig industry. PCV has four subgenotypes, with PCV4 being an emerging virus that requires investigation due to its potential for epidemic outbreaks. Therefore, there is a need to develop a method that can detect all four PCV strains simultaneously. In this study, four pairs of specific primers and TaqMan probes were designed based on the conserved sequence of the PCV1-4 ORF2 gene to establish a PCV1-4 TaqMan multiplex real-time quantitative PCR method. The novel method was compared to six commercial testing kits for its efficacy. Then, a total of 595 mixed samples of spleen and lymph node collected from 12 districts in Chengdu from July to December 2021 were tested using the novel method. The results showed that the novel PCV1-4 TaqMan multiplex real-time quantitative PCR detection method has satisfied specificity, sensitivity, and repeatability. The positive rates of PCV1, PCV2, and PCV3 in Chengdu were 2.18%, 31.60%, and 15.29%, respectively, while no positive PCV4 was detected. The mixed infection rate of PCV2 and PCV3 was 5.21%. Our novel method may be as a potential method for PCV1-4 detection. Currently, PCV2 is the main epidemic PCV subtype in Chengdu, while the potential threat of PCV4 should also be considered.

Establishment of real-time viral pathogen detection method-surface-enhanced Raman scattering system for porcine circovirus type II

The transition towards large-scale intensive pig farming methods has resulted in an increase in cases of complex, mixed infections, and secondary infections. Porcine circovirus (PCV), the causative agent of PCV disease (PCVD), is a small nonenveloped DNA virus containing a single-stranded circular genome. Previously, PCV was even considered nonpathogenic to pigs. However, a novel strain of PCV, designated PCV-2, has been associated with various disease syndromes in pigs over the last 5 years. PCV-2 primarily induces post-weaning multisystemic wasting syndrome, characterized by clinical signs such as debility, dyspnea, palpable lymphadenopathy, diarrhea, and pallor or icterus. PCV-2 is now recognized as an important emerging pathogen. Early diagnosis and disease prevention have become crucial for effective pig farm management. However, clinical diagnosis may be complicated by antibiotic treatment and atypical disease symptoms. To ensure accurate pathology diagnosis, it is essential to integrate robust laboratory diagnostics with traditional methods. Surface-enhanced Raman Scattering (SERS) spectroscopy has emerged as a potentially powerful technique for whole-organism fingerprinting, enabling rapid identification of bacteria. Biosensors utilizing SERS offer promising capabilities for sensitive and quick detection of bacterial pathogens, thereby reducing diagnosis time. In this study, our aim was to characterize and evaluate a SERS-based diagnostic system for detecting and identifying PCV-2 present in pooled swine sera and feces. We compared the spectra of PCV-2 recovered from the specimens to those of pure PCV-2 to determine the PCV-2 molecular fingerprint. Our results demonstrated successful detection, identification, and classification of PCV-2 in swine sera and feces using SERS. SERS provided reproducible molecular spectroscopic signatures suitable for analytical applications. This approach presents a new and potent tool for real-time surveillance of animal viral pathogens in clinical settings.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Porcine circovirus 3: a new challenge to explore.

The intensification of production processes, resulting from the rise in pork production, contributes to environmental changes and increased interaction between humans, animals, and wildlife. This favorable scenario promotes the spread of potent viral species, such as PCV3, increasing the potential for the emergence of new pathogenic agents and variants. These changes in the epidemiology and manifestation of PCV3 highlight the need for enhanced understanding and control. The current literature presents challenges in the classification of PCV3, with different groups proposing diverse criteria. Establishing common terminology is crucial to facilitate comparisons between studies. While consensus among experts is valuable, new approaches must be transparent and comparable to existing literature, ensuring reproducible results and proper interpretation, and positively impacting public health. This study aims to review the literature on PCV3 infection, exploring its key aspects and highlighting unanswered questions.

Frequency of seropositivity against porcine circovirus type 2 (PCV2) in the metropolitan area of Monterrey, Nuevo León, and its peripheral area

El circovirus porcino tipo 2 (PCV2) es un virus de tipo DNA que tiene afinidad por células del sistema inmune y que genera depleción de linfocitos por lo que favorece el desarrollo de enfermedades causadas por otros agentes oportunistas, así mismo está relacionado con la generación de diferentes síndromes. Por lo anterior, este virus produce importantes afecciones a la industria porcícola, sin embargo, fácilmente se pueden prevenir los síndromes asociados a PCV2 mediante la adecuada aplicación de medidas de bioseguridad y vacunación. Por otro lado, las unidades de producción a pequeña escala (UPPs) suelen carecer de este tipo de manejo preventivo, así como de vigilancia rutinaria por parte de un médico veterinario. Si bien, el PCV2 se considera un virus ampliamente distribuido, no existen reportes de su presencia en las UPPs en Nuevo León. Se determinó la presencia de anticuerpos contra PCV2 mediante el uso de un kit comercial y se realizó la biometría hemática de los animales. Se localizaron 48 UPPs en las que se encontró un 91.67% de positividad, así como un 89.7% de seropositividad en los animales. En la biometría se encontró que la HGB y el HCT se presentaron disminuidos en los individuos que resultaron positivos a anticuerpos comparados contra los negativos (P=0.03 y P=0.01, respectivamente), por el contrario, el valor de células blancas totales se encontró disminuido en los individuos que resultaron negativos a la presencia de anticuerpo contra PCV2 (P=0.01).

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.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis.The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Effect of PCV-2 Vaccination on Cytokines Gene Expression Profile in Wild Boar Peripheral Blood Mononuclear Cells after Stimulation with Mycobacteria Antigens

Wild boar (Sus scrofa) is a common wild ungulate known as the most important reservoir of tuberculosis (TB) in Spain. The severity of TB lesions in this species and the high prevalence of porcine circovirus type 2 (PCV-2) have been related. PCV-2 is ubiquitous in swine populations, being usual for the free-living ones the contact with this agent. Recent studies found a correlation between a decrease of generalised TB prevalence in wild boar populations and the PCV-2-vaccination. The aim of this study was to find out if PCV-2 vaccination modulates the gene expression of cytokines from immune cells after its exposition with mycobacterial antigens using an in vitro methodology. A total of 46 wild boars from a PCV-2 infection endemic area were blood-sampled before and after the PCV-2 vaccination of 22 of them. Peripheral blood mononuclear cells (PBMC) were obtained and isolated from these samples. Aliquots of the cells were in vitro cultured and respectively stimulated with PPDa, PPDb, and a mitogen. A complete analysis of the gene expression of cytokines from the cultured PBMC was carried out. Also, Mycobacterium bovis and PCV-2 contacts were revealed by ELISA and/or qPCR. The results demonstrated that the animals which have had contact with PCV-2 and had been vaccinated, manifested a significant decrease in gene expression of proinflammatory cytokines, like interleukin 1 beta, interleukin 6, and tumour necrosis factor-alpha, possibly related with the severity of TB lesions, and also a significant decrease of interleukin 10, a key cytokine. In conclusion, in case of possible infection or contact events with the virus, PCV-2 vaccination could be an effective measure to reduce the TB severity in wild boar populations, which could decrease the intra and interspecies transmission of TB.

Assessment of sow herd frequency of PCV-2 using placental umbilical cord serum and serology in 18 breeding farms in Brazil.

Porcine circovirus type 2 (PCV-2) is the agent of one of the most important diseases in the swine industry. Although it has been controlled through vaccination, viremic piglets at birth may represent a risk by reducing vaccination efficacy. Since there are few reports on the viremic status of pre-suckling piglets regarding PCV-2 infection, we assessed the PCV-2 frequency in sows housed in 18 breeding farms with no history of clinical PCVAD in Brazil, using placental umbilical cord serum (PUCS). The selection criteria were: breeding farms with more than 1,000 sows; sows not vaccinated for PCV-2 at least for 2 years prior to the study; farms with no history of PCV-2 clinical disease in the last 12 months; and production systems with a maximum of two sites. Blood from the umbilical cords in sow placenta or directly from piglet's immediately after birth was collected from 30 litters on each farm for PCR. In addition, blood from 538 sows was collected for PCV-2 antibody detection. A total of 17.29% of the PUCS tested positive. The PCV-2 DNA was detected in PUCS from 94.4% of all farms. A total of 94.8% of the sows was positive for PCV-2 antibodies. However, seronegative sows were detected in 44.4% of farms. All 18 farms had at least 46.9% seropositive dams. A higher percentage of seronegative sows was observed for farms with more than 10% of PCV-2-positive litters compared to those with ≤10% of PCV-2 positive litters (8.9 +/-1.7% vs. 1.5 +/- 0.7%, p < 0.01, respectively).

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, β-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.

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

Identification of the Effects of 5-Azacytidine on Porcine Circovirus Type 2 Replication in Porcine Kidney Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.

Crystal structure of the ATPase domain of porcine circovirus type 2 Rep protein.

PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and β-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.

Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.

Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China. In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice. rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators. rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.

Humoral and Cellular Immune Responses Induced by Bivalent DNA Vaccines Expressing Fusion Capsid Proteins of Porcine Circovirus Genotypes 2a and 2b.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated disease (PCVAD) that profoundly impacts the swine industry worldwide. While most of the commercial PCV vaccines are developed based on PCV genotype 2a (PCV2a), PCV genotype 2b (PCV2b) has become predominant since 2003. In this study, we developed and evaluated DNA-based bivalent vaccines covering both PCV2a and PCV2b. We generated a new immunogen, PCV2b-2a, by combining consensus sequences of the PCV2a and PCV2b capsid proteins (Cap2a and Cap2b) in a form of fusion protein. We also examined whether modifications of the PCV2b-2a fusion protein with a signal sequence (SS) and granulocyte macrophage-colony stimulating factor (GM-CSF) fusing with interleukine-4 (IL-4) (GI) could further improve the vaccine immunogenicity. An immunogenicity study of BALB/cAJcl mice revealed that the DNA vector pVAX1 co-expressing PCV2b-2a and GI (pVAX1.PCV2b-2a-GI) was most potent at inducing both antibody and cellular immune responses against Cap2a and Cap2b. Interestingly, the vaccines skewed the immune response towards Th1 phenotype (IgG2a > IgG1). By performing ELISA and ELISpot with predicted epitope peptides, the three most immunogenic B cell epitopes and five putative T cell epitopes were identified on Cap2a and Cap2b. Importantly, our DNA vaccines elicited broad immune responses recognizing both genotype-specific and PCV2-conserved epitopes. Sera from mice immunized with the DNAs expressing PCV2b-2a and PCV2b-2a-GI significantly inhibited PCV2a cell entry at serum dilution 1:8. All these results suggest a great potential of our PCV2b-2a-based vaccines, which can be further developed for use in other vaccine platforms to achieve both vaccine efficacy and economical production cost.

Divergent clinical outcomes depending on the sequential infection order of porcine circovirus type 2 and Mycoplasma hyopneumoniae

This study compared the different sequential order of infection of porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae. Thirty-six pigs were allocated randomly across six different groups. Pigs underwent various inoculation sequences: M. hyopneumoniae administered 14 days before PCV2d, simultaneous PCV2d-M. hyopneumoniae, PCV2d given 14 days before M. hyopneumoniae, PCV2d only, M. hyopneumoniae only, or a mock inoculum. Overall, the pigs inoculated with M. hyopneumoniae 14 days prior to PCV2d (Mhyo-PCV2 group) and those inoculated simultaneously with PCV2d and M. hyopneumoniae (PCV2+Mhyo group) displayed notably higher clinical disease severity and experienced a significant decrease of their average daily weight gain than pigs inoculated with PCV2d 14 days prior to M. hyopneumoniae (PCV2-Mhyo group). M. hyopneumoniae infection potentiated PCV2 blood and lymph node viral loads, as well as PCV2-associated lesions, while the infection of PCV2d did not impact the intensity of M. hyopneumoniae infection. Tumor necrosis factor-α (TNF-α) sera levels were significantly increased in the Mhyo-PCV2 and PCV2+Mhyo groups as compared to the PCV2-Mhyo, PCV2, and Mhyo groups. The most important information was that the potentiation effect of M. hyopneumoniae on PCV2d was found only in pigs inoculated with either M. hyopneumoniae followed by PCV2d (Mhyo-PCV2 group) or a simultaneous inoculation of PCV2d and M. hyopneumoniae (PCV2+Mhyo group). The sequential infection order of PCV2d and M. hyopneumoniae resulted in divergent clinical outcomes.

Construction of prokaryotic system for expression of porcine circovirus type 2 ORF-2 gene fragment

Porcine circovirus-associated diseases (PCVDs) are among the most significant challenges for pig farming in developed countries. Porcine circovirus type 2 (PCV-2) is considered the main etiological agent of postweaning multisystemic wasting syndrome in piglets. Mass PCVD occurrence has been reported in most regions of the world, that results in serious economic consequences. Optimal PCVD prevention is known to be achieved through a set of veterinary and sanitary measures in combination with vaccination. High evolutionary virus variability facilitating new genotype and strain emergence requires development of new candidate recombinant vaccines against PCV-2 infection. The study was aimed at construction of prokaryotic system for PCV-2 ORF-2 gene fragment expression and its functionality assessment. A genetic insert constructed from the most immunogenic type-specific PCV-2 epitopes based on genotype 2a, 2b, 2d strain and isolate consensus sequence was cloned into the expression vector pET-22b(+) that was incorporated into the Escherichia coli strain Rosetta 2(DE3). The transformants were selected based on the marker gene of ampicillin resistance on a selective medium. Target gene expression was induced by adding of isopropyl-β-D-1-thiogalactopyranoside at different concentrations. As a result, Escherichia coli Rosetta 2(DE3)/pET-22b-ORF-2 strain, a producer of capsid protein fragment (92–233 amino acid residues), was constructed. It was found that in the presence of 1 mM isopropyl-β-D-1-thiogalactopyranoside, the expression level of soluble truncated rCap was 35–40 mg/L 6 hours after induction. The expression product was tested for its specificity with indirect ELISA using whole-virion PCV-2-hyperimmunized porcine serum. It was shown that the positivity coefficient of producer strain cell lysates averaged to 4.34 (p &lt; 0.005). The recombinant rCap protein is suitable for serological diagnosis and is also of interest as a vaccine component, which is the goal of our further studies.