Isolation of porcine circovirus 3 using primary porcine bone marrow-derived cells.

Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6-10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3-4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.

[An in vitro self-ligation-based method for constructing infectious DNA clone of porcine circovirus type 2].

The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

First detection of PCV4 in swine in the United States: codetection with PCV2 and PCV3 and direct detection within tissues.

Since PCV4 was first described in 2019, the virus has been identified in several countries in Southeast Asia and Europe. Most studies have been limited to detecting PCV4 by PCR. Thus, PCV4 has an unclear association with clinical disease. This study utilized 512 porcine clinical lung, feces, spleen, serum, lymphoid tissue, and fetus samples submitted to the ISU-VDL from June-September 2023. PCV4 was detected in 8.6% of samples with an average Ct value of 33. While detection rates among sample types were variable, lymphoid tissue had the highest detection rate (18.7%). Two ORF2 sequences were obtained from lymphoid tissue samples and had 96.36-98.98% nucleotide identity with reference sequences. Direct detection of PCV4 by RNAscope revealed viral replication in B lymphocytes and macrophages in lymph node germinal centers and histiocytic and T lymphocyte infiltration in the lamina propria of the small intestine. PCV4 detection was most commonly observed in nursery to finishing aged pigs displaying respiratory and enteric disease. Coinfection with PCV2, PCV3, and other endemic pathogens was frequently observed, highlighting the complex interplay between different PCVs and their potential roles in disease pathogenesis. This study provides insights into the frequency of detection, tissue distribution, and genetic characteristics of PCV4 in the US.

Molecular characterization of canine circovirus based on the Capsid gene in Thailand.

BACKGROUND: Canine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach. RESULTS: According to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution. CONCLUSIONS: Our study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.

Epidemiological and evolutionary analysis of canine circovirus from 1996 to 2023.

BACKGROUND: Canine circovirus (CanineCV), a non-enveloped virus with a circular DNA genome, has been identified in various avian and mammalian species, including domestic and wild canids. This study aimed to comprehensively analyze the prevalence of CanineCV across diverse animal species in 11 provinces of China. RESULTS: A total of 1,666 serum samples were collected, revealing a 5.82% prevalence of CanineCV in dogs, with the highest rates being observed in southern and eastern China. Phylogenetic analysis of 266 global CanineCV genomes sourced from the NCBI identified six distinct genotypes, elucidating the complex dynamics of their evolution. Evidence suggested a potential bat origin for CanineCV, with positive selection and high rates of evolution being observed. Recombination analysis revealed dynamic genetic exchange, highlighting the intricate nature of CanineCV evolution. Mutational analysis identified key amino acid substitutions likely to influence the virus's adaptation. Additionally, glycosylation, palmitoylation, and SUMOylation sites were predicted, shedding light on crucial functional properties of the virus. CONCLUSIONS: This study provides a global perspective on the origin, genetic diversity, and evolutionary dynamics of CanineCV. Understanding these factors is crucial for elucidating its epidemiology and potential health risks.

Sophora subprostrate polysaccharide targets LncRNA MSTRG.5823.1 to suppress PCV2-mediated immunosuppression via TNF/NF-κB signaling.

Current evidence suggests that porcine circovirus type 2 (PCV2) infection induces immunosuppression in piglets. Sophora subprostrate polysaccharide (SSP) exhibits various pharmacological activities, including immunoregulatory, anti-inflammatory, antiviral, and antioxidant properties. However, the acts of lncRNAs in regulating the therapeutic effects of SSP on PCV2-infected RAW264.7 cells remains poorly understood. This study aimed to investigate the molecular mechanisms by which lncRNAs regulate PCV2-induced immunosuppression during SSP treatment. Our findings revealed that 1699 mRNAs, 373 lncRNAs, and 129 miRNAs were differentially expressed in PCV2-infected RAW264.7 cells. Additionally, 359 mRNAs, 271 lncRNAs, and 79 miRNAs exhibited differential expression in SSP-treated PCV2-infected RAW264.7 cells. GO and KEGG analyses indicated that the candidate genes were enriched in the TNF/NF-κB signaling pathway. Furthermore, based on GO and KEGG pathway analysis, a ceRNA network involving chemokine (C-X-C motif) ligand 2 (CXCL2), miR-217-x, and MSTRG.5823.1 was constructed. We demonstrated that lncRNA MSTRG.5823.1 localized to the cytoplasm. Moreover, we found that silencing or overexpressing lncRNA MSTRG.5823.1 significantly modulated PCV2-induced immunosuppression by regulating the activation of the TNF/NF-κB signaling pathway. Specifically, lncRNA MSTRG.5823.1 overexpression increased the expression of TNF/NF-κB signaling pathway-related genes and proteins in PCV2-infected RAW264.7 cells. Conversely, silencing lncRNA MSTRG.5823.1 decreased their expression. Rescue assays further revealed that the suppressive effects of miR-217-x overexpression on TNF/NF-κB signaling pathway-related genes and proteins could be reversed by MSTRG.5823.1 overexpression. These findings highlight the critical role of lncRNA MSTRG.5823.1 in PCV2 infection progression and suggest a new strategy for the prevention and treatment of PCV2 infection.

The pigeon circovirus evolution, epidemiology and interaction with the host immune system under One Loft Race rearing conditions.

This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective.

Importance of sequential infection order of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus to divergent clinical outcomes.

This study was designed to investigate the different sequential order of infection for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). Thirty-six pigs were randomly assigned to six different treatment groups. The first (hereafter referred to as PRRSV-PCV2) group was inoculated with PRRSV first followed by PCV2d. The second (hereafter referred to as PCV2+PRRSV) group was co-infected with both viruses at the same timepoint (42 days of age). The third (hereafter referred to as PCV2-PRRSV) group was inoculated with PCV2d first followed by PRRSV. A fourth group was only inoculated with PCV2d at 42 days of age, while a fifth group was only inoculated with PRRSV at the same timepoint. The sixth group served as a negative control group. The most important observation discovered that PRRSV only had a potentiation effect on PCV2 in both PRRS-PCV2 and PCV2+PRRSV groups. Both PRRSV-PCV2 and PCV2+PRRSV groups experienced a significant reduction in growth performance compared with control pigs. In addition, PRRSV-PCV2 and PCV2+PRRSV groups exhibited a greater severity in their clinical signs, and/or had higher PCV2 blood and lymphoid viral loads that resulted in a stronger severity of lymphoid lesions compared with PCV2-PRRSV group. Serum TNF-α levels were significantly higher in both PRRS-PCV2 and PCV2+PRRSV groups compared with those in PCV2-PRRS, PCV2, and PRRSV groups. The results of this study demonstrated that divergent clinical outcomes are dependent on the sequential infection order of PCV2 and PRRSV.

Identification and characterization of a novel circovirus in Iberian lynx in Spain.

Circoviruses cause severe disease in pigs and birds. Canine circovirus has thus far only been associated with respiratory and gastrointestinal disorders and systemic disease in dogs. The Iberian lynx (Lynx pardinus) is one of the most endangered carnivores in Europe and the most endangered felid worldwide. Exploring the virome of these animals may be important in terms of virus discovery and assessing the interspecies-circulation of viruses from related carnivores. In this study, 162 spleen samples from Iberian lynx were screened for CRESS DNA viruses. Overall, 11 (6.8%) of 162 samples tested positive using a consensus PCR. Partial rep sequences were tightly related to each other (96.6-100%). Specific molecular protocols were designed on the partial rep sequences of the novel virus, Iberian lynx-associated circovirus-1 (ILCV-1). By screening a subset of 45 spleen samples, the infection rate of ILCV-1 in Iberian lynxes was 57.8% (26/45). ILCV-1 strains formed a separate cluster intermingled with bat, rodent, mongoose, and felid circoviruses. The genome of the novel virus displayed the highest nucleotide identity (64.3-65.3%) to mongoose circoviruses, thus representing a novel candidate circovirus species. The detection of these viruses in the spleen tissues could suggest systemic infection in the animal host. Overall, these findings suggest that this novel circovirus is common in the Iberian lynx. Further studies are warranted to assess the possible health implications of ILCV-1 in this endangered species.

First Molecular Characterisation of Porcine Parvovirus 7 (PPV7) in Italy.

Porcine parvoviruses (PPVs) are among the most important agents of reproductive failure in swine worldwide. PPVs comprise eight genetically different species ascribed to four genera: Protoparvovirus (PPV1, PPV8), Tetraparvovirus (PPV2-3), Copiparvovirus (PPV4-6), and Chaphamaparvovirus (PPV7). In 2016, PPV7 was firstly detected in the USA and afterwards in Europe, Asia, and South America. Recently, it was also identified in Italy in pig farms with reproductive failure. This study aimed to evaluate the circulation of PPV7 in domestic and wild pigs in Sardinia, Italy. In addition, its coinfection with Porcine Circovirus 2 (PCV2) and 3 (PCV3) was analysed, and PPV7 Italian strains were molecularly characterised. PPV7 was detected in domestic pigs and, for the first time, wild pigs in Italy. The PPV7 viral genome was detected in 20.59% of domestic and wild pig samples. PPV7 detection was significantly lower in domestic pigs, with higher PCV2/PCV3 co-infection rates observed in PPV7-positive than in PPV7-negative domestic pigs. Molecular characterisation of the NS1 gene showed a very high frequency of recombination that could presumably promote virus spreading.

Identification and genomic analysis of a pathogenic circovirus associated with maricultured Scophthalmus maximus L. in China.

In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture.

Comparative immunogenic and immunoprotective activities of PCV2d Cap and Rep antigens delivered by an efficient eukaryotic expression system engineered into a Salmonella vaccine vector.

Porcine circovirus type 2 (PCV2) stands as a predominant etiological agent in porcine circovirus-associated diseases. To manage the spread of the disease, it is necessary to develop a next-generation vaccine expressing PCV2 antigens that target the prevailing genotype such as PCV2d. A bacterial-mediated vaccine delivery by live-attenuated Salmonella has attracted interest for its low-cost production and highly effective vaccine delivery. Thus, in this study, we utilized the advantages of the Salmonella-mediated vaccine delivery by cloning PCV2d cap and rep into a eukaryotic expression plasmid pJHL204 and electroporation into an engineered live-attenuated Salmonella Typhimurium JOL2500 (Δlon, ΔcpxR, ΔsifA, Δasd). The eukaryotic antigen expression by JOL2995 (p204:cap) and JOL2996 (p204:rep) was confirmed in vitro and in vivo which showed efficient antigen delivery. Furthermore, vaccination of mice model with the vaccine candidates elicited humoral and cell-mediated immune responses as depicted by high levels of PCV2-specific antibodies, CD4+ and CD8+ T cells, and neutralizing antibodies, especially by JOL2995 (p204:cap) which correlated with the significant decrease in the viral load in PCV2d-challenged mice. Interestingly, JOL2996 (p204:rep) may not have elicited high levels of neutralizing antibodies and protective efficacy, but it elicited considerably higher cell-mediated immune responses. This study demonstrated Salmonella-mediated vaccine delivery system coupled with the eukaryotic expression vector can efficiently deliver and express the target PCV2d antigens for strong induction of immune response and protective efficacy in mice model, further supporting the potential application of the Salmonella-mediated vaccine delivery system as an effective novel approach in vaccine strategies for PCV2d.

First report of goose circovirus identified in ducks from China.

Goose circovirus (GoCV) is a common pathogen that causes immunosuppression and promotes secondary infections with other infectious agents in geese worldwide. In the present study, we identified GoCV in 2 out of 93 duck flocks from China and successfully sequenced the complete genomes of 2 strains (AH22du and HN20du). The whole genome of the two strains shared a high identity of 90.5 to 98.63% with China GoCV reference, and low identity of 58.98% with DuCV reference, respectively. Phylogenetic tree constructed on the two and other genome sequences of GoCV revealed three main branches. Both strains sequenced in this study were distributed on different sub-branches with most other Chinese GoCV strains, and AH22du clustered into an independent sub-branch within the cluster. Recombination analysis predicted that HN20du might potentially recombine from the major parent of yk4 (Zhejiang Province, China, 2007) and minor parent of GD/YJ/g2 (Guangdong Province, China, 2020). To the best of our knowledge, this is the first report of GoCV in ducks from China. This broadened host spectrum of GoCVs requires attention from the waterfowl industry and researchers.

Identification of a conserved B-cell epitope on the capsid protein of porcine circovirus type 4.

Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225PKQG228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies. IMPORTANCE: Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.

The porcine circovirus 3 humoral response: characterization of maternally derived antibodies and dynamic following experimental infection.

Since Porcine Circovirus 3 (PCV3) was first identified in 2016, our understanding of the humoral response is still relatively scarce. Current knowledge of the PCV3 humoral response is primarily based on field studies identifying the seroprevalence of PCV3 Cap-induced antibodies. Studies on the humoral response following experimental PCV3 infection have conflicting results where one study reports the development of the Cap IgG response 7 days postinfection with no concurrent Cap IgM response, while a second study shows a Cap IgM response at the same time point with no detection of Cap IgG. The dynamics of the PCV3 Cap and Rep IgG following maternal antibody transfer and experimental infection have not been well characterized. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected animals to serologic methods of the alternate PCV has limited evaluation. Here, we show that maternally derived antibodies were detectable in piglet serum 7-9 weeks postfarrowing for the Cap IgG and 5-weeks-post farrowing for the Rep IgG using Cap- and Rep-specific enzyme linked immunosorbent assays (ELISA) and immunofluorescent assays (IFA) methods. Following experimental inoculation, Cap IgG was detected at 2-weeks-post inoculation and Rep IgG detection was delayed until 4-weeks-post inoculation. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by serological methods against the other PCV. The information gained in this study highlights the development of both the Cap- and Rep-specific antibodies following experimental infection and through the transfer of maternal antibodies. The increased understanding of the dynamics of maternal antibody transfer and development of the humoral response following infection gained in the present study may aid in the establishment of husbandry practices and potential application of prophylactics to control PCV3 clinical disease. IMPORTANCE: Research on Porcine Circovirus 3 (PCV3) immunology is vital for understanding and controlling this virus. Previous studies primarily relied on field observations, but they have shown conflicting results about the immunological response against PCV3. This study helps fill those gaps by looking at how antibodies develop in pigs, especially those maternal-derived, and their impact in neonatal pigs preventing PCV3-associated disease in piglets. In addition, we look at the dynamics of antibodies in experimental infections mimicking infection in pigs in the grower-phase condition. Understanding this process can help to develop better strategies to prevent PCV3 infection. Also, this research found that PCV2 and PCV3 do not cross-react, which is crucial for serological test development and results interpretation. Overall, this work is essential for improving swine health and farming practices in the face of PCV3 infections.

Codon usage bias of goose circovirus and its adaptation to host.

Goose circovirus (GoCV), a potential immunosuppressive virus possessing a circular single-stranded DNA genome, is widely distributed in both domesticated and wild geese. This virus infection causes significant economic losses in the waterfowl industry. The codon usage patterns of viruses reflect the evolutionary history and genetic architecture, allowing them to adapt quickly to changes in the external environment, particularly to their hosts. In this study, we retrieved the coding sequences (Rep and Cap) and the genome of GoCV from GenBank, conducting comprehensive research to explore the codon usage patterns in 144 GoCV strains. The overall codon usage of the GoCV strains was relatively similar and exhibited a slight bias. The effective number of codons (ENC) indicated a low overall extent of codon usage bias (CUB) in GoCV. Combined with the base composition and relative synonymous codon usage (RSCU) analysis, the results revealed a bias toward A- and G-ending codons in the overall codon usage. Analysis of the ENC-GC3s plot and neutrality plot suggested that natural selection plays an important role in shaping the codon usage pattern of GoCV, with mutation pressure having a minor influence. Furthermore, the correlations between ENC and relative indices, as well as correspondence analysis (COA), showed that hydrophobicity and geographical distribution also contribute to codon usage variation in GoCV, suggesting the possible involvement of natural selection. In conclusion, GoCV exhibits comparatively slight CUB, with natural selection being the major factor shaping the codon usage pattern of GoCV. Our research contributes to a deeper understanding of GoCV evolution and its host adaptation, providing valuable insights for future basic studies and vaccine design related to GoCV.

Establishment and application of multiplex PCR for rapid detection of three mink diarrhea-associated viruses.

In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and enteritis virus (MEV). Three compatible sets of primers specific for each virus were designed respectively based on their conserved sequences. After optimization of the crucial factors such as primer concentration and annealing temperature in single and multiple amplification, three specific fragments were simultaneously amplified with the highest sensitivity and specificity in one PCR reaction. The fragments amplified were 259 bp (MCV)，455 bp (MBoV) and 671 bp (MEV). The sensibility of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There were no cross-reactions with some relevant pathogens like mink coronavirus, canine distemper virus, and aleutian mink disease virus. In our study we analyzed viral DNA in mink fecal samples by multiplex PCR assay from China, which revealed the occurrence of MCV, MBoV, and MEV as 3.1 %, 5.7 %, and 9.8 %, respectively. The testing results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100 %. These results indicated that the method could provide technical support for rapid detection of the three diarrhea-associated viruses, and epidemiological investigation of mink viral diarrhea.

Development a high-sensitivity sandwich ELISA for determining antigen content of porcine circovirus type 2 vaccines.

Porcine circovirus type 2 (PCV2) is intensely prevalent in global pig farms. The PCV2 vaccine is an important means of preventing and controlling PCV2. The quality control of PCV2 vaccines is predominantly based on detection techniques such as animal testing and neutralizing antibody titration. Measuring the content of effective proteins in vaccines to measure vaccine efficacy is an excellent alternative to traditional methods, which can greatly accelerate the development speed and testing time of vaccines. In this study, we screened a monoclonal antibody (mAb) that can effectively recognize not only the exogenous expression of PCV2 Cap protein but also PCV2 virus. The double antibody sandwich ELISA (DAS-ELISA) was developed using this mAb that specifically recognize PCV2 Cap. The minimum protein content detected by this method is 3.5 ng/mL. This method can be used for the quality control of PCV2 inactivated vaccine and subunit vaccine, and the detection results are consistent with the results of mice animal experiments. This method has the advantages of simple operation, good sensitivity, high specificity and wide application. It can detect the effective antigen Cap protein content of various types of PCV2 vaccines, which not only shorten the vaccine inspection time but also save costs.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.