Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.

Establishment and application of multiplex real-time PCR for simultaneous detection of four viruses associated with porcine reproductive failure.

Many pathogens cause reproductive failure in sows suffering a broad spectrum of sequelae, including abortions, stillbirth, mummification, embryonic death, and infertility. Although various detection methods, such as polymerase chain reaction (PCR) and real-time PCR, have been widely used for molecular diagnosis, mainly for a single pathogen. In this study, we developed a multiplex real-time PCR method for the simultaneous detection of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine parvovirus (PPV) and pseudorabies virus (PRV) associated with porcine reproductive failure. The R 2 values for the standard curve of multiplex real-time PCR of PCV2, PCV3, PPV, and PRV reached to 0.996, 0.997, 0.996, and 0.998, respectively. Importantly, the limit of detection (LoD) of PCV2, PCV3, PPV, and PRV, were 1, 10, 10, 10 copies/reaction, respectively. Meanwhile, specificity test results indicated that multiplex real-time PCR for simultaneous detection is specific for these four target pathogens and does not react with other pathogens, such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine epidemic diarrhea virus. Besides, this method had good repeatability with coefficients of variation of intra- and inter-assay less than 2%. Finally, this approach was further evaluated by 315 clinical samples for its practicality in the field. The positive rates of PCV2, PCV3, PPV, and PRV were 66.67% (210/315), 8.57% (27/315), 8.89% (28/315), and 4.13% (13/315), respectively. The overall co-infection rates of two or more pathogens were 13.65% (43/315). Therefore, this multiplex real-time PCR provides an accurate and sensitive method for the identification of those four underlying DNA viruses among potential pathogenic agents, allowing it to be applied in diagnostics, surveillance, and epidemiology.

IFITM3 is a host restriction factor that inhibits porcine transmissible gastroenteritis virus infection.

Interferon-induced transmembrane proteins (IFITMs) play an important role in the innate immune response triggered by viral infection. Transmissible gastroenteritis virus (TGEV) causes severe diarrhea, vomiting and dehydration in piglets, resulting in huge economic losses to the swine industry. In this study, we showed that IFITM3 inhibits the replication of TGEV and interferes with the binding of TGEV to PK15 cells. Moreover, the inhibitory effect of IFITM3 on TGEV circumvents the upregulation of inflammatory cytokines. Subsequently, we found that the M22A mutant loses part of the antiviral effect of IFITM3 on TGEV; in contrast, the K24A mutant enhances the antiviral effect of IFITM3. Notably, our data shows a synergistic effect between IFITM3 and CQ, which further amplifies the antiviral effect against TGEV.

Relationship of noncoding RNA and swine viruses.

Swine viruses are well known as a threat to the pig industry. Many signaling pathways and a number of proteins were discovered to participate in the immune responses to swine viruses. Noncoding RNAs (ncRNAs), comprising a different set of transcripts including housekeeping RNAs (for example, rRNAs and tRNAs) and regulatory RNAs (small RNAs and long non-coding RNAs), recently have been described as important regulators of viral infections regarding to swine. A growing number of studies have shown ncRNAs are an indispensable part in the responses to virus infection, involved not only in antiviral responses, but in many interactions between host and virus, some of which may benefit the certain types of swine virus. Here we review the relationship of noncoding RNA and viruses through several representative swine viruses. Further, we discuss the potential of using ncRNAs as antiviral biomarkers.

Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.

Prevalence and genetic diversity of porcine circovirus type 2 in northern Guangdong Province during 2016-2021.

The emergence and widespread of porcine circovirus-associated diseases (PCVADs), mainly caused by porcine circovirus type 2 (PCV2), threatens the Chinese swine industry. In this study, to investigate the recent prevalence of PCV2 in northern Guangdong Province of China, 573 tissue samples from 132 pig farms were collected during 2016-2021 and analyzed via PCR. Overall, 51.38% (297/573, 95%CI 47.74-55.92) samples were tested PCV2 positive. The detection rate of PCV2 was significantly lower in samples collected before 2016-2018 than after the outbreak of African Swine Fever (2019-2021), being 59.85% (158/264, 95%CI 53.94-65.76) and 41.47% (141/340, 95%CI 36.43-46.71), respectively. On the other end, the genetic characteristics of 26 PCV2 strains were further analyzed. These PCV2 strains belonged to three genotypes, including PCV2a, PCV2b, and PCV2d. Specifically, the predominant genotype prevalent during two periods (2016-2018 and 2019-2021) wasPCV2b (81.82%, 9/11) and PCV2d (80.0%, 12/15), respectively. The results above illustrated the high prevalence and the genetic evolution feature of PCV2 in Guangdong Province in recent years.

Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies.

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

Distribution and persistence of residual T-2 and HT-2 toxins from moldy feed in broiler chickens.

T-2 and HT-2 widely found in food products can seriously affect human and animal health. In this study, sterilized corn was inoculated with F. poae and incubated to allow fungal growth before being examined via liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of T-2/HT-2. Broilers were then fed with a mix of moldy corn and normal feed at different ratios to obtain different toxin doses. After 35 days, the contaminated feed was replaced with mycotoxin-free feed and the distribution and concentration of residual toxins in the tissues and organs of the chickens were examined at different time points. The results showed that at the time of feed replacement (0 h), T-2 residue was present at significantly higher concentrations in the lungs and small intestines than in other tissues (P < 0.05). In addition, T-2 concentrations increased in a dose-dependent manner in the tissues of chickens in the low-, medium-, and high-dose groups; however, the differences in concentration between the groups were not statistically significant. The HT-2 content (0 h) in the livers and small intestines was significantly higher than that in other tissues (P < 0.05). At 48 h post-feed replacement, the concentration of T-2 dropped below detectable levels in all tissues while HT-2 could still be detected at 192 h post-feed replacement. Thus, this study reveals the distribution and persistence of residual T-2/HT-2 from moldy feed in broilers, providing a reference for the detection of these toxins in animal-derived food products and a theoretical basis for formulating food-safety and quality standards.

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation.

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

Epidemic and genetic characterization of porcine epidemic diarrhea virus strains circulating in the regions around Hunan, China, during 2017-2018.

Outbreaks of porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) infection have caused high mortality of piglets and significant economic losses to the Chinese swine industry. In the current study, 184 specimens from pigs with or without signs of diarrhea were collected from 39 farms across eight provinces, mainly around Hunan, People's Republic of China, in 2017 to 2018 in order to obtain epidemiological information on PEDV infections in these regions. The results indicated an average PEDV-positive rate of 38.04% (70/184) and more-pronounced disease severity in diarrheic pigs (48.76%; 59/121) than in non-diarrheic pigs (17.46%; 11/63). Phylogenetic and sequence analysis demonstrated that 14 representative PEDV strains from 14 swine farms belonged to the G2 group (G2-a and G2-b subgroups) and displayed a high degree of genetic variation. In particular, two out of the 14 PEDV strains were found to have unique indels in the S1 gene. The strain HN-SY-2017-Oct had a 9-nucleotide (T1152GAAGCCAAT1160T) insertion, and the strain ZJ-2018-May had a 3-nucleotide (AAA) deletion at position 1126 in the S1 gene. A three-dimensional structural prediction revealed that these unique insertions might lengthen the loop on the surface or increase the likelihood of the surface protein being phosphorylated at 388Y, thereby affecting the virulence or pathogenicity of PEDV. Collectively, the data show that PED remains a severe threat to the pig industry and that variant PEDV stains are circulating in China. The updated PEDV epidemiological data will facilitate the design of PEDV vaccines and the application of effective measures for PED prevention.

Protective humoral immunity in guinea pigs induced by PCV2 virus-like particles displaying the B cell linear epitope (228QQITDA233) of PPV1.

Although PCV2 infections generally cause mild disease in pigs, concurrent co-infections with other pathogens can damage the immune system and cause more severe diseases, collectively termed porcine circovirus associated diseases (PCVAD). Involvement of porcine parvovirus (PPV, a common cause of reproductive failure in naïve dams) in PCVAD caused by PCV2, has been reported. As this co-infection can be difficult to eliminate, there is a critical need to develop an effective vaccine to protect against PPV or synergistic effects of PCV2 and PPV under field conditions. In this study, we designed chimeric PCV2 virus-like particles (cVLPs) displaying a B-cell epitope derived from PPV1 structural protein around the surface of the 2-fold axes of PCV2 VLPs, based on 3D-structure analysis of the PCV2 capsid. The cVLPs were successfully prepared, verified by transmission electron microscopy and chromatography, with robust antibody titers against PCV2 and PPV1 produced in mice and guinea pigs. In addition, in guinea pigs challenged with 106 TCID50 PCV2, cVLPs conferred more effective immune protection (based on viral load) than a commercial PCV2 vaccine. Finally, antibody responses and immune protection against PPV were also evaluated. In guinea pigs vaccinated with cVLPs, although PPV antibodies detected by a hemagglutination inhibition (HI) assay appeared later after vaccination in the PCV2 cVLPs group than in the commercial PPV vaccine group, there were fewer PPV genomic DNA copies in the PCV2 cVLPs group than in a PBS group. In conclusion, guinea pigs vaccinated with cVLPs developed effective protective immunity against PCV2 challenge, with some protective immunity against PPV. This study provided valuable research data to pursue molecular design of chimeric epitopes PCV2 VLPs.

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells.

BACKGROUND: Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G710 to A710) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively. RESULTS: Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID50), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal. CONCLUSIONS: These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

Genome-wide analysis of long non-coding RNA expression profile in porcine circovirus 2-infected intestinal porcine epithelial cell line by RNA sequencing.

Porcine circovirus-associated disease (PCVAD), which is induced by porcine circovirus type 2 (PCV2), is responsible for severe economic losses. Recently, the role of noncoding RNAs, and in particular microRNAs, in PCV2 infection has received great attention. However, the role of long noncoding RNA (lncRNA) in PCV2 infection is unclear. Here, for the first time, we describe the expression profiles of lncRNAs in an intestinal porcine epithelial cell line (IPEC-J2) after PCV2 infection, and analyze the features of differently expressed lncRNAs and their potential target genes. After strict filtering of approximately 150 million reads, we identified 13,520 lncRNAs, including 199 lncRNAs that were differentially expressed in non-infected and PCV2-infected cells. Furthermore, trans analysis found lncRNA-regulated target genes enriched for specific Gene Ontology terms (P < 0.05), such as DNA binding, RNA binding, and transcription factor activity, which are closely associated with PCV2 infection. In addition, we analyzed the predicted target genes of differentially expressed lncRNAs, including SOD2, TNFAIP3, and ARG1, all of which are involved in infectious diseases. Our study identifies many candidate lncRNAs involved in PCV2 infection and provides new insight into the mechanisms underlying the pathogenesis of PCVAD.

The protective effects of DL-Selenomethionine against T-2/HT-2 toxins-induced cytotoxicity and oxidative stress in broiler hepatocytes.

T-2 and HT-2 toxins can cause cytotoxicity and oxidative stress in animals, while DL-Selenomethionine plays an important role in preventing oxidative stress and improving cell viability. However, the role of DL-Selenomethionine in T-2/HT-2 toxins-induced cell damage is still unknown. In this study, we investigated whether DL-Selenomethionine plays a protective role against T-2/HT-2-induced cytotoxicity and oxidative stress in primary hepatocytes. Our results demonstrated that T-2/HT-2 toxins-exposed broiler hepatocytes exhibited significantly decreased cell viability and intracellular glutathione (GSH) concentration while increased Lacate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and catalase (CAT) levels, as well as elevated expression levels of genes related to oxidative stress, in a toxin dose-dependent manner (P < 0.05). However, the application of DL-Selenomethionine into T-2/HT-2 treated hepatocytes effectively alleviated the adverse effects of T-2/HT-2, as demonstrated by increased cell viability, decreased LDH leakage, declined intracellular ROS and MDA levels, increased expression of oxidative stress-related genes, as well as accordingly enhanced activities of GSH, GSH-PX, SOD and CAT as compared to the control groups (P < 0.05). Therefore, our in vitro data demonstrate that DL-Selenomethionine can function as an effectively protective agent against T-2/HT-2-induced cytotoxicity and oxidative stress.

First molecular detection of porcine circovirus type 3 in dogs in China.

Porcine circovirus type 3 (PCV3) has recently been isolated from diseased pigs within the USA. The objective was to detect the presence of PCV3 in dogs. Nested polymerase chain reactions (PCR) with PCV3-specific primers for the capsid gene were used to detect PCV3 genomic DNA in serum samples from dogs (n = 44) in China. There was PCV3 DNA detected in 4 of 44 dogs [all were negative for PCV2 and canine circovirus (CanineCV)]. Based on sequence analysis, positive sequences were grouped into PCV3 genotypes. However, these isolates had close evolutionary relationships with FoxCV (KP941114) and CanineCV (JQ821392). Further investigations of the epidemiology, evolutionary biology, and pathobiology of PCV3 to dogs are warranted.

Evidence of natural co-infection with PCV2b subtypes in vivo.

Porcine circovirus type 2 (PCV2) is the causative pathogen of porcine circovirus-associated diseases (PCVAD). This virus evolves mostly through point mutations and genome recombination between different PCV2 genotypes (e.g. PCV2a and PCV2b), as has been confirmed in swine herds. In the current work, the complete PCV2 genome sequences of 69 clones derived from various tissues (lymph node, spleen and lung,) of an infected individual, were subjected to phylogenetic and alignment analyses. The results not only demonstrate co-infection with distinct PCV2b subtypes (e.g. 1B and 1C) in the same animal, but also highlight another mechanism of evolution - diverse point mutations acquired during immune evasion by this virus.

Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

Generation and immunogenicity of porcine circovirus type 2 chimeric virus-like particles displaying porcine reproductive and respiratory syndrome virus GP5 epitope B.

Virus-like particles (VLPs) can be used as transfer vehicles carrying foreign proteins or antigen epitopes to produce chimeric VLPs for bivalent or multivalent vaccines. Based on the crystal structure of porcine circovirus type 2 (PCV2) capsid protein (Cap), in addition to alignment of the Cap sequences collected from various isolates of PCV2 and PCV1, we predicted that Loop CD of the PCV2 Cap should tolerate insertion of foreign epitopes, and furthermore that such an insertion could be presented on the surface of PCV2 VLPs. To validate this, the GP5 epitope B of porcine reproductive and respiratory syndrome virus (PRRSV) was inserted into Loop CD of the PCV2 Cap. The 3D structure of the recombinant PCV2 Cap (rCap) was simulated by homology modeling; it appeared that the GP5 epitope B was folded as a relatively independent unit, separated from the PCV2 Cap backbone. Furthermore, based on transmission electron microscopy, the purified PCV2 rCap self-assembled into chimeric VLPs which entered PK-15 cells. In addition, PCV2 chimeric VLPs induced strong humoral (neutralizing antibodies against PCV2 and PRRSV) and cellular immune responses in mice. We concluded that the identified insertion site in the PCV2 Cap had great potential to develop PCV2 VLPs-based bivalent or multivalent vaccines; furthermore, it would also facilitate development of a nano-device to present a functional peptide on the surface of the VLPs that could be used for therapeutic purposes.